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Aarattuthodi S, Kang D, Gupta SK, Chen P, Redel B, Matuha M, Mohammed H, Sinha AK. Cryopreservation of biological materials: applications and economic perspectives. In Vitro Cell Dev Biol Anim 2025:10.1007/s11626-025-01027-0. [PMID: 40266443 DOI: 10.1007/s11626-025-01027-0] [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: 10/23/2024] [Accepted: 02/09/2025] [Indexed: 04/24/2025]
Abstract
Cryopreservation is a transformative technology that allows for the long-term storage of biological materials by cooling them to extremely low temperatures at which metabolic and biochemical processes are effectively slowed or halted. Cryopreservation utilizes various techniques to minimize ice crystal formation and cellular damage during freezing and thawing processes. This technology has broad applications in the fields of medicine, agriculture, and conservation, spanning across stem cell research, reproductive and regenerative medicine, organ transplantation, and cell-based therapies, each with significant economic implications. While current techniques and their associated costs present certain challenges, ongoing research advancements related to cryoprotectants, cooling methods, and automation promise to enhance efficiency and accessibility, potentially broadening the technology's impact across various sectors. This review focuses on the applications of cryopreservation, research advancements, and economic implications, emphasizing the importance of continued research to overcome the current limitations.
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Affiliation(s)
- Suja Aarattuthodi
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA.
| | - David Kang
- Biological Control of Insects Research Laboratory, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Sanjay Kumar Gupta
- Indian Institute of Agricultural Biotechnology, Garhkhatanga, Ranchi, Jharkhand, 834003, India
| | - Paula Chen
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Bethany Redel
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Moureen Matuha
- Department of Agriculture and Environmental Sciences, Lincoln University of Missouri, Jefferson City, MO, 65101, USA
| | - Haitham Mohammed
- Department of Rangeland, Wildlife and Fisheries Management, Texas a&M University, College Station, TX, 77843, USA
| | - Amit Kumar Sinha
- Department of Aquaculture and Fisheries, University of Arkansas Pine Bluff, Pine Bluff, AR, 71601, USA
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Liu M, Liang L, Yu C, Guo B, Zhang H, Yao F, Zhang H, Li J. Enhancing cell cryopreservation with acidic polyamino acids integrated liquid marbles. Colloids Surf B Biointerfaces 2024; 241:114055. [PMID: 38936034 DOI: 10.1016/j.colsurfb.2024.114055] [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: 01/02/2024] [Revised: 05/16/2024] [Accepted: 06/22/2024] [Indexed: 06/29/2024]
Abstract
Cryopreservation is highly desired for long-term maintenance of the viability of living biosamples, while effective cell cryopreservation still relies heavily on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). However, the intrinsic toxicity of DMSO is still a bottleneck, which could not only cause the clinical side effect but also induce cell genetic variants. In the meantime, the addition of FBS may bring potentially the risk of pathogenic microorganism contamination. The liquid marbles (LMs), a novel biotechnology tool for cell cryopreservation, which not only have a small volume system that facilitated recovery, but the hydrophobic shell also resisted the harm to cells caused by adverse environments. Previous LM-based cell cryopreservation relied heavily on the addition of FBS. In this work, we introduced acidic polyaspartic acid and polyglutamic acid as cryoprotectants to construct LM systems. LMs could burst in an instant to facilitate and achieve ultrarapid recovery process, and the hydrophilic carboxyl groups of the cryoprotectants could form hydrogen bonds with water molecules and further inhibit ice growth/formation to protect cells from cryoinjuries. The L929 cells could be well cryopreserved by acidic polyamino acid-based LMs. This new biotechnology platform is expected to be widely used for cell cryopreservation, which has the potential to propel LMs for the preservation of various functional cells in the future.
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Affiliation(s)
- Min Liu
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Lei Liang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Chaojie Yu
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Bingyan Guo
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Haitao Zhang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
| | - Fanglian Yao
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, China
| | - Hong Zhang
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
| | - Junjie Li
- Department of Polymer Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, China.
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Tan Y, Salkhordeh M, Murray ABP, Souza-Moreira L, Stewart DJ, Mei SHJ. Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications. Front Bioeng Biotechnol 2024; 12:1412811. [PMID: 39148941 PMCID: PMC11324487 DOI: 10.3389/fbioe.2024.1412811] [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: 04/05/2024] [Accepted: 07/12/2024] [Indexed: 08/17/2024] Open
Abstract
Introduction Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. Method and result MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3 M/mL), 1:1 dilution (from 6 M/mL), or 1:2 dilution (from 9 M/mL) with PLA/5% HA, to achieve uniform concentration at 3 M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6 h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6 h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3 M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. Conclusion MSCs can be cryopreserved up to 9 M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.
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Affiliation(s)
- Yuan Tan
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
- Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Mahmoud Salkhordeh
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Aidan B P Murray
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Luciana Souza-Moreira
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Duncan J Stewart
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
- Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Shirley H J Mei
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
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Dos Santos FF, Nunes L, Martins C, Smith MA, Cardoso C. Single laboratory evaluation of umbilical cord blood units processing methodologies for banking. Lab Med 2024; 55:285-292. [PMID: 37566522 DOI: 10.1093/labmed/lmad073] [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] [Indexed: 08/13/2023] Open
Abstract
OBJECTIVE To compare the efficiency of 3 different processing methods (Sepax, AutoXpress [AXP], and manual processing with hydroxyethyl starch [HES] sedimentation) used at Stemlab during a 10-year period. METHODS Historical data were compiled and the analytical results obtained for the 3 different methods were compared. RESULTS The manual processing (HES) method yielded the highest level of total nucleated cell recovery after processing, and the AXP system yielded the highest CD34+ cell number. The red blood cell reduction was also significantly higher with the HES method. Also, HES showed comparable results to Toticyte technology for umbilical cord blood (UCB) processing. CONCLUSION These results show that the HES method is as effective as automated technologies for UCB volume reduction; hence, it is a suitable methodology for private and public UCB banks. The HES method also proved to be superior to Toticyte technology for medical applications, with higher recovery yields of total nucleated cells after thawing and equivalent CD34+ cell recovery and functionality.
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Bennett B, Hanotaux J, Pasala AR, Hasan T, Hassan D, Shor R, Allan DS, Maganti HB. Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies. Cytotherapy 2024; 26:482-489. [PMID: 38416086 DOI: 10.1016/j.jcyt.2024.02.006] [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: 11/10/2023] [Revised: 02/12/2024] [Accepted: 02/12/2024] [Indexed: 02/29/2024]
Abstract
BACKGROUND AIMS Cryopreservation of hematopoietic stem cells (HSCs) is crucial for autologous transplantation, cord blood banking and other special circumstances. Dimethyl sulfoxide (DMSO) is used most commonly for cryopreserving HSC products but can cause infusional toxicities and affect cell viability and engraftment after transplant. A systematic review of controlled studies using lower concentrations of DMSO to cryopreserve HSC products in clinical transplant studies is needed to determine the effect of reducing DMSO concentrations on post-thaw cell viability, initial engraftment and adverse effects on patient health. METHODS All studies identified in our systematic search (to July 11, 2023) examining the use of cryopreserved peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (AHCT) were included. Meta-analysis was performed to determine how varying the concentration of DMSO during cryopreservation effects post-thaw cell viability, initial engraftment and adverse effects on patient health. RESULTS A total of 1547 studies were identified in our systematic search, with seven published articles meeting eligibility for inclusion in meta-analysis. All patients underwent AHCT using (PBSCs) to treat hematologic malignancies. The viability of CD34+ cells post thaw was greater when cryopreserved with 5% DMSO compared with 10% DMSO, with lower rates of adverse side effects in patients. DMSO concentration had minimal impact on rates of initial engraftment. Significant heterogeneity in outcome reporting was observed and the potential for bias was identified in all studies. CONCLUSIONS Reducing the concentration of DMSO from 10% to 5% during cryopreservation of autologous PBSCs may improve cell viability and reduce DMSO-associated adverse effects in patients undergoing AHCT. Data from more studies with similar patients and standard outcome reporting are needed to increase confidence in our initial observations. PROTOCOL REGISTRATION PROSPERO; registration number CRD42023476809 registered November 8, 2023.
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Affiliation(s)
- Bryenah Bennett
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada
| | - Justine Hanotaux
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada
| | - Ajay Ratan Pasala
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada
| | - Tanvir Hasan
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada
| | - Dhuha Hassan
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada
| | - Risa Shor
- Information Services, The Ottawa Hospital, Ottawa, Canada
| | - David S Allan
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada; Clinical Epidemiology & Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada
| | - Harinad B Maganti
- Canadian Blood Services, Stem Cells and Centre for Innovation, Ottawa, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.
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Ashrafi E, Radisic M, Elliott JAW. Systematic cryopreservation study of cardiac myoblasts in suspension. PLoS One 2024; 19:e0295131. [PMID: 38446773 PMCID: PMC10917286 DOI: 10.1371/journal.pone.0295131] [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: 09/13/2023] [Accepted: 11/15/2023] [Indexed: 03/08/2024] Open
Abstract
H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.
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Affiliation(s)
- Elham Ashrafi
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada
| | - Milica Radisic
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
- Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
| | - Janet A. W. Elliott
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
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Li M, Morse B, Kassim S. Development and clinical translation considerations for the next wave of gene modified hematopoietic stem and progenitor cells therapies. Expert Opin Biol Ther 2022; 22:1177-1191. [PMID: 35833356 DOI: 10.1080/14712598.2022.2101361] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
INTRODUCTION Consistent and reliable manufacture of gene modified hematopoietic stem and progenitor cell (HPSC) therapies will be of the utmost importance as they become more mainstream and address larger populations. Robust development campaigns will be needed to ensure that these products will be delivered to patients with the highest quality standards. AREAS COVERED Through publicly available manuscripts, press releases, and news articles - this review touches on aspects related to HSPC therapy, development, and manufacturing. EXPERT OPINION Recent advances in genome modification technology coupled with the longstanding clinical success of HSPCs warrants great optimism for the next generation of engineered HSPC-based therapies. Treatments for some diseases that have thus far been intractable now appear within reach. Reproducible manufacturing will be of critical importance in delivering these therapies but will be challenging due to the need for bespoke materials and methods in combination with the lack of off-the-shelf solutions. Continued progress in the field will manifest in the form of industrialization which currently requires attention and resources directed toward the custom reagents, a focus on closed and automated processes, and safer and more precise genome modification technologies that will enable broader, faster, and safer access to these life-changing therapies.
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Affiliation(s)
| | - Brent Morse
- Dark Horse Consulting Group, Walnut Creek, CA, USA
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Ikeda K, Minakawa K, Yamahara K, Yamada-Fujiwara M, Okuyama Y, Fujiwara SI, Yamazaki R, Kanamori H, Iseki T, Nagamura-Inoue T, Kameda K, Nagai K, Fujii N, Ashida T, Hirose A, Takahashi T, Ohto H, Ueda K, Tanosaki R. Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events. Transfusion 2022; 62:1280-1288. [PMID: 35396716 DOI: 10.1111/trf.16877] [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: 12/16/2021] [Revised: 03/13/2022] [Accepted: 03/15/2022] [Indexed: 11/30/2022]
Abstract
BACKGROUND The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. STUDY DESIGN AND METHODS To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. RESULTS The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. CONCLUSIONS Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.
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Affiliation(s)
- Kazuhiko Ikeda
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - Keiji Minakawa
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - Kenichi Yamahara
- Laboratory of Medical Innovation, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan
| | - Minami Yamada-Fujiwara
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Division of Blood Transfusion and Cell Therapy, Tohoku University Hospital, Sendai, Japan
| | - Yoshiki Okuyama
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Division of Transfusion and Cell Therapy, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan
| | - Shin-Ichiro Fujiwara
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Division of Cell Transplantation and Transfusion, Jichi Medical University Hospital, Shimotsuke, Japan
| | - Rie Yamazaki
- Center for Transfusion Medicine and Cell Therapy, Keio University School of Medicine, Tokyo, Japan
| | - Heiwa Kanamori
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Department of Hematology, Kanagawa Cancer Center, Yokohama, Japan
| | - Tohru Iseki
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan
| | - Tokiko Nagamura-Inoue
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Institution of Medical Science, University of Tokyo, Tokyo, Japan
| | - Kazuaki Kameda
- Division of Hematology, Saitama Medical Center, Jichi Medical University, Saitama, Japan
| | - Kazuhiro Nagai
- Transfusion and Cell Therapy Unit, Nagasaki University Hospital, Nagasaki, Japan
| | - Nobuharu Fujii
- Department of Transfusion Medicine, Okayama University Hospital, Okayama, Japan
| | - Takashi Ashida
- Center for Transfusion and Cell Therapy, Kindai University Hospital, Osakasayama, Japan
| | - Asao Hirose
- Department of Hematology, Osaka City University, Osaka, Japan
| | - Tsutomu Takahashi
- Department of Oncology/Hematology, Shimane University Hospital, Shimane, Japan
| | - Hitoshi Ohto
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - Koki Ueda
- Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - Ryuji Tanosaki
- Cell Therapy Committee, Japan Society of Transfusion Medicine and Cell Therapy, Tokyo, Japan.,Center for Transfusion Medicine and Cell Therapy, Keio University School of Medicine, Tokyo, Japan
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Halpenny M, William N, Elmoazzen H, Giulivi A, Martin L, Perron D, Bredeson C, Hamelin L, Huebsch L, Yang L, Birch P, Acker JP. The importance of evaluating differences in HES formulations used in hematopoietic progenitor cell cryopreservation. Cytotherapy 2021; 24:223-224. [PMID: 34688545 DOI: 10.1016/j.jcyt.2021.09.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 09/06/2021] [Indexed: 11/27/2022]
Affiliation(s)
| | - Nishaka William
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
| | | | - Antonio Giulivi
- Canadian Blood Services, Ottawa, Ontario, Canada; The Ottawa Hospital, Ottawa, Ontario, Canada
| | - Lisa Martin
- Canadian Blood Services, Ottawa, Ontario, Canada
| | - Donna Perron
- Canadian Blood Services, Ottawa, Ontario, Canada
| | | | | | | | - Lin Yang
- Canadian Blood Services, Ottawa, Ontario, Canada
| | - Paul Birch
- Canadian Blood Services, Ottawa, Ontario, Canada
| | - Jason P Acker
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; Centre for Innovation, Canadian Blood Services, Edmonton, Alberta, Canada.
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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11
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Ma Y, Gao L, Tian Y, Chen P, Yang J, Zhang L. Advanced biomaterials in cell preservation: Hypothermic preservation and cryopreservation. Acta Biomater 2021; 131:97-116. [PMID: 34242810 DOI: 10.1016/j.actbio.2021.07.001] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Revised: 06/29/2021] [Accepted: 07/01/2021] [Indexed: 02/07/2023]
Abstract
Cell-based medicine has made great advances in clinical diagnosis and therapy for various refractory diseases, inducing a growing demand for cell preservation as support technology. However, the bottleneck problems in cell preservation include low efficiency and poor biocompatibility of traditional protectants. In this review, cell preservation technologies are categorized according to storage conditions: hypothermic preservation at 1 °C~35 °C to maintain short-term cell viability that is useful in cell diagnosis and transport, while cryopreservation at -196 °C~-80 °C to maintain long-term cell viability that provides opportunities for therapeutic cell product storage. Firstly, the background and developmental history of the protectants used in the two preservation technologies are briefly introduced. Secondly, the progress in different cellular protection mechanisms for advanced biomaterials are discussed in two preservation technologies. In hypothermic preservation, the hypothermia-induced and extracellular matrix-loss injuries to cells are comprehensively summarized, as well as the recent biomaterials dependent on regulation of cellular ATP level, stabilization of cellular membrane, balance of antioxidant defense system, and supply of mimetic ECM to prolong cell longevity are provided. In cryopreservation, cellular injuries and advanced biomaterials that can protect cells from osmotic or ice injury, and alleviate oxidative stress to allow cell survival are concluded. Last, an insight into the perspectives and challenges of this technology is provided. We envision advanced biocompatible materials for highly efficient cell preservation as critical in future developments and trends to support cell-based medicine. STATEMENT OF SIGNIFICANCE: Cell preservation technologies present a critical role in cell-based applications, and more efficient biocompatible protectants are highly required. This review categorizes cell preservation technologies into hypothermic preservation and cryopreservation according to their storage conditions, and comprehensively reviews the recently advanced biomaterials related. The background, development, and cellular protective mechanisms of these two preservation technologies are respectively introduced and summarized. Moreover, the differences, connections, individual demands of these two technologies are also provided and discussed.
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Affiliation(s)
- Yiming Ma
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China
| | - Lei Gao
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China
| | - Yunqing Tian
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China
| | - Pengguang Chen
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China
| | - Jing Yang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China.
| | - Lei Zhang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300350, PR China; Frontier Technology Research Institute, Tianjin University, Tianjin 300350, PR China.
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Belisário AR, da Costa Funes AP, Luz JR, de Almeida Costa L, Furtado MDSBS, Martins MC, Cruz NG, Pederzoli PRMP, de Andrade RK, Libânio MRIS, de Lima Prata K. Influence of laboratory procedures on postthawing cell viability and hematopoietic engraftment after autologous peripheral blood stem cell transplantation. Transfusion 2021; 61:1202-1214. [PMID: 33569783 DOI: 10.1111/trf.16289] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2020] [Revised: 12/17/2020] [Accepted: 12/23/2020] [Indexed: 12/31/2022]
Abstract
BACKGROUND The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.
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Affiliation(s)
- André Rolim Belisário
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Ana Paula da Costa Funes
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Junio Rocha Luz
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Luciana de Almeida Costa
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | | | - Nathália Gomide Cruz
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | - Roberta Kelly de Andrade
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | - Karen de Lima Prata
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
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Jahan S, Kaushal R, Pasha R, Pineault N. Current and Future Perspectives for the Cryopreservation of Cord Blood Stem Cells. Transfus Med Rev 2021; 35:95-102. [PMID: 33640254 DOI: 10.1016/j.tmrv.2021.01.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Revised: 01/22/2021] [Accepted: 01/24/2021] [Indexed: 12/29/2022]
Abstract
Hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of many blood related malignancies and disorders. Before transplantation, HSC are collected and cryopreserved until use. The method of cryopreservation should preserve both the number and function of HSC and downstream progenitors responsible for long- and short-term engraftment, respectively. This is especially critical for cord blood grafts, since the cell number associated with this stem cell source is often limiting. Loss of function in cryopreserved cells occurs following cryoinjuries due to osmotic shock, dehydration, solution effects and mechanical damage from ice recrystallization during freezing and thawing. However, cryoinjuries can be reduced by 2 mitigation strategies; the use of cryoprotectants (CPAs) and use of control rate cooling. Currently, slow cooling is the most common method used for the cryopreservation of HSC graft. Moreover, dimethyl-sulfoxide (DMSO) and dextran are popular intracellular and extracellular CPAs used for HSC grafts, respectively. Yet, DMSO is toxic to cells and can cause significant side effects in stem cells' recipients. However, new CPAs and strategies are emerging that may soon replace DMSO. The aim of this review is to summarise key concepts in cryobiology and recent advances in the field of HSC cryobiology. Other important issues that need to be considered are also discussed such as transient warming events and thawing of HSC grafts.
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Affiliation(s)
- Suria Jahan
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Richa Kaushal
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Roya Pasha
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada
| | - Nicolas Pineault
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada.
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14
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Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, Lowdell M, Mericka P, Petrenko A, Petrenko Y, Rogulska O, Stolzing A, Stacey GN. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen Med 2020; 15:1463-1491. [PMID: 32342730 DOI: 10.2217/rme-2019-0145] [Citation(s) in RCA: 116] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings.
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Affiliation(s)
- Maooz Awan
- Institute for Liver & Digestive Health, UCL Division of Medicine, Royal Free Hospital, UCL, London, NW3 2PF, UK
| | - Iryna Buriak
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Roland Fleck
- Centre for Ultrastructural Imaging, Kings College London, London, SE1 1UL, UK
| | - Barry Fuller
- Department of Surgical Biotechnology, UCL Division of Surgery, Royal Free Hospital, UCL, London, NW3 2QG, UK
| | - Anatoliy Goltsev
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Julie Kerby
- Cell & Gene Therapy Catapult, 12th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK
| | - Mark Lowdell
- Centre for Cell, Gene & Tissue Therapy, Royal Free London NHS FT & UCL, London, NW3 2PF, UK
| | - Pavel Mericka
- Tissue Bank, University Hospital Hradec Kralové, Czech Republic
| | - Alexander Petrenko
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Yuri Petrenko
- Department of Biomaterials & Biophysical Methods, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic
| | - Olena Rogulska
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Alexandra Stolzing
- University of Loughborough, Centre for Biological Engineering, Loughborough University, Holywell Park, Loughborough, UK
| | - Glyn N Stacey
- International Stem Cell Banking Initiative, 2 High Street, Barley, Hertfordshire, SG8 8HZ
- Beijing Stem Cell Bank, Institute of Zoology, Chinese Academy of Sciences, 25–2 Beishuan West, Haidan District, 100190 Beijing, China
- Institute of Stem Cells & Regeneration, Chinese Academy of Sciences, Beijing 100101, China
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15
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Yang J, Sui X, Li Q, Zhao W, Zhang J, Zhu Y, Chen P, Zhang L. In Situ Encapsulation of Postcryopreserved Cells Using Alginate Polymer and Zwitterionic Betaine. ACS Biomater Sci Eng 2019; 5:2621-2630. [DOI: 10.1021/acsbiomaterials.9b00249] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Affiliation(s)
- Jing Yang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Xiaojie Sui
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Qingsi Li
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Weiqiang Zhao
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Jiamin Zhang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Yingnan Zhu
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Pengguang Chen
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
| | - Lei Zhang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300350, China
- Qingdao Institute for Marine Technology of Tianjin University, Qingdao, 266235, China
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Hornberger K, Yu G, McKenna D, Hubel A. Cryopreservation of Hematopoietic Stem Cells: Emerging Assays, Cryoprotectant Agents, and Technology to Improve Outcomes. Transfus Med Hemother 2019; 46:188-196. [PMID: 31244587 DOI: 10.1159/000496068] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Accepted: 12/04/2018] [Indexed: 12/11/2022] Open
Abstract
Hematopoietic stem cell (HSC) therapy is widely used to treat a growing number of hematological and non-hematological diseases. Cryopreservation of HSCs allows for cells to be transported from the site of processing to the site of clinical use, creates a larger window of time in which cells can be administered to patients, and allows sufficient time for quality control and regulatory testing. Currently, HSCs and other cell therapies conform to the same cryopreservation techniques as cells used for research purposes: cells are cryopreserved in dimethyl sulfoxide (DMSO) at a slow cooling rate. As a result, HSC therapy can result in numerous adverse symptoms in patients due to the infusion of DMSO. Efforts are being made to improve the cryopreservation of HSCs for clinical use. This review discusses advances in the cryopreservation of HSCs from 2007 to the present. The preclinical development of new cryoprotectants and new technology to eliminate cryoprotectants after thawing are discussed in detail. Additional cryopreservation considerations are included, such as cooling rate, storage temperature, and cell concentration. Preclinical cell assessment and quality control are discussed, as well as clinical studies from the past decade that focus on new cryopreservation protocols to improve patient outcomes.
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Affiliation(s)
- Kathlyn Hornberger
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA
| | - Guanglin Yu
- Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA
| | - David McKenna
- Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Allison Hubel
- Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA
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17
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Haack-Sørensen M, Ekblond A, Kastrup J. Cryopreservation and Revival of Human Mesenchymal Stromal Cells. Methods Mol Biol 2017; 1416:357-74. [PMID: 27236683 DOI: 10.1007/978-1-4939-3584-0_21] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.
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Affiliation(s)
- Mandana Haack-Sørensen
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark.
| | - Annette Ekblond
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark
| | - Jens Kastrup
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark
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18
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Naaldijk Y, Johnson AA, Friedrich-Stöckigt A, Stolzing A. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants. BMC Biotechnol 2016; 16:85. [PMID: 27903244 PMCID: PMC5131400 DOI: 10.1186/s12896-016-0315-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Accepted: 10/23/2016] [Indexed: 01/30/2023] Open
Abstract
BACKGROUND Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. RESULTS We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. CONCLUSION We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.
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Affiliation(s)
- Yahaira Naaldijk
- Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.,Interdisciplinary Institute for Bioinformatics, University of Leipzig, Leipzig, Germany
| | - Adiv A Johnson
- Department of Ophthalmology, Mayo Clinic, Rochester, MN, USA
| | | | - Alexandra Stolzing
- Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. .,Interdisciplinary Institute for Bioinformatics, University of Leipzig, Leipzig, Germany. .,Centre for Biological Engineering, Wolfson School of Material and Manufacturing Engineering, Loughborough University, Loughborough, UK.
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Yang J, Cai N, Zhai H, Zhang J, Zhu Y, Zhang L. Natural zwitterionic betaine enables cells to survive ultrarapid cryopreservation. Sci Rep 2016; 6:37458. [PMID: 27874036 PMCID: PMC5118695 DOI: 10.1038/srep37458] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Accepted: 10/25/2016] [Indexed: 12/22/2022] Open
Abstract
Cryoprotectants (CPAs) play a critical role in cryopreservation because they can resist the cell damage caused by the freezing process. Current state-of-the-art CPAs are mainly based on an organic solvent dimethyl sulfoxide (DMSO), and several DMSO-cryopreserved cell products have been brought to market. However, the intrinsic toxicity and complex freezing protocol of DMSO still remain as the bottleneck of the wide use for clinical applications. Herein, we reported that betaine, a natural zwitterionic molecule, could serve as a nontoxic and high efficient CPA. At optimum concentration of betaine, different cell types exhibited exceptional post-thaw survival efficiency with ultrarapid freezing protocol, which was straightforward, cost efficient but difficult to succeed using DMSO. Moreover, betaine showed negligible cytotoxicity even after long-term exposure of cells. Mechanistically, we hypothesized that betaine could be ultra-rapidly taken up by cells for intracellular protection during the freezing process. This technology unlocks the possibility of alternating the traditional toxic CPAs and is applicable to a variety of clinical applications.
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Affiliation(s)
- Jing Yang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
| | - Nana Cai
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
| | - Hongwen Zhai
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
| | - Jiamin Zhang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
| | - Yingnan Zhu
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
| | - Lei Zhang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
- Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin University, Tianjin 300072, P. R. China
- Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China
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Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach. Sci Rep 2016; 6:34393. [PMID: 27708349 PMCID: PMC5052637 DOI: 10.1038/srep34393] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2016] [Accepted: 09/07/2016] [Indexed: 12/24/2022] Open
Abstract
Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.
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Lecchi L, Giovanelli S, Gagliardi B, Pezzali I, Ratti I, Marconi M. An update on methods for cryopreservation and thawing of hemopoietic stem cells. Transfus Apher Sci 2016; 54:324-36. [DOI: 10.1016/j.transci.2016.05.009] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Cloutier M, Simard C, Jobin C, Fournier D, Néron S. An alternative to dextran for the thawing of cord blood units. Transfusion 2016; 56:1786-91. [DOI: 10.1111/trf.13633] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Revised: 03/16/2016] [Accepted: 03/29/2016] [Indexed: 11/29/2022]
Affiliation(s)
- Marc Cloutier
- Department of Biochemistry, Microbiology and Bio-Informatics; Laval University; Québec City Québec
| | - Carl Simard
- Department of Biochemistry, Microbiology and Bio-Informatics; Laval University; Québec City Québec
| | - Christine Jobin
- Department of Biochemistry, Microbiology and Bio-Informatics; Laval University; Québec City Québec
- Department of Research and Development; Héma-Québec; Québec City Québec, Canada
| | - Diane Fournier
- Public Cord Blood Bank; Héma-Québec; Saint-Laurent Québec Canada
| | - Sonia Néron
- Department of Biochemistry, Microbiology and Bio-Informatics; Laval University; Québec City Québec
- Department of Research and Development; Héma-Québec; Québec City Québec, Canada
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Zhu F, Heditke S, Kurtzberg J, Waters-Pick B, Hari P, Margolis DA, Keever-Taylor CA. Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products. Cytotherapy 2015; 17:1813-9. [DOI: 10.1016/j.jcyt.2015.08.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2015] [Revised: 08/11/2015] [Accepted: 08/24/2015] [Indexed: 01/15/2023]
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Kang YH, Lee HJ, Jang SJ, Byun JH, Lee JS, Lee HC, Park WU, Lee JH, Rho GJ, Park BW. Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles. Differentiation 2015; 90:48-58. [PMID: 26493125 DOI: 10.1016/j.diff.2015.10.001] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2015] [Revised: 09/09/2015] [Accepted: 10/09/2015] [Indexed: 02/08/2023]
Abstract
In our previous study, dental follicle tissues from extracted wisdom teeth were successfully cryopreserved for use as a source of stem cells. The goals of the present study were to investigate the immunomodulatory properties of stem cells from fresh and cryopreserved dental follicles (fDFCs and cDFCs, respectively) and to analyze in vivo osteogenesis after transplantation of these DFCs into experimental animals. Third passage fDFCs and cDFCs showed similar expression levels of interferon-γ receptor (CD119) and major histocompatibility complex class I and II (MHC I and MHC II, respectively), with high levels of CD119 and MHC I and nearly no expression of MHC II. Both fresh and cryopreserved human DFCs (hDFCs) were in vivo transplanted along with a demineralized bone matrix scaffold into mandibular defects in miniature pigs and subcutaneous tissues of mice. Radiological and histological evaluations of in vivo osteogenesis in hDFC-transplanted sites revealed significantly enhanced new bone formation activities compared with those in scaffold-only implanted control sites. Interestingly, at 8 weeks post-hDFC transplantation, the newly generated bones were overgrown compared to the original size of the mandibular defects, and strong expression of osteocalcin and vascular endothelial growth factor were detected in the hDFCs-transplanted tissues of both animals. Immunohistochemical analysis of CD3, CD4, and CD8 in the ectopic bone formation sites of mice showed significantly decreased CD4 expression in DFCs-implanted tissues compared with those in control sites. These findings indicate that hDFCs possess immunomodulatory properties that involved inhibition of the adaptive immune response mediated by CD4 and MHC II, which highlights the usefulness of hDFCs in tissue engineering. In particular, long-term preserved dental follicles could serve as an excellent autologous or allogenic stem cell source for bone tissue regeneration as well as a valuable therapeutic agent for immune diseases.
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Affiliation(s)
- Young-Hoon Kang
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Hye-Jin Lee
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Si-Jung Jang
- OBS/Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju, Republic of Korea
| | - June-Ho Byun
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Jong-Sil Lee
- Department of Pathology, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea
| | - Hee-Chun Lee
- Department of Medical Imaging, College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea
| | - Won-Uk Park
- Department of Dental Technology, Jinju Health College, Jinju, Republic of Korea
| | - Jin-Ho Lee
- Department of Advanced Materials, College of Life Science and Nano Technology, Hannam University, Daejeon, Republic of Korea
| | - Gyu-Jin Rho
- OBS/Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Bong-Wook Park
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea.
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Yong KW, Wan Safwani WKZ, Xu F, Wan Abas WAB, Choi JR, Pingguan-Murphy B. Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges. Biopreserv Biobank 2015; 13:231-239. [PMID: 26280501 DOI: 10.1089/bio.2014.0104] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.
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Affiliation(s)
- Kar Wey Yong
- 1 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya , Kuala Lumpur, Malaysia
- 2 Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University , Xi'an, P.R. China
| | | | - Feng Xu
- 2 Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University , Xi'an, P.R. China
- 3 The Key Library of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University , Xi'an, P.R. China
| | - Wan Abu Bakar Wan Abas
- 1 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya , Kuala Lumpur, Malaysia
| | - Jane Ru Choi
- 1 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya , Kuala Lumpur, Malaysia
- 2 Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an Jiaotong University , Xi'an, P.R. China
| | - Belinda Pingguan-Murphy
- 1 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya , Kuala Lumpur, Malaysia
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26
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Reich-Slotky R, Bachegowda LS, Ancharski M, Mendeleyeva L, Rubinstein P, Rennert H, Shore T, van Besien K, Cushing M. How we handled the dextran shortage: an alternative washing or dilution solution for cord blood infusions. Transfusion 2015; 55:1147-53. [DOI: 10.1111/trf.13015] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 12/02/2014] [Accepted: 12/02/2014] [Indexed: 11/30/2022]
Affiliation(s)
- Ronit Reich-Slotky
- Department of Transfusion Medicine and Cellular Therapy; New York Presbyterian Hospital/Weill Cornell Medical Center; New York New York
| | - Lohith S. Bachegowda
- Department of Transfusion Medicine and Cellular Therapy; New York Presbyterian Hospital/Weill Cornell Medical Center; New York New York
- National Cord Blood Program; New York Blood Center; New York New York
| | - Michael Ancharski
- Department of Transfusion Medicine and Cellular Therapy; New York Presbyterian Hospital/Weill Cornell Medical Center; New York New York
| | - Lyubov Mendeleyeva
- Department of Transfusion Medicine and Cellular Therapy; New York Presbyterian Hospital/Weill Cornell Medical Center; New York New York
| | - Pablo Rubinstein
- National Cord Blood Program; New York Blood Center; New York New York
| | | | - Tsiporah Shore
- Department of Medicine; Weill Cornell Medical College; New York New York
| | - Koen van Besien
- Department of Medicine; Weill Cornell Medical College; New York New York
| | - Melissa Cushing
- Department of Transfusion Medicine and Cellular Therapy; New York Presbyterian Hospital/Weill Cornell Medical Center; New York New York
- Department of Pathology and Laboratory Medicine
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27
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Park BW, Jang SJ, Byun JH, Kang YH, Choi MJ, Park WU, Lee WJ, Rho GJ. Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells. J Tissue Eng Regen Med 2014; 11:489-500. [PMID: 25052907 DOI: 10.1002/term.1945] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Revised: 05/12/2014] [Accepted: 06/16/2014] [Indexed: 01/06/2023]
Abstract
The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate-buffered saline (PBS) was employed, with a slow-ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive-cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze-thawing process. Real-time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis- and transcription-related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long-term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright © 2014 John Wiley & Sons, Ltd.
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Affiliation(s)
- Bong-Wook Park
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Si-Jung Jang
- OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Republic of Korea
| | - June-Ho Byun
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Young-Hoon Kang
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Mun-Jeong Choi
- Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
| | - Won-Uk Park
- Department of Ceramic Engineering, Gyeongsang National University, Jinju, Republic of Korea
| | - Won-Jae Lee
- OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Republic of Korea
| | - Gyu-Jin Rho
- OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Republic of Korea.,Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
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28
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Morris C, de Wreede L, Scholten M, Brand R, van Biezen A, Sureda A, Dickmeiss E, Trneny M, Apperley J, Chiusolo P, van Imhoff GW, Lenhoff S, Martinelli G, Hentrich M, Pabst T, Onida F, Quinn M, Kroger N, de Witte T, Ruutu T. Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide. Transfusion 2014; 54:2514-22. [DOI: 10.1111/trf.12759] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2013] [Revised: 02/18/2014] [Accepted: 02/20/2014] [Indexed: 12/30/2022]
Affiliation(s)
- Curly Morris
- Centre for Cancer Research and Cell Biology; Queen's University of Belfast; Belfast UK
| | - Liesbeth de Wreede
- EBMT, Department of Medical Statistics and Bioinformatics; Leiden University Medical Center; Leiden The Netherlands
| | - Marijke Scholten
- EBMT, Department of Medical Statistics and Bioinformatics; Leiden University Medical Center; Leiden The Netherlands
| | - Ronald Brand
- EBMT, Department of Medical Statistics and Bioinformatics; Leiden University Medical Center; Leiden The Netherlands
| | - Anja van Biezen
- EBMT, Department of Medical Statistics and Bioinformatics; Leiden University Medical Center; Leiden The Netherlands
| | - Anna Sureda
- Department of Haematology, Addenbrooke's Hospital; Cambridge University; Cambridge UK
| | - Ebbe Dickmeiss
- Cell Therapy Section, Department of Clinical Immunology; Rigshospitalet; Copenhagen Denmark
| | - Marek Trneny
- Charles University Hospital; Prague Czech Republic
| | - Jane Apperley
- Department of Haematology; Hammersmith Hospital; London UK
| | | | - Gustaaf W. van Imhoff
- Department of Hematology; University Medical Center Groningen; Groningen The Netherlands
| | - Stig Lenhoff
- Department of Hematology; University Hospital; Lund Sweden
| | | | | | | | - Francesco Onida
- Department of Hematology and Oncology; University of Milan; Milan Italy
| | - Michael Quinn
- Department of Haematology; Belfast City Hospital; Belfast UK
| | - Nicolaus Kroger
- Department of Stem Cell Transplantation; University Hospital Hamburg-Eppendorf; Hamburg Germany
| | - Theo de Witte
- Department of Hematology; Radboud University Nijmegen Medical Centre; Nijmegen The Netherlands
| | - Tapani Ruutu
- Department of Medicine; Helsinki University Central Hospital; Helsinki Finland
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29
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Essential requirements for setting up a stem cell processing laboratory. Bone Marrow Transplant 2014; 49:1098-105. [DOI: 10.1038/bmt.2014.104] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2013] [Revised: 01/24/2014] [Accepted: 03/13/2014] [Indexed: 11/08/2022]
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30
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Gladstone DE, Davis-Sproul J, Campian J, Lemas MV, Malatyali S, Borrello I, King K, Grossman SA. Infusion of cryopreserved autologous lymphocytes using a standard peripheral i.v. catheter. Bone Marrow Transplant 2014; 49:1119-20. [PMID: 24842526 DOI: 10.1038/bmt.2014.98] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- D E Gladstone
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - J Davis-Sproul
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - J Campian
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - M V Lemas
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - S Malatyali
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - I Borrello
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - K King
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - S A Grossman
- The Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
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31
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Demirci S, Doğan A, Şişli B, Sahin F. Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation. Cryobiology 2014; 68:139-46. [DOI: 10.1016/j.cryobiol.2014.01.010] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2013] [Revised: 01/14/2014] [Accepted: 01/15/2014] [Indexed: 12/17/2022]
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32
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Holbro A, Graf L, Topalidou M, Bucher C, Passweg JR, Tsakiris DA. Cryopreserved stem cell products containing dimethyl sulfoxide lead to activation of the coagulation system without any impact on engraftment. Transfusion 2013; 54:1508-14. [PMID: 24304039 DOI: 10.1111/trf.12511] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Revised: 10/14/2013] [Accepted: 10/22/2013] [Indexed: 11/30/2022]
Abstract
BACKGROUND Dimethyl sulfoxide (DMSO) is extensively used as a cryoprotectant in stem cell preservation. Little is known on direct hemostatic changes in recipients of hematopoietic stem cell transplantation (HSCT), immediately after DMSO administration. The objectives of the current study were to measure hemostatic changes during HSCT. STUDY DESIGN AND METHODS In this prospective analysis, changes in plasma biomarkers, platelets (PLTs), or endothelial cells (D-dimers, thrombin-antithrombin complex [TAT], microparticle activity as thrombin-generation potential [MPA], whole blood aggregation, von Willebrand factor) were measured before and immediately after HSCT. Furthermore, associations with clinical complications were recorded. RESULTS A total of 54 patients were included in the study. Mean MPA and TAT increased significantly immediately after HSCT, returning to baseline the day after the procedure (p<0.01). No significant differences in engraftment for neutrophils and PLTs were found in patients presenting a high increase of TAT or MPA compared with those presenting with a smaller increase. Patients with a high increase in TAT and MPA had received a greater number of total mononucleated cells (p<0.001) and higher transplant volumes (p=0.002). CONCLUSIONS Infusion of stem cells containing DMSO reversibly activated coagulation, measured as thrombin generation. This finding was not associated with acute adverse events and did not influence engraftment. Further studies are needed to compare variable DMSO concentrations as well as DMSO-free products, to better address the influence of DMSO on hemostasis.
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Affiliation(s)
- Andreas Holbro
- Department of Hematology and Diagnostic Hematology, University Hospital Basel, Basel, Switzerland; Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland
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Shu Z, Heimfeld S, Gao D. Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant 2013; 49:469-76. [PMID: 24076548 DOI: 10.1038/bmt.2013.152] [Citation(s) in RCA: 140] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2013] [Accepted: 05/15/2013] [Indexed: 12/23/2022]
Abstract
Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large number of clinical indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, preparation, quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been associated with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurological complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used-which is typically dimethyl sulfoxide (DMSO)-is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concentration or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise dilution-centrifugation using rotating syringe, diffusion-based DMSO extraction in microfluidic channels, dialysis and dilution-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, dilution-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with minimum cell loss and damage remains an unfilled need.
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Affiliation(s)
- Z Shu
- Department of Mechanical Engineering and Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - S Heimfeld
- Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
| | - D Gao
- Department of Mechanical Engineering and Department of Bioengineering, University of Washington, Seattle, WA, USA
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Bogoslovsky T, Wang D, Maric D, Scattergood-Keepper L, Spatz M, Auh S, Hallenbeck J. Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials. JOURNAL OF BLOOD DISORDERS & TRANSFUSION 2013; 4:158. [PMID: 25309814 PMCID: PMC4193669 DOI: 10.4172/2155-9864.1000158] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
BACKGROUND Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC. PATIENTS/METHODS EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45- CD34+CD133+/-VEGFR2+/- and EC were gated for CD45-CD146+CD34+/-VEGFR2+/-. RESULTS Cryopreservation for 7 days at -80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at -65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45- CD34+CD133+VEGFR2+) were enumerated as 0.2 ± 0.06%, late EPC(CD45-CD146+CD34+VEGFR2+) as 0.6 ± 0.1% and mature EC (CD45-CD146+CD34-VEGFR2+) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at -80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant. CONCLUSIONS Our data indicate that cryopreservation at -80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.
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Affiliation(s)
- T Bogoslovsky
- Center for Neuroscience & Regenerative Medicine, Uniformed Services University of Health Sciences, Bethesda, USA
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA
| | - D Wang
- National Institute of Nursing Research, Bethesda, USA
| | - D Maric
- National Institute of Neurological Disorders and Stroke, Flow Cytometry Core Facility, Bethesda, USA
| | | | - M Spatz
- Stroke Branch, National Institute of Neurological Disorders and Stroke, Bethesda, USA
| | - S Auh
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA
| | - J Hallenbeck
- Stroke Branch, National Institute of Neurological Disorders and Stroke, Bethesda, USA
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35
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Naaldijk Y, Friedrich-Stöckigt A, Sethe S, Stolzing A. Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation. J Tissue Eng Regen Med 2013; 10:E354-E364. [PMID: 23963809 DOI: 10.1002/term.1815] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2013] [Revised: 07/18/2013] [Accepted: 07/24/2013] [Indexed: 11/11/2022]
Abstract
Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method. Copyright © 2013 John Wiley & Sons, Ltd.
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Affiliation(s)
- Yahaira Naaldijk
- Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.,Translation Centre for Regenerative Medicine, University of Leipzig, Germany
| | | | - Sebastian Sethe
- Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany
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36
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Scerpa MC, Rossi C, Daniele N, Lanti A, Adorno G, Picardi A, Arcese W, Amadori S, Isacchi G, Zinno F. A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant. Transfusion 2013; 54:522-31. [PMID: 23789937 DOI: 10.1111/trf.12307] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Revised: 05/09/2013] [Accepted: 05/10/2013] [Indexed: 12/29/2022]
Abstract
BACKGROUND In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. STUDY DESIGN AND METHODS This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." RESULTS The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. CONCLUSION The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.
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Affiliation(s)
- Maria Cristina Scerpa
- Cryolab Center of Biotechnology and Cryobiology, Immunohematology Section, SIMT, Department of Hematology, Tor Vergata University, Rome, Italy; Rome Transplant Network, Department of Hematology, Tor Vergata University, Rome, Italy
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Naaldijk Y, Staude M, Fedorova V, Stolzing A. Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide. BMC Biotechnol 2012; 12:49. [PMID: 22889198 PMCID: PMC3465236 DOI: 10.1186/1472-6750-12-49] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2012] [Accepted: 07/31/2012] [Indexed: 11/21/2022] Open
Abstract
Background Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. Results The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. Conclusion A 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC.
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Affiliation(s)
- Yahaira Naaldijk
- Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, Leipzig, 04103, Germany
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Calvet L, Cabrespine A, Boiret-Dupré N, Merlin E, Paillard C, Berger M, Bay JO, Tournilhac O, Halle P. Hematologic, immunologic reconstitution, and outcome of 342 autologous peripheral blood stem cell transplantations after cryopreservation in a -80°C mechanical freezer and preserved less than 6 months. Transfusion 2012; 53:570-8. [PMID: 22804351 DOI: 10.1111/j.1537-2995.2012.03768.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND Controlled-rate freezing and storage in nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHPCs) but presents high cost and dimethyl sulfoxide (DMSO) toxicity. Cryopreservation at -80°C, by uncontrolled rate freezing with only 3.5% DMSO, preserves the functional capacities of PHPCs, produces successful engraftment, and reduces toxicity during infusion. STUDY DESIGN AND METHODS Long-term hematopoietic and immunologic reconstitution for 342 autografts (311 adults, 31 children) after PHPCs were cryopreserved at -80°C was studied at 3, 6, and 12 months. The median (range) storage time of PHPCs cryopreserved was 1.7 (0.1-5.99) months. RESULTS Hemoglobin (Hb), white blood cells, and platelets (PLTs) reach normal values to trilineage at 12 months for 39% patients. Multivariate analysis shows a significant impact on CD34+ infused and on conditioning regimen for PLTs. Hb was influenced by growth factor administration at 3 months. Long-term recovery is also highly dependent on blood counts (Hb, PLT, and neutrophil) at start of high-dose chemotherapy. Only 43% of patients had reached normal lymphocyte values at 12 months after transplant, and a profound CD4+ T-lymphocyte deficit remained, as others reported. CONCLUSION Transplantation with PHPCs cryopreserved at -80°C for no more than 6 months is satisfactory for long-term hematopoietic and immunologic reconstitution, even if a profound CD4+ T lymphocyte deficit persists at 1 year. This easier and cheaper cryopreservation method also leads to successful engraftment.
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Affiliation(s)
- Laure Calvet
- Department of Clinical Hematology and Cell Therapy, EA3846, CIC 501, Auvergne University, France
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Culturing with trehalose produces viable endothelial cells after cryopreservation. Cryobiology 2012; 64:240-4. [DOI: 10.1016/j.cryobiol.2012.02.006] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2011] [Revised: 12/12/2011] [Accepted: 02/07/2012] [Indexed: 11/22/2022]
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Sánchez-Salinas A, Cabañas-Perianes V, Blanquer M, Majado MJ, Insausti CL, Monserrat J, Sánchez-Ibáñez MV, Menchón P, García-Hernández A, Gómez-Espuch J, Morales A, Moraleda JM. An automatic wash method for dimethyl sulfoxide removal in autologous hematopoietic stem cell transplantation decreases the adverse effects related to infusion. Transfusion 2012; 52:2382-6. [DOI: 10.1111/j.1537-2995.2012.03585.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Stolzing A, Naaldijk Y, Fedorova V, Sethe S. Hydroxyethylstarch in cryopreservation - mechanisms, benefits and problems. Transfus Apher Sci 2012; 46:137-47. [PMID: 22349548 DOI: 10.1016/j.transci.2012.01.007] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2011] [Revised: 12/19/2011] [Accepted: 01/23/2012] [Indexed: 12/20/2022]
Abstract
As the progress of regenerative medicine places ever greater attention on cryopreservation of (stem) cells, tried and tested cryopreservation solutions deserve a second look. This article discusses the use of hydroxyethyl starch (HES) as a cryoprotectant. Charting carefully the recorded uses of HES as a cryoprotectant, in parallel to its further clinical use, indicates that some HES subtypes are a useful supplement to dimethysulfoxide (DMSO) in cryopreservation. However, we suggest that the most common admixture ratio of HES and DMSO in cryoprotectant solutions has been established by historical happenstance and requires further investigation and optimization.
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Affiliation(s)
- A Stolzing
- Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
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Abstract
BACKGROUND Autologous, and in some cases allogeneic, hemopoietic stem cells (HSC) are stored for varying periods of time prior to infusion. For periods of greater than 48 h, storage requires cryopreservation. It is essential to optimize cell storage and ensure the quality of the product for subsequent reinfusion. METHODS A number of important variables may affect the subsequent quality of infused HSC and therapeutic cells (TC). This review discusses these and also reviews the regulatory framework that now aims to ensure the quality of stem cells and TC for transplantation. RESULTS Important variables included cell concentration, temperature, interval from collection to cryopreservation, manipulations performed. They also included rate of freezing and whether controlled-rate freezing was employed. Parameters studied were type of cryoprotectant utilized [dimethyl sulphoxide (DMSO) is most commonly used, sometimes in combination with hydroxyethyl starch (HES)]; and storage conditions. It is also important to assess the quality of stored stem cells. Measurements employed included the total cell count (TNC), mononuclear cell count (MNC), CD34+ cells and colony-forming units - granulocyte macrophage (CFU-GM). Of these, TNC and CD34+ are the most useful. However, the best measure of the quality of stored stem cells is their subsequent engraftment. The quality systems used in stem cell laboratories are described in the guidance of the Joint Accreditation Committee of ISCT (Europe) and the EBMT (JACIE) and the EU Directive on Tissues and Cells plus its supporting commission directives. Inspections of facilities are carried out by the appropriate national agencies and JACIE. CONCLUSION For high-quality storage of HSC and TC, processing facilities should use validated procedures that take into account critical variables. The quality of all products must be assessed before and after storage.
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Affiliation(s)
- Derwood Pamphilon
- Bristol Institute for Transfusion Sciences, University of Bristol, and English National Blood Service, UK
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Wu J, Lu Z, Nie M, Zhou H, Sun X, Xue X, Bi J, Fang G. Optimization of cryopreservation procedures for porcine endothelial progenitor cells. J Biosci Bioeng 2011; 113:117-23. [PMID: 22036230 DOI: 10.1016/j.jbiosc.2011.09.012] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Revised: 09/20/2011] [Accepted: 09/20/2011] [Indexed: 11/15/2022]
Abstract
Endothelial progenitor cells (EPCs) provide a powerful option for therapeutic use in ischemic diseases. The cell therapy-induced vasculogenesis requires sufficient homogeneous cells, and cryopreservation is a prerequisite for long-term storage and quality assurance of EPCs. The aim of this study was to optimize cryopreservation protocols of EPCs derived from porcine bone marrow. Bone marrow-derived mononuclear cells (MNCs) were isolated by density centrifugation and differentiated into EPCs. The first passage EPCs were frozen by using different methodologies, and after cryopreservation the thawed cells were cultured to the fourth passage. The recovery efficiency and functions of these cells were evaluated by determination of cell viability, proliferation and migration. We found the optimal conditions for cryopreservation of EPCs as follows: (i) a cryopreservation medium consisting of 10% dimethylsulphoxide (DMSO) in combination with 50% fetal bovine serum (FBS); (ii) using a controlled freezing rate at 5°C/min; (iii) at an optimal density of 5×10⁶/ml for cryopreserved EPCs; (iv) a storage temperature of -156°C. Under these conditions we demonstrated that EPCs could be stored in mechanical freezer for up to 18 months after cryopreservation without losing their phenotypic characteristics and biological functions.
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Affiliation(s)
- Jianguo Wu
- Department of General Surgery, Changhai Hospital, 168 Changhai Road, Shanghai 200433, China
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Orange Interventions for Symptoms Associated With Dimethyl Sulfoxide During Stem Cell Reinfusions. Cancer Nurs 2011; 34:361-8. [DOI: 10.1097/ncc.0b013e31820641a5] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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Akkök ÇA, Liseth K, Melve GK, Ersvær E, Hervig T, Bruserud Ø. Is there a scientific basis for a recommended standardization of collection and cryopreservation of peripheral blood stem cell grafts? Cytotherapy 2011; 13:1013-24. [DOI: 10.3109/14653249.2011.574117] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
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Ock SA, Rho GJ. Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cells (pMSCs). Cell Transplant 2011; 20:1231-9. [PMID: 21294964 DOI: 10.3727/096368910x552835] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Dimethyl sulfoxide (DMSO), a commonly used cryoprotectant in cryopreservation procedures, is detrimental to viability of cells. In this view point, a comparative study was carried out to evaluate the effect of DMSO on porcine mesenchymal stem cells (pMSCs). We compared the viability, colony forming unit-fibroblast (CFU-F) assay, expression of Bak and Bcl2 genes, Bcl2 protein antigen, and CD90 in pMSCs cryopreserved with 5%, 10%, and 20% DMSO. pMSCs isolated from bone marrow were characterized by alkaline phosphatase activity and the expression of transcription factors, such as Oct 3/4, Nanog, and Sox2. The cells were then cryopreserved by cooling at a rate of -1°C/min in a programmable freezer and stored in liquid nitrogen. The results of survival of pMSCs cryopreserved at 5% DMSO were comparable to control group (fresh pMSCs). The survival and the number of colonies formed in cryopreserved pMSCs were inversely proportional to the concentration of DMSO. The number of colonies formed in pMSCs cryopreserved with all concentrations of DMSO was significantly (p < 0.05) lower than the control group. An increased tendency for Bak and Bcl2 gene expression was noticed in cryopreserved pMSCs at 3 h postthawing compared to control group. There was a close resemblance in higher level of expression of CD90 between control and cryopreserved pMSCs. Because there was no considerable difference in the results of pMSCs cryopreserved at 5% and 10% DMSO, this study strongly suggests the use of 5% DMSO in cryopreservation of pMSCs as an alternative to conventional 10% DMSO.
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Affiliation(s)
- Sun-A Ock
- College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea
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Abstract
Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation is the lethality associated with the cooling and thawing processes. The major objective is to minimize damage to cells during low temperature freezing and storage and the use of a suitable cryoprotectant. The detrimental effects of cellular cryopreservation can be minimized by controlling the cooling rate, using better cryoprotective agents, maintaining appropriate storage temperatures, and controlling the cell thawing rate. As is described in this chapter, human MSCs can either be frozen in cryovials or in freezing bags together with cryopreserve solutions containing dimethyl sulfoxide (DMSO).
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Hayakawa J, Joyal EG, Gildner JF, Washington KN, Phang OA, Uchida N, Hsieh MM, Tisdale JF. 5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO. Transfusion 2010; 50:2158-66. [PMID: 20492608 DOI: 10.1111/j.1537-2995.2010.02684.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system. STUDY DESIGN AND METHODS We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice. RESULTS CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. CONCLUSION Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.
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Affiliation(s)
- Jun Hayakawa
- Molecular and Clinical Hematology Branch (MCHB), National Institutes of Diabetes and Digestive and Kidney Disorders (NIDDK), National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland, USA
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Majado MJ, Salgado-Cecilia G, Blanquer M, Funes C, González-García C, Insausti CL, Parrado A, Morales A, Minguela A, Moraleda JM. Cryopreservation impact on blood progenitor cells: influence of diagnoses, mobilization treatments, and cell concentration. Transfusion 2010; 51:799-807. [DOI: 10.1111/j.1537-2995.2010.02885.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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Martín-Henao GA, Resano PM, Villegas JMS, Manero PP, Sánchez JM, Bosch MP, Codins AE, Bruguera MS, Infante LR, Oyarzabal AP, Soldevila RN, Caiz DC, Bosch LM, Barbeta EC, Ronda JRG. Adverse reactions during transfusion of thawed haematopoietic progenitor cells from apheresis are closely related to the number of granulocyte cells in the leukapheresis product. Vox Sang 2010; 99:267-73. [DOI: 10.1111/j.1423-0410.2010.01341.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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