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1
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Schneider BM, Hamurcu HI, Salzbrunn A, von Kopylow K. Microfluidic systems in testicular in vitro culture: a powerful model tool for spermatogenesis and reprotoxicity studies. Asian J Androl 2025:00129336-990000000-00307. [PMID: 40260644 DOI: 10.4103/aja20254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2024] [Accepted: 01/22/2025] [Indexed: 04/23/2025] Open
Abstract
ABSTRACT As prepubertal boys do not yet produce spermatozoa, they cannot rely on sperm cryopreservation for fertility preservation before gonadotoxic therapy, such as high-dose alkylating agents or radiotherapy in the case of childhood cancers. According to the current guidelines, cryopreservation of testicular biopsies containing spermatogonial stem cells (SSCs) may be proposed to high-risk patients for potential later therapeutic use to fulfill the patients' wish for a biological child. One promising technique for human in vitro spermatogenesis and in vitro propagation of human SSCs is microfluidic (MF) culture, in which cells or tissues are subjected to a continuous flow of medium. This provides exact control over such parameters as nutrient content and gradients, as well as the removal of waste metabolites. While MF has been shown to maintain tissues and cell populations of organs for longer than conventional in vitro culture techniques, it has not been widely used for testicular in vitro culture. MF could advance human testicular in vitro culture and is also applicable to reprotoxicity studies. This review summarizes the findings and achievements of testis-on-chip (ToC) setups to date and discusses the benefits and limitations of these for spermatogenesis in vitro and toxicity assessment.
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Affiliation(s)
- Botho Maximilian Schneider
- Clinic and Polyclinic for Dermatology and Venerology, Andrological Section, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany
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Heisser RH, Bawa M, Shah J, Bu A, Raman R. Soft Biological Actuators for Meter-Scale Homeostatic Biohybrid Robots. Chem Rev 2025; 125:3976-4007. [PMID: 40138615 DOI: 10.1021/acs.chemrev.4c00785] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/29/2025]
Abstract
Skeletal muscle's elegant protein-based architecture powers motion throughout the animal kingdom, with its constituent actomyosin complexes driving intra- and extra-cellular motion. Classical motors and recently developed soft actuators cannot match the packing density and contractility of individual muscle fibers that scale to power the motion of ants and elephants alike. Accordingly, the interdisciplinary fields of robotics and tissue engineering have combined efforts to build living muscle actuators that can power a new class of robots to be more energy-efficient, dexterous, and safe than existing motor-powered and hydraulic paradigms. Doing so ethically and at scale─creating meter-scale tissue constructs from sustainable muscle progenitor cell lines─has inspired innovations in biomaterials and tissue culture methodology. We weave discussions of muscle cell biology, materials chemistry, tissue engineering, and biohybrid design to review the state of the art in soft actuator biofabrication. Looking forward, we outline a vision for meter-scale biohybrid robotic systems and tie discussions of recent progress to long-term research goals.
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Affiliation(s)
- Ronald H Heisser
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, United States of America
| | - Maheera Bawa
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, United States of America
| | - Jessica Shah
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, United States of America
- Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 45 Carleton St., Cambridge, Massachusetts 02142, United States of America
| | - Angel Bu
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, United States of America
| | - Ritu Raman
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, United States of America
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3
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Robinson MA, Kung SHY, Youssef KYM, Scheck KM, Bell RH, Sar F, Haegert AM, Asmae MM, Cheng C, Yeack SV, Mathur BT, Jiang F, Collins CC, Hach F, Willerth SM, Flannigan RK. 3D Bioprinted Coaxial Testis Model Using Human Induced Pluripotent Stem Cells:A Step Toward Bicompartmental Cytoarchitecture and Functionalization. Adv Healthc Mater 2025; 14:e2402606. [PMID: 39955738 PMCID: PMC12004438 DOI: 10.1002/adhm.202402606] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 02/04/2025] [Indexed: 02/17/2025]
Abstract
Fertility preservation following pediatric cancer therapy programs has become a major avenue of infertility research. In vitro spermatogenesis (IVS) aims to generate sperm from banked prepubertal testicular tissues in a lab setting using specialized culture conditions. While successful using rodent tissues, progress with human tissues is limited by the scarcity of human prepubertal testicular tissues for research. This study posits that human induced pluripotent stem cells (hiPSCs) can model human prepubertal testicular tissue to facilitate the development of human IVS conditions. Testicular cells derived from hiPSCs are characterized for phenotype markers and profiled transcriptionally. HiPSC-derived testicular cells are bioprinted into core-shell constructs representative of testis cytoarchitecture and found to capture functional aspects of prepubertal testicular tissues within 7 days under xeno-free conditions. Moreover, hiPSC-derived Sertoli cells illustrate the capacity to mature under pubertal-like conditions. The utility of the model is tested by comparing 2 methods of supplementing retinoic acid (RA), the vitamin responsible for inducing spermatogenesis. The model reveals a significant gain in activity under microsphere-released RA compared to RA medium supplementation, indicating that the fragility of free RA in vitro may be a contributing factor to the molecular dysfunction observed in human IVS studies to date.
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Affiliation(s)
| | - Sonia HY Kung
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | | | - Kali M Scheck
- Axolotl BiosciencesVictoriaBritish ColumbiaV8W 2Y2Canada
| | - Robert H Bell
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - Funda Sar
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - Anne M Haegert
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - M Mahdi Asmae
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - Changfeng Cheng
- Faculty of ForestryUniversity of British ColumbiaVancouverBritish ColumbiaV6T 1Z4Canada
| | - Salina V Yeack
- Axolotl BiosciencesVictoriaBritish ColumbiaV8W 2Y2Canada
| | - Bhairvi T Mathur
- Faculty of MedicineUniversity of British ColumbiaVancouverBritish ColumbiaV6T 1Z4Canada
| | - Feng Jiang
- Faculty of ForestryUniversity of British ColumbiaVancouverBritish ColumbiaV6T 1Z4Canada
| | - Colin C Collins
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - Faraz Hach
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
| | - Stephanie M Willerth
- Faculty of MedicineUniversity of British ColumbiaVancouverBritish ColumbiaV6T 1Z4Canada
- Department of Mechanical EngineeringUniversity of VictoriaVictoriaBritish ColumbiaV8P 5C2Canada
- Division of Medical SciencesUniversity of VictoriaVictoriaBritish ColumbiaV8P 5C2Canada
| | - Ryan K Flannigan
- Vancouver Prostate CentreVancouverBritish ColumbiaV6H 3Z6Canada
- Department of Urologic SciencesUniversity of British ColumbiaVancouverBritish ColumbiaV6T 1Z4Canada
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Shen J, Wang X, Yang C, Ren G, Wang L, Piao S, Zhang B, Sun W, Ge X, Jing J, Xiang Y, He Z, Wang L, Yao B, Liu Z. Development and evaluation of a microfluidic human testicular tissue chip: a novel in vitro platform for reproductive biology and pharmacology studies. LAB ON A CHIP 2025; 25:577-589. [PMID: 39820994 DOI: 10.1039/d4lc00780h] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2025]
Abstract
Organ-on-a-chip culture systems using human organ tissues provide invaluable preclinical insights into systemic functions in vitro. This study aimed to develop a novel human testicular tissue chip within a microfluidic device employing computer-aided design software and photolithography technology. Polydimethylsiloxane was used as the primary material to ensure marked gas permeability and no biotoxicity, enabling effective mimicry of the in vivo testicular microenvironment. This biochip preserved the structural integrity and cellular composition of human testicular tissue, as well as part of its functionality, over an extended period in vitro. Moreover, compared to traditional static culture methods, it more effectively maintained tissue viability and endocrine function. The chip maintained cellular components, histological morphology, and an ultrastructure similar to those in vivo. Notably, the addition of gonadotropins to the human testis tissue on the chip resulted in consistent and steady in vitro production of testosterone and inhibin B. Additionally, the chip displayed sensitivity to the reproductive toxicity of the chemotherapeutic drug busulfan. The results demonstrate the successful establishment of a novel human testicular tissue chip culture system, providing a novel in vitro approach enabling the exploration of human reproductive biology, reproductive pharmacology, toxicology, individual diagnosis, and treatment strategies.
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Affiliation(s)
- Jiaming Shen
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Xinlong Wang
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Chenghua Yang
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Guanyu Ren
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Lei Wang
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Shuguang Piao
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Boyang Zhang
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Weihao Sun
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
| | - Xie Ge
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Jun Jing
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Yijian Xiang
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Zhaowanyue He
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Linhui Wang
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Bing Yao
- Department of Reproductive Medicine, Nanjing Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Zhiyong Liu
- Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.
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Önen S, Gizer M, Çolak İÖ, Korkusuz P. Bioengineering Approaches for Male Infertility: From Microenvironmental Regeneration to in vitro Fertilization. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1479:59-72. [PMID: 39881052 DOI: 10.1007/5584_2024_844] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/31/2025]
Abstract
Male factor accounts for 30-50% of infertility cases and may occur due to congenital anomalies or acquired disorders. In such infertility cases where a limited number of mature sperm is produced, a solution is offered to patients with ART applications; however, these methods are inadequate in patients with germ cell aplasia due to damaged microenvironment. Since monolayer cell culture and static culture conditions do not provide the physical conditions of the 3D microenvironment, they have a limited effect on ensuring the execution of in vitro spermatogenesis properly. For this reason, current treatment approaches turn to biomaterial-implemented, microfluidic, and bioreactor systems where 3D physical conditions are provided. This book chapter focuses on static and dynamic culture conditions, as well as the use of biomaterials to increase the success of ex vivo spermatogenesis and microfluidic device-assisted sperm selection in ART.
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Affiliation(s)
| | | | - İmran Özge Çolak
- Department of Stem Cell Sciences, Graduate School of Health Sciences, Hacettepe University, Ankara, Turkey
| | - Petek Korkusuz
- METU MEMS Center, Ankara, Turkey.
- Department of Stem Cell Sciences, Graduate School of Health Sciences, Hacettepe University, Ankara, Turkey.
- Department of Histology and Embryology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
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6
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Aydin S, Yaşlı M, Yildiz Ş, Urman B. Advancements in three-dimensional bioprinting for reproductive medicine: a systematic review. Reprod Biomed Online 2024; 49:104273. [PMID: 39033691 DOI: 10.1016/j.rbmo.2024.104273] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 05/14/2024] [Accepted: 05/14/2024] [Indexed: 07/23/2024]
Abstract
Reproductive failure due to age, genetics and disease necessitates innovative solutions. While reproductive tissue transplantation has advanced, ongoing research seeks superior approaches. Biomaterials, bioengineering and additive manufacturing, such as three-dimensional (3D) bioprinting, are harnessed to restore reproductive function. 3D bioprinting uses materials, cells and growth factors to mimic natural tissues, proving popular for tissue engineering, notably in complex scaffold creation with cell distribution. The versatility which is brought to reproductive medicine by 3D bioprinting allows more accurate and on-site applicability to various problems that are encountered in the field. However, in the literature, there is a lack of studies encompassing the valuable applications of 3D bioprinting in reproductive medicine. This systematic review aims to improve understanding, and focuses on applications in several branches of reproductive medicine. Advancements span the restoration of ovarian function, endometrial regeneration, vaginal reconstruction, and male germ cell bioengineering. 3D bioprinting holds untapped potential in reproductive medicine.
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Affiliation(s)
- Serdar Aydin
- Department of Obstetrics and Gynaecology, Koc University Hospital, Zeytinburnu, Istanbul, Turkey; Koc University School of Medicine, Koc University, Sariyer, Istanbul, Turkey.
| | - Mert Yaşlı
- Koc University School of Medicine, Koc University, Sariyer, Istanbul, Turkey
| | - Şule Yildiz
- Department of Obstetrics and Gynaecology, Koc University Hospital, Zeytinburnu, Istanbul, Turkey; Koc University School of Medicine, Koc University, Sariyer, Istanbul, Turkey
| | - Bulent Urman
- Department of Obstetrics and Gynaecology, Koc University Hospital, Zeytinburnu, Istanbul, Turkey; Koc University School of Medicine, Koc University, Sariyer, Istanbul, Turkey; Department of Obstetrics and Gynaecology, American Hospital, Tesvikiye, Sisli, Istanbul, Turkey
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7
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Nakagiri H, Ogawa T, Ikeda N, Terasaka S, Nukada Y, Miyazawa M. Application of testicular organ culture system for the evaluation of spermatogenesis impairment. Sci Rep 2024; 14:21581. [PMID: 39285184 PMCID: PMC11405715 DOI: 10.1038/s41598-024-71561-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Accepted: 08/29/2024] [Indexed: 09/20/2024] Open
Abstract
Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.
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Affiliation(s)
- Hideaki Nakagiri
- Safety Science Research Laboratories, Kao Corporation, Haga, Tochigi, 321-3497, Japan.
| | - Takehiko Ogawa
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-Ku, Yokohama, Kanagawa, 236-0004, Japan
| | - Naohiro Ikeda
- Safety Science Research Laboratories, Kao Corporation, Haga, Tochigi, 321-3497, Japan
| | - Shimpei Terasaka
- Safety Science Research Laboratories, Kao Corporation, Haga, Tochigi, 321-3497, Japan
| | - Yuko Nukada
- Safety Science Research Laboratories, Kao Corporation, Haga, Tochigi, 321-3497, Japan
| | - Masaaki Miyazawa
- Safety Science Research Laboratories, Kao Corporation, Haga, Tochigi, 321-3497, Japan
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8
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Bashiri Z, Hosseini SJ, Salem M, Koruji M. In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration. Clin Exp Reprod Med 2024; 51:171-180. [PMID: 38525520 PMCID: PMC11372308 DOI: 10.5653/cerm.2023.06569] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Accepted: 12/14/2023] [Indexed: 03/26/2024] Open
Abstract
Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.
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Affiliation(s)
- Zahra Bashiri
- Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Omid Fertility and Infertility Clinic, Hamedan, Iran
| | - Seyed Jamal Hosseini
- Biomedical Engineering Department, Amirkabir University of Technology, Tehran, Iran
- Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
| | - Maryam Salem
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Morteza Koruji
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
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9
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Shoji JY, Davis RP, Mummery CL, Krauss S. Global Literature Analysis of Organoid and Organ-on-Chip Research. Adv Healthc Mater 2024; 13:e2301067. [PMID: 37479227 DOI: 10.1002/adhm.202301067] [Citation(s) in RCA: 12] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 07/14/2023] [Indexed: 07/23/2023]
Abstract
Organoids and cells in organ-on-chip platforms replicate higher-level anatomical, physiological, or pathological states of tissues and organs. These technologies are widely regarded by academia, the pharmacological industry and regulators as key biomedical developments. To map advances in this emerging field, a literature analysis of 16,000 article metadata based on a quality-controlled text-mining algorithm is performed. The analysis covers titles, keywords, and abstracts of categorized academic publications in the literature and preprint databases published after 2010. The algorithm identifies and tracks 149 and 107 organs or organ substructures modeled as organoids and organ-on-chip, respectively, stem cell sources, as well as 130 diseases, and 16 groups of organisms other than human and mouse in which organoid/organ-on-chip technology is applied. The analysis illustrates changing diversity and focus in organoid/organ-on-chip research and captures its geographical distribution. The downloadable dataset provided is a robust framework for researchers to interrogate with their own questions.
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Affiliation(s)
- Jun-Ya Shoji
- Hybrid Technology Hub, Center of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0372, Norway
| | - Richard P Davis
- Department of Anatomy & Embryology, Leiden University Medical Center, Leiden, 2300RC, the Netherlands
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden, 2300RC, the Netherlands
| | - Christine L Mummery
- Department of Anatomy & Embryology, Leiden University Medical Center, Leiden, 2300RC, the Netherlands
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden, 2300RC, the Netherlands
- Department of Applied Stem Cell Technologies, University of Twente, Enschede, 7522NB, the Netherlands
| | - Stefan Krauss
- Hybrid Technology Hub, Center of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0372, Norway
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10
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Lopez I, Truskey GA. Multi-cellular engineered living systems to assess reproductive toxicology. Reprod Toxicol 2024; 127:108609. [PMID: 38759876 PMCID: PMC11179964 DOI: 10.1016/j.reprotox.2024.108609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 05/07/2024] [Accepted: 05/09/2024] [Indexed: 05/19/2024]
Abstract
Toxicants and some drugs can negatively impact reproductive health. Many toxicants haven't been tested due to lack of available models. The impact of many drugs taken during pregnancy to address maternal health may adversely affect fetal development with life-long effects and clinical trials do not examine toxicity effects on the maternal-fetal interface, requiring indirect assessment of safety and efficacy. Due to current gaps in reproductive toxicological knowledge and limitations of animal models, multi-cellular engineered living systems may provide solutions for modeling reproductive physiology and pathology for chemical and xenobiotic toxicity studies. Multi-cellular engineered living systems, such as microphysiological systems (MPS) and organoids, model of functional units of tissues. In this review, we highlight the key functions and structures of human reproductive organs and well-known representative toxicants afflicting these systems. We then discuss current approaches and specific studies where scientists have used MPS or organoids to recreate in vivo markers and cellular responses of the female and male reproductive system, as well as pregnancy-associated placenta formation and embryo development. We provide specific examples of organoids and organ-on-chip that have been used for toxicological purposes with varied success. Finally, we address current issues related to usage of MPS, emerging techniques for improving upon these complications, and improvements needed to make MPS more capable in assessing reproductive toxicology. Overall, multi-cellular engineered living systems have considerable promise to serve as a suitable, alternative reproductive biological model compared to animal studies and 2D culture.
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Affiliation(s)
- Isabella Lopez
- Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States
| | - George A Truskey
- Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States.
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11
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Galdon G, Zarandi NP, Deebel NA, Zhang S, Cornett O, Lyalin D, Pettenati MJ, Lue Y, Wang C, Swerdloff R, Shupe TD, Bishop C, Stogner K, Kogan SJ, Howards S, Atala A, Sadri-Ardekani H. In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System. Bioengineering (Basel) 2024; 11:677. [PMID: 39061759 PMCID: PMC11274239 DOI: 10.3390/bioengineering11070677] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2024] [Revised: 06/21/2024] [Accepted: 06/22/2024] [Indexed: 07/28/2024] Open
Abstract
Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.
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Affiliation(s)
- Guillermo Galdon
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Facultad de Medicina, Universidad de Barcelona, 08036 Barcelona, Spain
| | - Nima Pourhabibi Zarandi
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Internal Medicine, University of Pittsburgh Medical Center, Harrisburg, PA 17101, USA
| | - Nicholas A. Deebel
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
| | - Sue Zhang
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
| | - Olivia Cornett
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
| | - Dmitry Lyalin
- Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
- Department of Pathology, Molecular Diagnostics Division, Virginia Commonwealth University, Richmond, VA 23284, USA
| | - Mark J. Pettenati
- Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
| | - YanHe Lue
- Division of Endocrinology, Department of Medicine, The Lundquist Institute, Harbor-University of California Los Angeles (UCLA) Medical Center, Los Angeles, CA 90502, USA
| | - Christina Wang
- Division of Endocrinology, Department of Medicine, The Lundquist Institute, Harbor-University of California Los Angeles (UCLA) Medical Center, Los Angeles, CA 90502, USA
| | - Ronald Swerdloff
- Division of Endocrinology, Department of Medicine, The Lundquist Institute, Harbor-University of California Los Angeles (UCLA) Medical Center, Los Angeles, CA 90502, USA
| | - Thomas D. Shupe
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
| | - Colin Bishop
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
| | - Kimberly Stogner
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
| | - Stanley J. Kogan
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
| | - Stuart Howards
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
| | - Anthony Atala
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
| | - Hooman Sadri-Ardekani
- Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
- Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
- Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
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12
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Park SR, Kook MG, Kim SR, Lee CM, Lee JW, Park JK, Park CH, Oh BC, Jung Y, Hong IS. Development of a novel testis-on-a-chip that demonstrates reciprocal crosstalk between Sertoli and Leydig cells in testicular tissue. Exp Mol Med 2024; 56:1591-1605. [PMID: 38945952 PMCID: PMC11297247 DOI: 10.1038/s12276-024-01258-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2023] [Revised: 02/19/2024] [Accepted: 03/19/2024] [Indexed: 07/02/2024] Open
Abstract
The reciprocal crosstalk between testicular Sertoli and Leydig cells plays a vital role in supporting germ cell development and maintaining testicular characteristics and spermatogenesis. Conventional 2D and the recent 3D assay systems fail to accurately replicate the dynamic interactions between these essential endocrine cells. Furthermore, most in vitro testicular tissue models lack the ability to capture the complex multicellular nature of the testis. To address these limitations, we developed a 3D multicellular testis-on-a-chip platform that effectively demonstrates the reciprocal crosstalk between Sertoli cells and the adjacent Leydig cells while incorporating various human testicular tissue constituent cells and various natural polymers infused with blood coagulation factors. Additionally, we identified SERPINB2 as a biomarker of male reproductive toxicity that is activated in both Sertoli and Leydig cells upon exposure to various toxicants. Leveraging this finding, we designed a fluorescent reporter-conjugated toxic biomarker detection system that enables both an intuitive and quantitative assessment of material toxicity by measuring the converted fluorescence intensity. By integrating this fluorescent reporter system into the Sertoli and Leydig cells within our 3D multicellular chip platform, we successfully developed a testis-on-chip model that can be utilized to evaluate the male reproductive toxicity of potential drug candidates. This innovative approach holds promise for advancing toxicity screening and reproductive research.
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Affiliation(s)
- Se-Ra Park
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Myung Geun Kook
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Soo-Rim Kim
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Choon-Mi Lee
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Jin Woo Lee
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Jung-Kyu Park
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea
| | - Chan Hum Park
- Department of Otolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, Hallym University College of Medicine, Chuncheon, Republic of Korea
| | - Byung-Chul Oh
- Department of Physiology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University College of Medicine, Incheon, 21999, Republic of Korea
| | - YunJae Jung
- Department of Microbiology, College of Medicine, Gachon University, Incheon, 21999, Korea
| | - In-Sun Hong
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea.
- Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 406-840, Republic of Korea.
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13
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Hashimoto K, Arakawa H, Imamura R, Nishimura T, Kitajima S, Sato T, Makiyama K, Ogawa T, Yokota S. A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system. J Appl Toxicol 2024; 44:784-793. [PMID: 38262615 DOI: 10.1002/jat.4584] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 12/27/2023] [Accepted: 01/08/2024] [Indexed: 01/25/2024]
Abstract
Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.
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Affiliation(s)
- Kiyoshi Hashimoto
- Department of Urology, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan
| | - Hiroshi Arakawa
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Rikako Imamura
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Takuya Nishimura
- Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
| | - Satoshi Kitajima
- Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
| | - Takuya Sato
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan
| | - Kazuhide Makiyama
- Department of Urology, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan
| | - Takehiko Ogawa
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan
| | - Satoshi Yokota
- Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
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14
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Ogawa T, Matsumura T, Yao T, Kimura H, Hashimoto K, Ishikawa-Yamauchi Y, Sato T. Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control. J Reprod Dev 2024; 70:1-9. [PMID: 38143077 PMCID: PMC10902634 DOI: 10.1262/jrd.2023-093] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Accepted: 11/22/2023] [Indexed: 12/26/2023] Open
Abstract
Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.
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Affiliation(s)
- Takehiko Ogawa
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
| | - Takafumi Matsumura
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
| | - Tatsuma Yao
- Research and Development Center, Fuso Pharmaceutical Industries, Ltd., Osaka 536-8523, Japan
| | - Hiroshi Kimura
- Micro/Nano Technology Center, Tokai University, Hiratsuka 259-1292, Japan
| | - Kiyoshi Hashimoto
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
| | - Yu Ishikawa-Yamauchi
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
| | - Takuya Sato
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
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15
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Bashiri Z, Gholipourmalekabadi M, Khadivi F, Salem M, Afzali A, Cham TC, Koruji M. In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility. Cell Tissue Res 2023; 394:393-421. [PMID: 37721632 DOI: 10.1007/s00441-023-03824-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 08/14/2023] [Indexed: 09/19/2023]
Abstract
Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.
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Affiliation(s)
- Zahra Bashiri
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Omid Fertility & Infertility Clinic, Hamedan, Iran.
| | - Mazaher Gholipourmalekabadi
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Farnaz Khadivi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Department of Anatomy, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Maryam Salem
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Azita Afzali
- Hajar Hospital, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Tat-Chuan Cham
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada
| | - Morteza Koruji
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
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16
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Despicht C, Munkboel CH, Chou HN, Ertl P, Rothbauer M, Kutter JP, Styrishave B, Kretschmann A. Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip. Anal Bioanal Chem 2023; 415:5421-5436. [PMID: 37438566 PMCID: PMC10444685 DOI: 10.1007/s00216-023-04816-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Revised: 06/16/2023] [Accepted: 06/19/2023] [Indexed: 07/14/2023]
Abstract
The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.
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Affiliation(s)
- Caroline Despicht
- Toxicology and Drug Metabolism Group, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen OE, Denmark
| | - Cecilie H Munkboel
- Toxicology and Drug Metabolism Group, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen OE, Denmark
| | - Hua Nee Chou
- Toxicology and Drug Metabolism Group, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen OE, Denmark
| | - Peter Ertl
- Institute of Applied Synthetic Chemistry, Institute of Chemical Technologies and Analytics, Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060, Vienna, Austria
| | - Mario Rothbauer
- Institute of Applied Synthetic Chemistry, Institute of Chemical Technologies and Analytics, Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060, Vienna, Austria
- Karl Chiari Lab for Orthopaedic Biology, Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Währinger Gürtel 18-22, 1090, Vienna, Austria
| | - Jörg P Kutter
- Microscale Analytical Systems, Department of Pharmacy, Faculty of Health and Medical Sciences, Univeristy of Copenhagen, Copenhagen, OE, Denmark
| | - Bjarne Styrishave
- Toxicology and Drug Metabolism Group, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen OE, Denmark.
| | - Andreas Kretschmann
- Toxicology and Drug Metabolism Group, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen OE, Denmark
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17
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Li CY, Liu SP, Dai XF, Lan DF, Song T, Wang XY, Kong QH, Tan J, Zhang JD. The emerging role of exosomes in the development of testicular. Asian J Androl 2023; 25:547-555. [PMID: 37040218 PMCID: PMC10521952 DOI: 10.4103/aja2022126] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Accepted: 02/04/2023] [Indexed: 04/12/2023] Open
Abstract
The mechanisms of testicular development in mammals are complex. Testis is an organ that produces sperm and secretes androgens. It is rich in exosomes and cytokines that mediate signal transduction between tubule germ cells and distal cells, promoting testicular development and spermatogenesis. Exosomes are nanoscale extracellular vesicles that transmit information between cells. By transmitting information, exosomes play an important role in male infertility diseases such as azoospermia, varicocele, and testicular torsion. However, due to the wide range of sources of exosomes, extraction methods are numerous and complex. Therefore, there are many difficulties in studying the mechanisms of exosomal effects on normal development and male infertility. Therefore, in this review, first, we introduce the formation of exosomes and methods for culturing testis and sperm. Then, we introduce the effects of exosomes on different stages of testicular development. Finally, we summarize the prospects and shortcomings of exosomes when used in clinical applications. We lay the theoretical foundation for the mechanism of the influence of exosomes on normal development and male infertility.
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Affiliation(s)
- Chun-Yang Li
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
| | - Song-Po Liu
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
| | - Xiao-Fang Dai
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
| | - Dong-Feng Lan
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
| | - Tao Song
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
- Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine, Zunyi Medical University, Zunyi 563000, China
| | - Xian-Yao Wang
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
- Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine, Zunyi Medical University, Zunyi 563000, China
| | - Qing-Hong Kong
- Guizhou Provincial College-Based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi 563000, China
| | - Jun Tan
- Department of Histology and Embryology, Zunyi Medical University, Zunyi 563000, China
| | - Ji-Dong Zhang
- Department of Immunology, Zunyi Medical University, Zunyi 563000, China
- Special Key Laboratory of Gene Detection and Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China
- Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine, Zunyi Medical University, Zunyi 563000, China
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18
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Hau RK, Wright SH, Cherrington NJ. In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier. Drug Metab Dispos 2023; 51:1157-1168. [PMID: 37258305 PMCID: PMC10449102 DOI: 10.1124/dmd.123.001288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 05/10/2023] [Accepted: 05/25/2023] [Indexed: 06/02/2023] Open
Abstract
The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.
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Affiliation(s)
- Raymond K Hau
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Stephen H Wright
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Nathan J Cherrington
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
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19
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Younis N, Caldeira-Brant AL, Chu T, Abdalla S, Orwig KE. Human immature testicular tissue organ culture: a step towards fertility preservation and restoration. Front Endocrinol (Lausanne) 2023; 14:1242263. [PMID: 37701899 PMCID: PMC10494240 DOI: 10.3389/fendo.2023.1242263] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2023] [Accepted: 08/07/2023] [Indexed: 09/14/2023] Open
Abstract
Background Cryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may not be safe or appropriate for all patients. Therefore, methods to mature ITT ex vivo are needed. Objectives Aim to investigate the feasibility of inducing in vitro spermatogenesis from ITT cryopreserved for pediatric patients prior to initiation of gonadotoxic therapy. Materials and methods Cryopreserved-thawed ITT from prepubertal and peripubertal patients were cultured for 7, 16, and 32 days in medium with no hormones or supplemented with 5 IU/L FSH, 1 IU/L hCG, or 5IU/L FSH+1 IU/L hCG. Samples were evaluated histologically to assess tissue integrity, and immunofluorescence staining was performed to identify VASA (DDX4)+ germ cells, UCHL1+ spermatogonia, SYCP3+ spermatocytes, CREM+ spermatids, SOX9+ Sertoli cells. Proliferation (KI67) and apoptosis (CASPASE3) of germ cells and Sertoli cells were also analyzed. Sertoli and Leydig cell maturation was evaluated by AR and INSL3 expression as well as expression of the blood testis barrier protein, CLAUDIN11, and testosterone secretion in the culture medium. Results Integrity of seminiferous tubules, VASA+ germ cells and SOX9+ Sertoli cells were maintained up to 32 days. The number of VASA+ germ cells was consistently higher in the peripubertal groups. UCHL1+ undifferentiated spermatogonia and SOX9+ Sertoli cell proliferation was confirmed in most samples. SYCP3+ primary spermatocytes began to appear by day 16 in both age groups. Sertoli cell maturation was demonstrated by AR expression but the expression of CLAUDIN11 was disorganized. Presence of mature and functional Leydig cells was verified by INSL3 expression and secretion of testosterone. Gonadotropin treatments did not consistently impact the number or proliferation of germ cells or somatic cells, but FSH was necessary to increase testosterone secretion over time in prepubertal samples. Conclusion ITT were maintained in organotypic culture for up to 32 days and spermatogonia differentiated to produce primary spermatocytes in both pre- and peripubertal age groups. However, complete spermatogenesis was not observed in either group.
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Affiliation(s)
- Nagham Younis
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
- Department of Biological Sciences, School of Science, University of Jordan, Amman, Jordan
| | - Andre L. Caldeira-Brant
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - Tianjiao Chu
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
| | - Shtaywy Abdalla
- Department of Biological Sciences, School of Science, University of Jordan, Amman, Jordan
| | - Kyle E. Orwig
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
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20
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Lee S, Ahn J, Kim SM, Kim D, Yeom J, Kim J, Park JY, Ryu BY. Fluid dynamic design for mitigating undesired cell effects and its application to testis cell response testing to endocrine disruptors. J Biol Eng 2023; 17:51. [PMID: 37550751 PMCID: PMC10408176 DOI: 10.1186/s13036-023-00369-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Accepted: 07/16/2023] [Indexed: 08/09/2023] Open
Abstract
Microfluidic devices have emerged as powerful tools for cell-based experiments, offering a controlled microenvironment that mimic the conditions within the body. Numerous cell experiment studies have successfully utilized microfluidic channels to achieve various new scientific discoveries. However, it has been often overlooked that undesired and unnoticed propagation of cellular molecules in such bio-microfluidic channel systems can have a negative impact on the experimental results. Thus, more careful designing is required to minimize such unwanted issues through deeper understanding and careful control of chemically and physically predominant factors at the microscopic scale. In this paper, we introduce a new approach to improve microfluidic channel design, specifically targeting the mitigation of the aforementioned challenges. To minimize the occurrence of undesired cell positioning upstream from the main test section where a concentration gradient field locates, an additional narrow port structure was devised between the microfluidic upstream channel and each inlet reservoir. This port also functioned as a passive lock that hold the flow at rest via fluid-air surface tension, which facilitated manual movement of the device even when cell attachment was not achieved completely. To demonstrate the practicability of the system, we conducted experiments and diffusion simulations on the effect of endocrine disruptors on germ cells. To this end, a bisphenol-A (BPA) concentration gradient was generated in the main channel of the system at BPA concentrations ranging from 120.8 μM to 79.3 μM, and the proliferation of GC-1 cells in the BPA gradient environment was quantitatively evaluated. The features and concepts of the introduced design is to minimize unexpected and ignored error sources, which will be one of the issues to be considered in the development of microfluidic systems to explore extremely delicate cellular phenomena.
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Affiliation(s)
- Seungjin Lee
- School of Mechanical Engineering, College of Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Jinseop Ahn
- Present address: Columbia Center for Translational Immunology, Department of Medicine, Columbia University Irving Medical Center, New York, NY, 10032, USA
- Department of Animal Science and Technology, BET Research Institute, Chung-Ang University, Anseong, 17546, Republic of Korea
| | - Seok-Man Kim
- Department of Animal Science and Technology, BET Research Institute, Chung-Ang University, Anseong, 17546, Republic of Korea
| | - Daehan Kim
- School of Mechanical Engineering, College of Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Jiun Yeom
- School of Mechanical Engineering, College of Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Jeongmok Kim
- School of Mechanical Engineering, College of Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Joong Yull Park
- School of Mechanical Engineering, College of Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea.
- Department of Intelligent Energy and Industry, Graduate School, Chung-Ang University, Seoul, 06974, Republic of Korea.
| | - Buom-Yong Ryu
- Department of Animal Science and Technology, BET Research Institute, Chung-Ang University, Anseong, 17546, Republic of Korea.
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21
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Ferraz MDAMM, Ferronato GDA. Opportunities involving microfluidics and 3D culture systems to the in vitro embryo production. Anim Reprod 2023; 20:e20230058. [PMID: 37638255 PMCID: PMC10449241 DOI: 10.1590/1984-3143-ar2023-0058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/29/2023] [Indexed: 08/29/2023] Open
Abstract
Traditional methods of gamete handling, fertilization, and embryo culture often face limitations in efficiency, consistency, and the ability to closely mimic in vivo conditions. This review explores the opportunities presented by microfluidic and 3D culture systems in overcoming these challenges and enhancing in vitro embryo production. We discuss the basic principles of microfluidics, emphasizing their inherent advantages such as precise control of fluid flow, reduced reagent consumption, and high-throughput capabilities. Furthermore, we delve into microfluidic devices designed for gamete manipulation, in vitro fertilization, and embryo culture, highlighting innovations such as droplet-based microfluidics and on-chip monitoring. Next, we explore the integration of 3D culture systems, including the use of biomimetic scaffolds and organ-on-a-chip platforms, with a particular focus on the oviduct-on-a-chip. Finally, we discuss the potential of these advanced systems to improve embryo production outcomes and advance our understanding of early embryo development. By leveraging the unique capabilities of microfluidics and 3D culture systems, we foresee significant advancements in the efficiency, effectiveness, and clinical success of in vitro embryo production.
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Affiliation(s)
- Marcia de Almeida Monteiro Melo Ferraz
- Faculty of Veterinary Medicine, Ludwig-Maximilians University of Munich, Oberschleißheim, Germany
- Gene Center, Ludwig-Maximilians University of Munich, Munich, Germany
| | - Giuliana de Avila Ferronato
- Faculty of Veterinary Medicine, Ludwig-Maximilians University of Munich, Oberschleißheim, Germany
- Gene Center, Ludwig-Maximilians University of Munich, Munich, Germany
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22
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Hashimoto K, Odaka H, Ishikawa-Yamauchi Y, Nagata S, Nakamura H, Kimura H, Sato T, Makiyama K, Ogawa T. Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice. Sci Rep 2023; 13:12354. [PMID: 37524742 PMCID: PMC10390558 DOI: 10.1038/s41598-023-39323-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2023] [Accepted: 07/24/2023] [Indexed: 08/02/2023] Open
Abstract
The classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.
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Affiliation(s)
- Kiyoshi Hashimoto
- Department of Urology, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
- Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan
| | - Hisakazu Odaka
- Department of Urology, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
- Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan
| | | | - Shino Nagata
- Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan
| | - Hiroko Nakamura
- Micro/Nano Technology Center, Tokai University, Hiratsuka, Japan
| | - Hiroshi Kimura
- Micro/Nano Technology Center, Tokai University, Hiratsuka, Japan
| | - Takuya Sato
- Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan
| | - Kazuhide Makiyama
- Department of Urology, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
| | - Takehiko Ogawa
- Department of Regenerative Medicine, Yokohama City University, Yokohama, Japan.
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23
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Matsumura T, Katagiri K, Yao T, Ishikawa-Yamauchi Y, Nagata S, Hashimoto K, Sato T, Kimura H, Shinohara T, Sanbo M, Hirabayashi M, Ogawa T. Generation of rat offspring using spermatids produced through in vitro spermatogenesis. Sci Rep 2023; 13:12105. [PMID: 37495678 PMCID: PMC10372019 DOI: 10.1038/s41598-023-39304-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Accepted: 07/23/2023] [Indexed: 07/28/2023] Open
Abstract
An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.
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Affiliation(s)
- Takafumi Matsumura
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Kumiko Katagiri
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Tatsuma Yao
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
- Research and Development Center, Fuso Pharmaceutical Industries, Ltd., 2-3-30 Morinomiya, Joto-ku, Osaka, 536-8523, Japan
| | - Yu Ishikawa-Yamauchi
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Shino Nagata
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama, Kanagawa, 230-0045, Japan
| | - Kiyoshi Hashimoto
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
- Department of Urology, Yokohama City University School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Takuya Sato
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Hiroshi Kimura
- Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, 259-1292, Japan
| | - Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
| | - Makoto Sanbo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan.
| | - Takehiko Ogawa
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan.
- Department of Urology, Yokohama City University School of Medicine, Yokohama, Kanagawa, 236-0004, Japan.
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Juraski AC, Sharma S, Sparanese S, da Silva VA, Wong J, Laksman Z, Flannigan R, Rohani L, Willerth SM. 3D bioprinting for organ and organoid models and disease modeling. Expert Opin Drug Discov 2023; 18:1043-1059. [PMID: 37431937 DOI: 10.1080/17460441.2023.2234280] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Accepted: 07/05/2023] [Indexed: 07/12/2023]
Abstract
INTRODUCTION 3D printing, a versatile additive manufacturing technique, has diverse applications ranging from transportation, rapid prototyping, clean energy, and medical devices. AREAS COVERED The authors focus on how 3D printing technology can enhance the drug discovery process through automating tissue production that enables high-throughput screening of potential drug candidates. They also discuss how the 3D bioprinting process works and what considerations to address when using this technology to generate cell laden constructs for drug screening as well as the outputs from such assays necessary for determining the efficacy of potential drug candidates. They focus on how bioprinting how has been used to generate cardiac, neural, and testis tissue models, focusing on bio-printed 3D organoids. EXPERT OPINION The next generation of 3D bioprinted organ model holds great promises for the field of medicine. In terms of drug discovery, the incorporation of smart cell culture systems and biosensors into 3D bioprinted models could provide highly detailed and functional organ models for drug screening. By addressing current challenges of vascularization, electrophysiological control, and scalability, researchers can obtain more reliable and accurate data for drug development, reducing the risk of drug failures during clinical trials.
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Affiliation(s)
- Amanda C Juraski
- Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada
- Division of Medical Sciences, University of Victoria, Victoria BC, Canada
- Department of Chemical Engineering, Polytechnic School, University of Sao Paulo, Sao Paulo, Brazil
| | - Sonali Sharma
- Faculty of Medicine, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | - Sydney Sparanese
- Faculty of Medicine, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
- Department of Urologic Sciences, University of British Columbia, Vancouver BC, Canada
| | - Victor A da Silva
- Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada
- Division of Medical Sciences, University of Victoria, Victoria BC, Canada
| | - Julie Wong
- Department of Urologic Sciences, University of British Columbia, Vancouver BC, Canada
| | - Zachary Laksman
- Faculty of Medicine, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | - Ryan Flannigan
- Department of Urologic Sciences, University of British Columbia, Vancouver BC, Canada
| | - Leili Rohani
- Faculty of Medicine, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | - Stephanie M Willerth
- Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada
- Division of Medical Sciences, University of Victoria, Victoria BC, Canada
- Faculty of Medicine, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
- Centre for Advanced Materials and Related Technology (CAMTEC), University of Victoria, Victoria, BC, Canada
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25
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Dufva M. A quantitative meta-analysis comparing cell models in perfused organ on a chip with static cell cultures. Sci Rep 2023; 13:8233. [PMID: 37217582 DOI: 10.1038/s41598-023-35043-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 05/11/2023] [Indexed: 05/24/2023] Open
Abstract
As many consider organ on a chip for better in vitro models, it is timely to extract quantitative data from the literature to compare responses of cells under flow in chips to corresponding static incubations. Of 2828 screened articles, 464 articles described flow for cell culture and 146 contained correct controls and quantified data. Analysis of 1718 ratios between biomarkers measured in cells under flow and static cultures showed that the in all cell types, many biomarkers were unregulated by flow and only some specific biomarkers responded strongly to flow. Biomarkers in cells from the blood vessels walls, the intestine, tumours, pancreatic island, and the liver reacted most strongly to flow. Only 26 biomarkers were analysed in at least two different articles for a given cell type. Of these, the CYP3A4 activity in CaCo2 cells and PXR mRNA levels in hepatocytes were induced more than two-fold by flow. Furthermore, the reproducibility between articles was low as 52 of 95 articles did not show the same response to flow for a given biomarker. Flow showed overall very little improvements in 2D cultures but a slight improvement in 3D cultures suggesting that high density cell culture may benefit from flow. In conclusion, the gains of perfusion are relatively modest, larger gains are linked to specific biomarkers in certain cell types.
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Affiliation(s)
- Martin Dufva
- Department of Health Technology, Technical University of Denmark, 2800, Kgs Lyngby, Denmark.
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26
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Önen S, Atik AC, Gizer M, Köse S, Yaman Ö, Külah H, Korkusuz P. A pumpless monolayer microfluidic device based on mesenchymal stem cell-conditioned medium promotes neonatal mouse in vitro spermatogenesis. Stem Cell Res Ther 2023; 14:127. [PMID: 37170113 PMCID: PMC10173473 DOI: 10.1186/s13287-023-03356-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 04/27/2023] [Indexed: 05/13/2023] Open
Abstract
BACKGROUND Childhood cancer treatment-induced gonadotoxicity causes permanent infertility/sub-infertility in nearly half of males. The current clinical and experimental approaches are limited to cryopreservation of prepubertal testicular strips and in vitro spermatogenesis which are inadequate to achieve the expanded spermatogonial stem/progenitor cells and spermatogenesis in vitro. Recently, we reported the supportive effect of bone marrow-derived mesenchymal cell co-culture which is inadequate after 14 days of culture in static conditions in prepubertal mouse testis due to lack of microvascular flow and diffusion. Therefore, we generated a novel, pumpless, single polydimethylsiloxane-layered testis-on-chip platform providing a continuous and stabilized microfluidic flow and real-time cellular paracrine contribution of allogeneic bone marrow-derived mesenchymal stem cells. METHODS We aimed to evaluate the efficacy of this new setup in terms of self-renewal of stem/progenitor cells, spermatogenesis and structural and functional maturation of seminiferous tubules in vitro by measuring the number of undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and tubular growth by histochemical, immunohistochemical, flow cytometric and chromatographic techniques. RESULTS Bone marrow-derived mesenchymal stem cell-based testis-on-chip platform supported the maintenance of SALL4(+) and PLZF(+) spermatogonial stem/progenitor cells, for 42 days. The new setup improved in vitro spermatogenesis in terms of c-Kit(+) differentiating spermatogonia, VASA(+) total germ cells, the meiotic cells including spermatocytes and spermatids and testicular maturation by increasing testosterone concentration and improved tubular growth for 42 days in comparison with hanging drop and non-mesenchymal stem cell control. CONCLUSIONS Future fertility preservation for male pediatric cancer survivors depends on the protection/expansion of spermatogonial stem/progenitor cell pool and induction of in vitro spermatogenesis. Our findings demonstrate that a novel bone marrow-derived mesenchymal stem cell-based microfluidic testis-on-chip device supporting the maintenance of stem cells and spermatogenesis in prepubertal mice in vitro. This new, cell therapy-based microfluidic platform may contribute to a safe, precision-based cell and tissue banking protocols for prepubertal fertility restoration in future.
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Affiliation(s)
- Selin Önen
- Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey
- Department of Medical Biology, Atilim University, Ankara, Turkey
| | - Ali Can Atik
- Department of Electrical and Electronics Engineering, Middle East Technical University, Ankara, Turkey
- METU MEMS Center, Ankara, Turkey
| | - Merve Gizer
- Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey
| | - Sevil Köse
- Department of Plastic, Reconstructive, and Aesthetic Surgery, Akdeniz University, Antalya, Turkey
| | - Önder Yaman
- Department of Urology, Ankara University, Ankara, Turkey
| | - Haluk Külah
- Department of Electrical and Electronics Engineering, Middle East Technical University, Ankara, Turkey
- METU MEMS Center, Ankara, Turkey
| | - Petek Korkusuz
- Department of Histology and Embryology, Faculty of Medicine, Hacettepe University, Sihhiye, Ankara, 06100, Turkey.
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27
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Naeemi S, Sabetkish S, Kiani MJ, Dehghan A, Kajbafzadeh AM. Ex-Vivo and In-Vivo Expansion of Spermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model. Cell Tissue Bank 2023; 24:153-166. [PMID: 35792989 DOI: 10.1007/s10561-022-10024-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 06/23/2022] [Indexed: 11/24/2022]
Abstract
AIMS This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs). METHODS For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation. RESULTS Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds. CONCLUSION Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.
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Affiliation(s)
- Sahar Naeemi
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Shabnam Sabetkish
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Mohammad Javad Kiani
- School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran
| | - Amin Dehghan
- School of Mechanical Engineering, Iran University of Science and Technology, Tehran, Iran
| | - Abdol-Mohammad Kajbafzadeh
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
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28
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Patrício D, Santiago J, Mano JF, Fardilha M. Organoids of the male reproductive system: Challenges, opportunities, and their potential use in fertility research. WIREs Mech Dis 2023; 15:e1590. [PMID: 36442887 DOI: 10.1002/wsbm.1590] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 10/17/2022] [Accepted: 11/12/2022] [Indexed: 11/30/2022]
Abstract
Organoids are units of function of a given organ able to reproduce, in culture, a biological structure similar in architecture and function to its counterpart in vivo. Today, it is possible to develop an organoid from a fragment of tissue, a stem cell located in an adult organ, an embryonic stem cell, or an induced pluripotent stem cell. In the past decade, many organoids have been developed which mimic stomach, pancreas, liver and brain tissues, optic cups, among many others. Additionally, different male reproductive system organs have already been developed as organoids, including the prostate and testis. These 3D cultures may be of great importance for urological cancer research and have the potential to be used in fertility research for the study of spermatozoa production and maturation, germ cells-somatic cells interactions, and mechanisms of disease. They also provide an accurate preclinical pipeline for drug testing and discovery, as well as for the study of drug resistance. In this work, we revise the current knowledge on organoid technology and its use in healthcare and research, describe the male reproductive system organoids and other biomaterials already developed, and discuss their current application. Finally, we highlight the research gaps, challenges, and opportunities in the field and propose strategies to improve the use of organoids for the study of male infertility situations. This article is categorized under: Reproductive System Diseases > Stem Cells and Development Reproductive System Diseases > Biomedical Engineering.
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Affiliation(s)
- Daniela Patrício
- Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal.,Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
| | - Joana Santiago
- Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal
| | - João F Mano
- Department of Chemistry, CICECO - Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
| | - Margarida Fardilha
- Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal
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29
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Human in vitro spermatogenesis as a regenerative therapy - where do we stand? Nat Rev Urol 2023:10.1038/s41585-023-00723-4. [PMID: 36750655 DOI: 10.1038/s41585-023-00723-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/04/2023] [Indexed: 02/09/2023]
Abstract
Spermatogenesis involves precise temporal and spatial gene expression and cell signalling to reach a coordinated balance between self-renewal and differentiation of spermatogonial stem cells through various germ cell states including mitosis, and meiosis I and II, which result in the generation of haploid cells with a unique genetic identity. Subsequently, these round spermatids undergo a series of morphological changes to shed excess cytoplast, develop a midpiece and tail, and undergo DNA repackaging to eventually form millions of spermatozoa. The goal of recreating this process in vitro has been pursued since the 1920s as a tool to treat male factor infertility in patients with azoospermia. Continued advances in reproductive bioengineering led to successful generation of mature, functional sperm in mice and, in the past 3 years, in humans. Multiple approaches to study human in vitro spermatogenesis have been proposed, but technical and ethical obstacles have limited the ability to complete spermiogenesis, and further work is needed to establish a robust culture system for clinical application.
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30
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Aydos OS, Yukselten Y, Ozkan T, Ozkavukcu S, Tuten Erdogan M, Sunguroglu A, Aydos K. Co-Culture of Cryopreserved Healthy Sertoli Cells with Testicular Tissue of Non-Obstructive Azoospermia (NOA) Patients in Culture Media Containing Follicle-Stimulating Hormone (FSH)/Testosterone Has No Advantage in Germ Cell Maturation. J Clin Med 2023; 12:jcm12031073. [PMID: 36769720 PMCID: PMC9917953 DOI: 10.3390/jcm12031073] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Revised: 01/11/2023] [Accepted: 01/26/2023] [Indexed: 01/31/2023] Open
Abstract
Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient's testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; p < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% (p < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively (p < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; p > 0.05). Our results suggest that the presence of the patient's own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing-thawing process would not impair the viability and proliferation of SCs.
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Affiliation(s)
- O. Sena Aydos
- Department of Medical Biology, School of Medicine, Ankara University, Ankara 06230, Turkey
- Correspondence: (O.S.A.); (Y.Y.); Tel.: +90-3125958050 (O.S.A.)
| | - Yunus Yukselten
- Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06520, USA
- Correspondence: (O.S.A.); (Y.Y.); Tel.: +90-3125958050 (O.S.A.)
| | - Tulin Ozkan
- Department of Medical Biology, School of Medicine, Ankara University, Ankara 06230, Turkey
| | - Sinan Ozkavukcu
- Center for Assisted Reproduction, School of Medicine, Ankara University, Ankara 06230, Turkey
- Postgraduate Medicine, School of Medicine, University of Dundee, Dundee DD1 4HN, UK
| | - Meltem Tuten Erdogan
- Department of Medical Biology, School of Medicine, Ankara University, Ankara 06230, Turkey
| | - Asuman Sunguroglu
- Department of Medical Biology, School of Medicine, Ankara University, Ankara 06230, Turkey
| | - Kaan Aydos
- Department of Urology, School of Medicine, Ankara University, Ankara 06230, Turkey
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Munyoki SK, Orwig KE. Perspectives: Methods for Evaluating Primate Spermatogonial Stem Cells. Methods Mol Biol 2023; 2656:341-364. [PMID: 37249880 DOI: 10.1007/978-1-0716-3139-3_18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Mammalian spermatogenesis is a complex, highly productive process generating millions of sperm per day. Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and can either self-renew, producing more SSCs, or differentiate to initiate spermatogenesis and produce sperm. The biological potential of SSCs to produce and maintain spermatogenesis makes them a promising tool for the treatment of male infertility. However, translating knowledge from rodents to higher primates (monkeys and humans) is challenged by different vocabularies that are used to describe stem cells and spermatogenic lineage development in those species. Furthermore, while rodent SSCs are defined by their biological potential to produce and maintain spermatogenesis in a transplant assay, there is no equivalent routine and accessible bioassay to test monkey and human SSCs or replicate their functions in vitro. This chapter describes progress characterizing, isolating, culturing, and transplanting SSCs in higher primates.
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Affiliation(s)
- Sarah K Munyoki
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Integrative Systems Biology Graduate Program, Magee-Women's Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Integrative Systems Biology Graduate Program, Magee-Women's Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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Ramsoomair CK, Alver CG, Flannigan R, Ramasamy R, Agarwal A. Spermatogonial Stem Cells and In Vitro Spermatogenesis: How Far Are We from a Human Testis on a Chip? Eur Urol Focus 2023; 9:46-48. [PMID: 36396561 PMCID: PMC11221572 DOI: 10.1016/j.euf.2022.11.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 10/25/2022] [Accepted: 11/02/2022] [Indexed: 11/16/2022]
Abstract
An in vitro testis model will provide a superior platform for studying the testis microenvironment and molecular mechanisms that affect male fertility. The ultimate aim is to provide reproductive hope for children diagnosed with cancer who were sterilized by aggressive gonadotoxic therapies.
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Affiliation(s)
- Christian K Ramsoomair
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Charles G Alver
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, USA; Department of Biomedical Engineering, University of Miami, Coral Gables, FL, USA
| | - Ryan Flannigan
- Department of Urologic Sciences, The University of British Columbia, Vancouver, BC, Canada
| | - Ranjith Ramasamy
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Ashutosh Agarwal
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, USA; Department of Biomedical Engineering, University of Miami, Coral Gables, FL, USA.
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33
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Kulibin AY, Malolina EA. In vitro spermatogenesis: In search of fully defined conditions. Front Cell Dev Biol 2023; 11:1106111. [PMID: 36910153 PMCID: PMC9998899 DOI: 10.3389/fcell.2023.1106111] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Accepted: 02/08/2023] [Indexed: 03/14/2023] Open
Abstract
A complete reconstruction of spermatogenesis in vitro under fully defined conditions still has not been achieved. However, many techniques have been proposed to get closer to that aim. Here we review the current progress in the field. At first, we describe the most successful technique, the organ culture method, which allows to produce functional haploid cells. However, this method is based on the culturing of intact testis tissue with unknown factors acting inside it. Then we discuss different types of 3D-cultures where specific testicular cell populations may be aggregated and the impact of each cell population may be examined. Unfortunately, germ cell development does not proceed further than the pachytene stage of meiosis there, with rare exceptions. Finally, we describe recent studies that focus on germ cells in a conventional adherent cell culture. Such studies thoroughly examine issues with in vitro meiosis and provide insight into the mechanisms of meiotic initiation.
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Affiliation(s)
- A Yu Kulibin
- Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
| | - E A Malolina
- Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
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34
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Ibtisham F, Cham TC, Fayaz MA, Honaramooz A. Long-Term In Vitro Maintenance of Piglet Testicular Tissue: Effects of Tissue Fragment Size, Preparation Method, and Serum Source. Animals (Basel) 2022; 13:ani13010128. [PMID: 36611737 PMCID: PMC9817678 DOI: 10.3390/ani13010128] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2022] [Revised: 12/22/2022] [Accepted: 12/26/2022] [Indexed: 12/30/2022] Open
Abstract
Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks.
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35
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Chan CJ, Hirashima T. Tissue hydraulics in reproduction. Semin Cell Dev Biol 2022; 131:124-133. [PMID: 35606275 DOI: 10.1016/j.semcdb.2022.05.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 05/12/2022] [Accepted: 05/13/2022] [Indexed: 12/14/2022]
Abstract
The development of functional eggs and sperm are critical processes in mammalian development as they ensure successful reproduction and species propagation. While past studies have identified important genes that regulate these processes, the roles of luminal flow and fluid stress in reproductive biology remain less well understood. Here, we discuss recent evidence that support the diverse functions of luminal fluid in oogenesis, spermatogenesis and embryogenesis. We also review emerging techniques that allow for precise quantification and perturbation of tissue hydraulics in female and male reproductive systems, and propose new questions and approaches in this field. We hope this review will provide a useful resource to inspire future research in tissue hydraulics in reproductive biology and diseases.
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Affiliation(s)
- Chii Jou Chan
- Mechanobiology Institute, National University of Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore.
| | - Tsuyoshi Hirashima
- Mechanobiology Institute, National University of Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; The Hakubi Center/Graduate School of Biostudies, Kyoto University, Kyoto, Japan; Japan Science and Technology Agency, PRESTO, Kawaguchi, Japan.
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36
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Kouthouridis S, Robson E, Hartung A, Raha S, Zhang B. Se(XY) matters: the importance of incorporating sex in microphysiological models. Trends Biotechnol 2022; 40:1284-1298. [PMID: 35597689 DOI: 10.1016/j.tibtech.2022.04.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 04/06/2022] [Accepted: 04/09/2022] [Indexed: 01/21/2023]
Abstract
The development of microphysiological models is currently at the forefront of preclinical research. Although these 3D tissue models are being developed to mimic physiological organ function and diseases, which are often sexually dimorphic, sex is usually neglected as a biological variable. For decades, national research agencies have required government-funded clinical trials to include both male and female participants as a means of eliminating male bias. However, this is not the case in preclinical trials, which have been shown to favor male rodents in animal studies and male cell types in in vitro studies. In this Opinion, we highlight the importance of considering sex as a biological variable and outline five approaches for incorporating sex-specific features into current microphysiological models.
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Affiliation(s)
- Sonya Kouthouridis
- Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada
| | - Eleanor Robson
- Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada
| | - Alicia Hartung
- Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada; School of Biomedical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada
| | - Sandeep Raha
- Department of Pediatrics, McMaster University, Hamilton, ON, L8S 4L8, Canada; Graduate Program in Medical Sciences, McMaster University, Hamilton, ON, L8S 4L8, Canada.
| | - Boyang Zhang
- Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada; School of Biomedical Engineering, McMaster University, Hamilton, ON, L8S 4L8, Canada.
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37
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Liu X, Wu K, Gao L, Wang L, Shi X. Biomaterial strategies for the application of reproductive tissue engineering. Bioact Mater 2022; 14:86-96. [PMID: 35310354 PMCID: PMC8892081 DOI: 10.1016/j.bioactmat.2021.11.023] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 11/13/2021] [Accepted: 11/22/2021] [Indexed: 02/06/2023] Open
Abstract
Human reproductive organs are of vital importance to the life of an individual and the reproduction of human populations. So far, traditional methods have a limited effect in recovering the function and fertility of reproductive organs and tissues. Thus, aim to replace and facilitate the regrowth of damaged or diseased tissue, various biomaterials are developed to offer hope to overcome these difficulties and help gain further research progress in reproductive tissue engineering. In this review, we focus on the biomaterials and their four main applications in reproductive tissue engineering: in vitro generation and culture of reproductive cells; development of reproductive organoids and models; in vivo transplantation of reproductive cells or tissues; and regeneration of reproductive tissue. In reproductive tissue engineering, designing biomaterials for different applications with different mechanical properties, structure, function, and microenvironment is challenging and important, and deserves more attention.
Various biomaterials have been developed and used in reproductive tissue engineering. 3D culture systems can lead to better cell-cell interactions for in vitro production of reproductive cells. Reproductive organoids and models are formed by biomaterials to simulate the environment of natural reproductive organs. Biomaterials should promote vascular regeneration and resist inflammation for in-situ reproductive tissue regeneration.
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38
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Mesenchymal stem cells promote spermatogonial stem/progenitor cell pool and spermatogenesis in neonatal mice in vitro. Sci Rep 2022; 12:11494. [PMID: 35798781 PMCID: PMC9263145 DOI: 10.1038/s41598-022-15358-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Accepted: 06/16/2022] [Indexed: 12/02/2022] Open
Abstract
Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air–liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.
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Kanbar M, de Michele F, Poels J, Van Loo S, Giudice MG, Gilet T, Wyns C. Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study. Front Physiol 2022; 13:884122. [PMID: 35721544 PMCID: PMC9201455 DOI: 10.3389/fphys.2022.884122] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Accepted: 05/16/2022] [Indexed: 11/24/2022] Open
Abstract
Background:In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.
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Affiliation(s)
- Marc Kanbar
- Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Francesca de Michele
- Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Jonathan Poels
- Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Stéphanie Van Loo
- Microfluidics Lab, Department of Aerospace and Mechanical Engineering, University of Liege, Liege, Belgium
| | - Maria Grazia Giudice
- Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Tristan Gilet
- Microfluidics Lab, Department of Aerospace and Mechanical Engineering, University of Liege, Liege, Belgium
| | - Christine Wyns
- Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
- *Correspondence: Christine Wyns,
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40
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Lüönd F, Santacroce N, Beisel C, Guérard L, Bürglin TR, Christofori G, Sugiyama N. Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer. STAR Protoc 2022; 3:101438. [PMID: 35707685 PMCID: PMC9189628 DOI: 10.1016/j.xpro.2022.101438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).
A dual recombinase lineage tracing system for cancer cells undergoing EMT Enables live imaging of cancer cells undergoing early or late EMT Supports flow-cytometry-mediated isolation of cancer cells in various stages of EMT Enables single-cell transcriptomic studies of cancer cells undergoing EMT Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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Affiliation(s)
- Fabiana Lüönd
- Department of Biomedicine, University of Basel, 4058 Basel, Switzerland
| | - Natascha Santacroce
- Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland
| | - Christian Beisel
- Department of Biosystems Science and Engineering, ETH Zürich, 4058 Basel, Switzerland
| | | | - Thomas R. Bürglin
- Department of Biomedicine, University of Basel, 4058 Basel, Switzerland
| | | | - Nami Sugiyama
- Department of Biomedicine, University of Basel, 4058 Basel, Switzerland
- Corresponding author
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Diao L, Turek PJ, John CM, Fang F, Reijo Pera RA. Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration. Front Endocrinol (Lausanne) 2022; 13:895528. [PMID: 35634498 PMCID: PMC9135128 DOI: 10.3389/fendo.2022.895528] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 03/31/2022] [Indexed: 01/21/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are a group of adult stem cells in the testis that serve as the foundation of continuous spermatogenesis and male fertility. SSCs are capable of self-renewal to maintain the stability of the stem cell pool and differentiation to produce mature spermatozoa. Dysfunction of SSCs leads to male infertility. Therefore, dissection of the regulatory network of SSCs is of great significance in understanding the fundamental molecular mechanisms of spermatogonial stem cell function in spermatogenesis and the pathogenesis of male infertility. Furthermore, a better understanding of SSC biology will allow us to culture and differentiate SSCs in vitro, which may provide novel stem cell-based therapy for assisted reproduction. This review summarizes the latest research progress on the regulation of SSCs, and the potential application of SSCs for fertility restoration through in vivo and in vitro spermatogenesis. We anticipate that the knowledge gained will advance the application of SSCs to improve male fertility. Furthermore, in vitro spermatogenesis from SSCs sets the stage for the production of SSCs from induced pluripotent stem cells (iPSCs) and subsequent spermatogenesis.
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Affiliation(s)
- Lei Diao
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
| | | | | | - Fang Fang
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
| | - Renee A. Reijo Pera
- McLaughlin Research Institute, Touro College of Osteopathic Medicine – Montana (TouroCOM-MT), Great Falls, MT, United States
- Research Division, Touro College of Osteopathic Medicine – Montana (TouroCOM-MT), Great Falls, MT, United States
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Delessard M, Stalin L, Rives-Feraille A, Moutard L, Saulnier J, Dumont L, Rives N, Rondanino C. Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy. Sci Rep 2022; 12:7407. [PMID: 35523907 PMCID: PMC9076692 DOI: 10.1038/s41598-022-11286-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Accepted: 04/18/2022] [Indexed: 02/08/2023] Open
Abstract
The assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry.
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Affiliation(s)
- Marion Delessard
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Laura Stalin
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Aurélie Rives-Feraille
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Laura Moutard
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Justine Saulnier
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Ludovic Dumont
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Nathalie Rives
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France
| | - Christine Rondanino
- INSERM, U1239, Team Adrenal and Gonadal Pathophysiology, Laboratory of Neuroendocrine Endocrine and Germinal Differentiation and Communication, Rouen University Hospital, Rouen Normandy University, 76000, Rouen, France.
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Robinson M, Bedford E, Witherspoon L, Willerth SM, Flannigan R. Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report. F&S SCIENCE 2022; 3:130-139. [PMID: 35560010 DOI: 10.1016/j.xfss.2022.02.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/24/2021] [Revised: 02/10/2022] [Accepted: 02/11/2022] [Indexed: 06/15/2023]
Abstract
OBJECTIVE To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA). DESIGN A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded in vitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted in vitro control. SETTING Academic medical center. PATIENT(S) A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome. INTERVENTION(S) Three-dimensional bioprinting and in vitro culturing of patient-derived testis cells. MAIN OUTCOME MEASURE(S) Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of in vitro culture. RESULT(S) Testicular cultures were expandable in vitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold. CONCLUSION(S) This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of in vitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.
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Affiliation(s)
- Meghan Robinson
- Vancouver Prostate Centre, Vancouver, British Columbia, Canada
| | - Erin Bedford
- Aspect Biosystems, Vancouver, British Columbia, Canada
| | - Luke Witherspoon
- Vancouver Prostate Centre, Vancouver, British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada; Department of Urology, The Ottawa Hospital, Ottawa, Ontario, Canada
| | - Stephanie M Willerth
- Division of Medical Sciences, University of Victoria, Victoria, British Columbia, Canada; Department of Mechanical Engineering, University of Victoria, Victoria, British Columbia, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia, Canada
| | - Ryan Flannigan
- Vancouver Prostate Centre, Vancouver, British Columbia, Canada; Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada; Department of Urology, Weill Cornell Medicine, New York, New York.
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Amirkhani Z, Movahedin M, Baheiraei N, Ghiaseddin A. Mini Bioreactor Can Support In Vitro Spermatogenesis of Mouse Testicular Tissue. CELL JOURNAL 2022; 24:277-284. [PMID: 35717571 PMCID: PMC9445517 DOI: 10.22074/cellj.2022.8053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Accepted: 07/11/2021] [Indexed: 11/05/2022]
Abstract
Objective It was in the early 20th century when the quest for in vitro spermatogenesis started. In vitro spermatogenesis is critical for male cancer patients undergoing gonadotoxic treatment. Dynamic culture system creates in vivo-like conditions. In this study, it was intended to evaluate the progression of spermatogenesis after testicular tissue culture in mini-perfusion bioreactor. Materials and Methods In this experimental study, 12 six-day postpartum neonatal mouse testes were removed and fragmented, placed on an agarose gel in parallel to bioreactor culture, and incubated for 8 weeks. Histological, molecular and immunohistochemical evaluations were carried out after 8 weeks. Results Histological analysis suggested successful maintenance of spermatogenesis in tissues grown in the bioreactor but not on agarose gel, possibly because the central region did not receive sufficient oxygen and nutrients, which led to necrotic or degenerative changes. Molecular analysis indicated that Plzf, Tekt1 and Tnp1 were expressed and that their expression did not differ significantly between the bioreactor and agarose gel. Immunohistochemical evaluation of testis fragments showed that PLZF, SCP3 and ACRBP proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa. PLZF expression after 8 weeks was significantly lower (P<0.05) in tissues incubated on agarose gel than in the bioreactor, but there was no significant difference between SCP3 and ACRBP expression among the bioreactor and agarose gel culture systems. Conclusion This three-dimensional (3D) dynamic culture system can provide somewhat similar conditions to the physiological environment of the testis. Our findings suggest that the perfusion bioreactor supports induction of spermatogenesis for generation of haploid cells. Further studies will be needed to address the fertility of the sperm generated in the bioreactor system..
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Affiliation(s)
- Zahra Amirkhani
- Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mansoureh Movahedin
- Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran,P.O.Box: 14115-331Department of Anatomical SciencesFaculty of Medical SciencesTarbiat Modares UniversityTehranIran
| | - Nafiseh Baheiraei
- Tissue Engineering and Applied Cell Sciences Division, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat
Modares University, Tehran, Iran
| | - Ali Ghiaseddin
- Adjunct Research Associate Professor at Chemistry Department, Michigan State University, East Lansing, MI, USA
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Tran KTD, Valli-Pulaski H, Colvin A, Orwig KE. Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†. Biol Reprod 2022; 107:382-405. [PMID: 35403667 PMCID: PMC9382377 DOI: 10.1093/biolre/ioac072] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 03/29/2022] [Accepted: 04/04/2022] [Indexed: 11/22/2022] Open
Abstract
Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.
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Affiliation(s)
- Kien T D Tran
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Hanna Valli-Pulaski
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Amanda Colvin
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Correspondence: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 412-641-2460; E-mail:
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Male Infertility in the XXI Century: Are Obesogens to Blame? Int J Mol Sci 2022; 23:ijms23063046. [PMID: 35328463 PMCID: PMC8948702 DOI: 10.3390/ijms23063046] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 03/06/2022] [Accepted: 03/09/2022] [Indexed: 11/17/2022] Open
Abstract
The permanent exposure to environmental contaminants promoting weight gain (i.e., obesogens) has raised serious health concerns. Evidence suggests that obesogens are one of the leading causes of the marked decline in male fertility and are key players in shaping future health outcomes, not only for those who are directly exposed to them, but also for upcoming generations. It has been hypothesized that obesogens affect male fertility. By using an interdisciplinary strategy, combining in silico, in vitro, in vivo and epidemiological findings, this review aims to contribute to the biological understanding of the molecular transformations induced by obesogens that are the basis of male infertility. Such understanding is shaped by the use of Adverse Outcomes Pathways, a new approach that may shift the paradigm of reproductive toxicology, contributing to the improvement of the diagnosis and management of the adverse effects of obesogens in male fertility.
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Bonner MG, Gudapati H, Mou X, Musah S. Microfluidic systems for modeling human development. Development 2022; 149:274363. [PMID: 35156682 PMCID: PMC8918817 DOI: 10.1242/dev.199463] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
The proper development and patterning of organs rely on concerted signaling events emanating from intracellular and extracellular molecular and biophysical cues. The ability to model and understand how these microenvironmental factors contribute to cell fate decisions and physiological processes is crucial for uncovering the biology and mechanisms of life. Recent advances in microfluidic systems have provided novel tools and strategies for studying aspects of human tissue and organ development in ways that have previously been challenging to explore ex vivo. Here, we discuss how microfluidic systems and organs-on-chips provide new ways to understand how extracellular signals affect cell differentiation, how cells interact with each other, and how different tissues and organs are formed for specialized functions. We also highlight key advancements in the field that are contributing to a broad understanding of human embryogenesis, organogenesis and physiology. We conclude by summarizing the key advantages of using dynamic microfluidic or microphysiological platforms to study intricate developmental processes that cannot be accurately modeled by using traditional tissue culture vessels. We also suggest some exciting prospects and potential future applications of these emerging technologies.
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Affiliation(s)
- Makenzie G. Bonner
- Developmental and Stem Cell Biology Program, Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA,Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA,Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC 27708, USA
| | - Hemanth Gudapati
- Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA
| | - Xingrui Mou
- Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA
| | - Samira Musah
- Developmental and Stem Cell Biology Program, Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA,Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA,Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC 27708, USA,Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA,Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA,MEDx Investigator and Faculty Member at the Duke Regeneration Center, Duke University, Durham, NC 27710, USA,Author for correspondence ()
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Gholami K, Solhjoo S, Aghamir SMK. Application of Tissue-Specific Extracellular Matrix in Tissue Engineering: Focus on Male Fertility Preservation. Reprod Sci 2022; 29:3091-3099. [PMID: 35028926 DOI: 10.1007/s43032-021-00823-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Accepted: 12/03/2021] [Indexed: 11/28/2022]
Abstract
In vitro spermatogenesis and xenotransplantation of the immature testicular tissues (ITT) are the experimental approaches that have been developed for creating seminiferous tubules-like functional structures in vitro and keeping the integrity of the ITTs in vivo, respectively. These strategies are rapidly developing in response to the growing prevalence of infertility in adolescent boys undergoing cancer treatment, by the logic that there is no sperm cryopreservation option for them. Recently, with the advances made in the field of tissue engineering and biomaterials, these methods have achieved promising results for fertility preservation. Due to the importance of extracellular matrix for the formation of vascular bed around the grafted ITTs and also the creation of spatial arrangements between Sertoli cells and germ cells, today it is clear that the scaffold plays a very important role in the success of these methods. Decellularized extracellular matrix (dECM) as a biocompatible, functionally graded, and biodegradable scaffold with having tissue-specific components and growth factors can support reorganization and physiologic processes of originated cells. This review discusses the common protocols for the tissue decellularization, sterilization, and hydrogel formation of the decellularized and lyophilized tissues as well as in vitro and in vivo studies on the use of the testis-derived dECM for testicular organoids.
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Affiliation(s)
- Keykavos Gholami
- Urology Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Somayeh Solhjoo
- Department of Anatomy, Kerman University of Medical Sciences, Kerman, Iran
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Richer G, Hobbs RM, Loveland KL, Goossens E, Baert Y. Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation. Front Physiol 2022; 12:757565. [PMID: 35002756 PMCID: PMC8739976 DOI: 10.3389/fphys.2021.757565] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Accepted: 11/18/2021] [Indexed: 01/15/2023] Open
Abstract
Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.
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Affiliation(s)
- Guillaume Richer
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
| | - Robin M Hobbs
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia
| | - Katherine L Loveland
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Molecular and Translational Sciences, School of Clinical Sciences, Monash Medical Centre, Monash University, Clayton, VIC, Australia
| | - Ellen Goossens
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
| | - Yoni Baert
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
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Mendoza-Martinez AK, Loessner D, Mata A, Azevedo HS. Modeling the Tumor Microenvironment of Ovarian Cancer: The Application of Self-Assembling Biomaterials. Cancers (Basel) 2021; 13:5745. [PMID: 34830897 PMCID: PMC8616551 DOI: 10.3390/cancers13225745] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 11/07/2021] [Accepted: 11/11/2021] [Indexed: 02/06/2023] Open
Abstract
Ovarian cancer (OvCa) is one of the leading causes of gynecologic malignancies. Despite treatment with surgery and chemotherapy, OvCa disseminates and recurs frequently, reducing the survival rate for patients. There is an urgent need to develop more effective treatment options for women diagnosed with OvCa. The tumor microenvironment (TME) is a key driver of disease progression, metastasis and resistance to treatment. For this reason, 3D models have been designed to represent this specific niche and allow more realistic cell behaviors compared to conventional 2D approaches. In particular, self-assembling peptides represent a promising biomaterial platform to study tumor biology. They form nanofiber networks that resemble the architecture of the extracellular matrix and can be designed to display mechanical properties and biochemical motifs representative of the TME. In this review, we highlight the properties and benefits of emerging 3D platforms used to model the ovarian TME. We also outline the challenges associated with using these 3D systems and provide suggestions for future studies and developments. We conclude that our understanding of OvCa and advances in materials science will progress the engineering of novel 3D approaches, which will enable the development of more effective therapies.
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Affiliation(s)
- Ana Karen Mendoza-Martinez
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK;
- Institute of Bioengineering, Queen Mary University of London, Mile End Road, London E1 4NS, UK
| | - Daniela Loessner
- Department of Chemical Engineering, Faculty of Engineering, Monash University, Melbourne, VIC 3800, Australia;
- Department of Materials Science and Engineering, Faculty of Engineering, Monash University, Melbourne, VIC 3800, Australia
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, VIC 3800, Australia
- Max Bergmann Center of Biomaterials Dresden, Leibniz Institute of Polymer Research Dresden e.V., 01069 Dresden, Germany
| | - Alvaro Mata
- School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK;
- Department of Chemical and Environmental Engineering, University of Nottingham, Nottingham NG7 2RD, UK
- Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK
| | - Helena S. Azevedo
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK;
- Institute of Bioengineering, Queen Mary University of London, Mile End Road, London E1 4NS, UK
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