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Uehara K, Oshiro E, Ochiai A, Takagi R, Yamato M, Kato A. Lessons learned from contamination with endotoxin originated from the supplement in the cell culture medium. Regen Ther 2024; 27:230-233. [PMID: 38596824 PMCID: PMC11002528 DOI: 10.1016/j.reth.2024.03.022] [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: 01/16/2024] [Revised: 03/13/2024] [Accepted: 03/20/2024] [Indexed: 04/11/2024] Open
Abstract
Introduction Endotoxin is a typical pyrogen derived from the outer membrane of Gram-negative bacteria. In fabricating cell-based medicinal products, it is necessary to control endotoxin in the process and the products. In the quality control tests of our clinical study, endotoxin concentration in the culture supernatant of autologous oral mucosal epithelial cell sheets exceeded the criterion value. Therefore, endotoxin measurements were conducted to clarify the cause of the endotoxin contamination. Methods The reagents used to prepare the culture medium, the unused culture medium, and the culture supernatants were diluted with pure water. Endotoxin concentrations in the diluted samples were measured. Results Endotoxin was detected in both the unused culture medium and the culture supernatant of the epithelial cell sheets at higher concentrations than the criterion value. Therefore, endotoxin concentrations in the reagents used to prepare the culture medium were measured and were found to be below the criterion value, except for cholera toxin. On the other hand, three lots of cholera toxin products were used for the measurement, and the endotoxin concentrations were higher than the criterion value. The results indicate that the endotoxin contamination is caused by the cholera toxin product. Conclusions To prevent endotoxin contamination in cell-based medicinal products, endotoxin concentrations in reagents used for the fabrication should be measured in the facility conducting clinical research or confirmed by an adequate certificate of analysis from the manufacturers of the reagents.
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Affiliation(s)
- Koaki Uehara
- Social Medical Corporation Yuuaikai, Yuuai Medical Center, Advanced Medical Research Center, 50-5, Yone, Tomigusuku-shi, Okinawa 901-0224, Japan
| | - Eriko Oshiro
- Social Medical Corporation Yuuaikai, Yuuai Medical Center, Advanced Medical Research Center, 50-5, Yone, Tomigusuku-shi, Okinawa 901-0224, Japan
| | - Atsushi Ochiai
- Social Medical Corporation Yuuaikai, Yuuai Medical Center, Advanced Medical Research Center, 50-5, Yone, Tomigusuku-shi, Okinawa 901-0224, Japan
| | - Ryo Takagi
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Atsunaga Kato
- Social Medical Corporation Yuuaikai, Yuuai Medical Center, Advanced Medical Research Center, 50-5, Yone, Tomigusuku-shi, Okinawa 901-0224, Japan
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Takizawa H, Karakawa A, Suzawa T, Chatani M, Ikeda M, Sakai N, Azetsu Y, Takahashi M, Urano E, Kamijo R, Maki K, Takami M. Neural crest-derived cells possess differentiation potential to keratinocytes in the process of wound healing. Biomed Pharmacother 2021; 146:112593. [PMID: 34968925 DOI: 10.1016/j.biopha.2021.112593] [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: 12/10/2021] [Revised: 12/23/2021] [Accepted: 12/23/2021] [Indexed: 11/02/2022] Open
Abstract
Neural crest-derived cells (NCDCs), which exist as neural crest cells during the fetal stage and differentiate into palate cells, also exist in adult palate tissues, though with unknown roles. In the present study, NCDCs were labeled with EGFP derived from P0-Cre/CAG-CAT-EGFP (P0-EGFP) double transgenic mice, then their function in palate mucosa wound healing was analyzed. As a palate wound healing model, left-side palate mucosa of P0-EGFP mice was resected, and stem cell markers and keratinocyte markers were detected in healed areas. NCDCs were extracted from normal palate mucosa and precultured with stem cell media for 14 days, then were differentiated into keratinocytes or osteoblasts to analyze pluripotency. The wound healing process started with marginal mucosal regeneration on day two and the entire wound area was lined by regenerated mucosa with EGFP-positive cells (NCDCs) on day 28. EGFP-positive cells comprised approximately 60% of cells in healed oral mucosa, and 65% of those expressed stem cell markers (Sca-1+, PDGFRα+) and 30% expressed a keratinocyte marker (CK13+). In tests of cultured palate mucosa cells, approximately 70% of EGFP-positive cells expressed stem cell markers (Sca-1+, PDGFRα+). Furthermore, under differentiation inducing conditions, cultured EGFP-positive cells were successfully induced to differentiate into keratinocytes and osteoblasts. We concluded that NCDCs exist in adult palate tissues as stem cells and have potential to differentiate into various cell types during the wound healing process.
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Affiliation(s)
- Hideomi Takizawa
- Department of Orthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan; Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Akiko Karakawa
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
| | - Tetsuo Suzawa
- Department of Biochemistry, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Masahiro Chatani
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Megumi Ikeda
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Division of Endodontology, Department of Conservative Dentistry, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo, 145-8515, Japan
| | - Nobuhiro Sakai
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Yuki Azetsu
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Masahiro Takahashi
- Department of Orthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan
| | - Eri Urano
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan
| | - Ryutaro Kamijo
- Department of Biochemistry, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
| | - Koutaro Maki
- Department of Orthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan
| | - Masamichi Takami
- Department of Pharmacology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Pharmacological Research Center, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
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Takagi R, Tanuma-Takahashi A, Akiyama S, Kaneko W, Miura C, Yamato M, Shimizu T, Umezawa A. Laminin-511-derived recombinant fragment and Rho kinase inhibitor Y-27632 facilitate serial cultivation of keratinocytes differentiated from human embryonic stem cells. Regen Ther 2021; 18:242-252. [PMID: 34409136 PMCID: PMC8342860 DOI: 10.1016/j.reth.2021.07.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2021] [Revised: 07/07/2021] [Accepted: 07/13/2021] [Indexed: 11/17/2022] Open
Abstract
INTRODUCTION Keratinocytes derived from pluripotent stem cells have a short proliferative lifespan under conventional culture conditions that are optimized for keratinocytes. Recently, a Rho kinase inhibitor, Y-27632, had been used as a standard supplement for culture medium in which the proliferative lifespan of postnatal keratinocytes was markedly expanded. In addition, recombinant human laminin-511 was demonstrated to be an adhesive ligand for promoting proliferation of cultured epidermal keratinocytes. Based on this knowledge, efficacies of Y-27632 and a laminin511-derived recombinant fragment, known as laminin-511 E8 fragment (LN-511-E8), were evaluated for establishing cultivation methods of keratinocyte differentiated from human embryonic stem cells (hESC). METHODS Differentiated cells from hESCs, which were established with clinical grade in previous study, were seeded onto culture dishes coated with LN-511-E8 and co-cultured with a mouse feeder layer in serum-free medium supplemented with Y-27632. Before serial cultivation, hESC-derived keratinocytes were separated from other differentiated cells by trypsinization. The isolated hESC-derived keratinocytes were used for evaluating clonogenicity, gene expression analysis for keratinocyte markers, potency of terminal differentiation by air-lifting culture, and long-term proliferation activity by serial cultivation. Moreover, efficacies of Y-27632, LN-511-E8, and mouse feeder layer were evaluated on proliferation of hESC-derived keratinocytes. RESULTS hESC-derived keratinocytes with activity of clonal growth were successfully isolated by trypsinization and exhibited potency of differentiation to form stratified epidermal equivalents with expressions of progenitor and differentiation markers of epidermal keratinocyte. Y-27632 and LN-511-E8 were required for maintaining the proliferative activity of the hESC-derived keratinocytes in serially cultivation using mouse feeder layer with stable doubling time during logarithmic growth phase. CONCLUSIONS These results indicate the utility of Y-27632 and LN-511-E8 for serial cultivation of hESC-derived keratinocytes, which have a potential for fabricating allogeneic cellular products in clinical situations for regeneration of stratified epithelial tissues.
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Affiliation(s)
- Ryo Takagi
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Akiko Tanuma-Takahashi
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
| | - Saeko Akiyama
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
| | - Wakana Kaneko
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
| | - Chika Miura
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Tatsuya Shimizu
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Akihiro Umezawa
- Center of Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan
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Guérin LP, Le-Bel G, Desjardins P, Couture C, Gillard E, Boisselier É, Bazin R, Germain L, Guérin SL. The Human Tissue-Engineered Cornea (hTEC): Recent Progress. Int J Mol Sci 2021; 22:ijms22031291. [PMID: 33525484 PMCID: PMC7865732 DOI: 10.3390/ijms22031291] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 01/18/2021] [Accepted: 01/19/2021] [Indexed: 12/11/2022] Open
Abstract
Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.
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Affiliation(s)
- Louis-Philippe Guérin
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Gaëtan Le-Bel
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
- Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Pascale Desjardins
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
- Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Camille Couture
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
- Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Elodie Gillard
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Élodie Boisselier
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Richard Bazin
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Lucie Germain
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
- Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
| | - Sylvain L. Guérin
- CUO-Recherche, Médecine Régénératrice—Centre de Recherche du CHU de Québec, Université Laval, Québec, QC G1S 4L8, Canada; (L.-P.G.); (G.L.-B.); (P.D.); (C.C.); (E.G.); (É.B.); (R.B.); (L.G.)
- Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC G1J 1Z4, Canada
- Département d’Ophtalmologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada
- Correspondence: ; Tel.: +1-418-682-7565
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Benchaprathanphorn K, Sakulaue P, Siriwatwechakul W, Muangman P, Chinaroonchai K, Viravaidya-Pasuwat K. Preparation and characterization of human keratinocyte-fibroblast cell sheets constructed using PNIAM-co-AM grafted surfaces for burn wound healing. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2020; 31:126. [PMID: 33247815 DOI: 10.1007/s10856-020-06469-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 11/04/2020] [Indexed: 05/13/2023]
Abstract
Autologous skin grafting, the standard treatment for severe burns, is sometimes not possible due to the limited available skin surfaces for the procedure. With advances in tissue engineering, various cell-based skin substitutes have been developed to serve as skin replacements and to promote tissue regeneration and healing. In this work, we propose the use of cell sheet technology to fabricate keratinocyte-fibroblast tissue constructs from the temperature-responsive poly(N-isoproprylacrylamide-co-acrylamide) (PNIAM-co-AM) grafted surfaces for the treatment of burn wounds. The characteristics of the human keratinocyte and fibroblast cell sheets harvested using PNIAM-co-AM grafted surfaces were similar to those cell sheets detached from the commercially-available UpCellTM plates. Upon lowering the incubation temperature, confluent keratinocytes and fibroblasts could be detached as intact sheets, consisting of biologically active cells, as indicated by their high cell viability and their reattachment, migratory, and proliferative activities. A histological analysis of the stratified keratinocyte-fibroblast cell sheets revealed the evidence of cell migration and tissue reorganization to form two distinct epidermal and dermal layers, quite similar to the skin tissue's structure. In addition, the keratinocyte-fibroblast sheets could synthesize and release significant amounts of essential cytokines and growth factors involved in regulating the wound healing process, including IL-1α, IL-6, TNF-α, VEGF, and bFGF, implying the therapeutic effect of these cell sheets, which could be beneficial to accelerate tissue repair and regeneration, leading to faster wound healing.
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Affiliation(s)
- Kanokaon Benchaprathanphorn
- Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand
| | - Phongphot Sakulaue
- School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University, Pathumthani, 12120, Thailand
| | - Wanwipa Siriwatwechakul
- School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University, Pathumthani, 12120, Thailand
| | - Pornprom Muangman
- Department of Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Kusuma Chinaroonchai
- Department of Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Kwanchanok Viravaidya-Pasuwat
- Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
- Department of Chemical Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
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Ghauri AK, Wahid M, Mirza T, Uddin JAA. Direct differentiation of cord blood derived mesenchymal stem cells into keratinocytes without feeder layers and cAMP inducers. Pak J Med Sci 2020; 36:946-951. [PMID: 32704269 PMCID: PMC7372670 DOI: 10.12669/pjms.36.5.1566] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Objectives The purpose of our study was isolation of umbilical cord blood derived mesenchymal stem (UCB-MSCs), their direct differentiation towards keratinocytes without using feeder layers, cAMP inducers and hormones known for morphological maintenance and proliferation of keratinocytes and characterization of UCB-MSCs through flowcytometry and keratinocytes through immunofluorescence. Methods We have isolated and cultured UCB-MSCs (n=4) following critical parameters for successful isolation like sample processing within an hour of collection, gestational age not more than 38 weeks, no co-morbid and blood volume at least 80 ml. Cord blood mononuclear cells were isolated through ficoll based density-gradient centrifugation then cultured to isolate MSCs, defined by minimum criteria of International Society for Cellular Therapy. UCB-MSCs were then differentiated directly into keratinocytes. Differentiation was confirmed by morphology and characterized through immunofluorescence staining. UCB samples were collected from gynae/obstetric ward of OJHA campus under sterile conditions and processed at Stem cells and Regenerative medicine Lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Ojha campus. The total duration of study was approximately 12 months. Results We have successfully isolated UCB-MSCs that were plastic adherent, spindle shaped, showed trilineage mesodermal differentiation potential and were positive for CD90, CD73 and CD105 and negative for CD34 markers. UCB-MSCs were directly differentiated towards keratinocytes without using cAMP inducers, hormones or feeder layers. Differentiated keratinocytes attained typical honeycomb morphology and were stained positive on immunofluorescence for anti-pan cytokeratin antibody. Conclusion Our study concludes possibility of direct differentiation of isolated and cultured UCB-MSCs into keratinocytes without using feeder layers and conventional keratinocyte culture media.
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Affiliation(s)
- Ayesha Kashmala Ghauri
- Ayesha Kashmala Ghauri, Stem Cells and Regenerative Medicine Lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Karachi, Pakistan
| | - Mohsin Wahid
- Mohsin Wahid, Stem Cells and Regenerative Medicine Lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Department of Pathology, Dow International Medical College, Dow University of Health Sciences (OJHA Campus), Karachi, Pakistan
| | - Talat Mirza
- Talat Mirza, Department of Research, Ziauddin Medical University, Karachi, Pakistan
| | - Jahan Ara Ain Uddin
- Jahan Ara Ain Uddin, Department of Gynecology and Obstetrics, Dow University Hospital, Karachi, Pakistan
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Ohki T, Yamamoto M. Esophageal regenerative therapy using cell sheet technology. Regen Ther 2020; 13:8-17. [PMID: 33490318 PMCID: PMC7794050 DOI: 10.1016/j.reth.2020.04.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2019] [Revised: 03/20/2020] [Accepted: 04/19/2020] [Indexed: 12/13/2022] Open
Abstract
We have been conducting research on esophageal regenerative therapy using cell sheet technology. In particular, in the endoscopic field, we have pushed forward clinical research on endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to esophageal ulcer after endoscopic submucosal dissection (ESD). We started research in this direction using animal models in 2004 and performed clinical research in 2012 in collaboration with Nagasaki University and Karolinska Institute. Although in full-circumferential cases it was difficult to prevent esophageal stricture after ESD, there were no complications and stricture could be suppressed. The cell sheet technology is still in its infancy. However, we are convinced that it has a high potential for application in various areas of gastrointestinal science. In this review, we focus on the pre-clinical and clinical trial results obtained and on the theoretical aspects of (1) stricture prevention, (2) esophageal tissue engineering research, and (3) endoscopic transplantation, and review the esophageal regenerative therapy by cell sheet technology.
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Key Words
- CMC, carboxymethyl cellulose
- CPC, cell-processing center
- Cell sheet technology
- EBD, endoscopic balloon dilation
- ECM, extracellular matrix
- EMR, endoscopic mucosal dissection
- ESD, endoscopic submucosal dissection
- Endoscopic submucosal dissection (ESD)
- Endoscopic transplantation
- Esophageal stricture
- GMP, good manufacturing practice
- OMECS, oral mucosal epithelial cell sheet
- PGA, polyglycolic acid
- PIPAAm, poly(N-isopropylacrylamide)
- PVDF, polyvinylidene difluoride
- Regenerative medicine
- SEMS, self-expandable metallic stent
- TAC, triamcinolone
- Tissue-engineered oral mucosal
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Affiliation(s)
- Takeshi Ohki
- Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Masakazu Yamamoto
- Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan
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Cortez Ghio S, Cantin-Warren L, Guignard R, Larouche D, Germain L. Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes' Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers? Int J Mol Sci 2018; 19:E2174. [PMID: 30044428 PMCID: PMC6121595 DOI: 10.3390/ijms19082174] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2018] [Accepted: 07/18/2018] [Indexed: 12/12/2022] Open
Abstract
Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells' phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes' proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.
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Affiliation(s)
- Sergio Cortez Ghio
- Université Laval Research Center on Experimental Organogenesis/LOEX, Québec, QC G1J 1Z4, Canada.
- Regenerative Medicine Division, CHU de Québec-Université Laval Research Centre, Québec, QC G1J 1Z4, Canada.
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada.
| | - Laurence Cantin-Warren
- Université Laval Research Center on Experimental Organogenesis/LOEX, Québec, QC G1J 1Z4, Canada.
- Regenerative Medicine Division, CHU de Québec-Université Laval Research Centre, Québec, QC G1J 1Z4, Canada.
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada.
| | - Rina Guignard
- Université Laval Research Center on Experimental Organogenesis/LOEX, Québec, QC G1J 1Z4, Canada.
- Regenerative Medicine Division, CHU de Québec-Université Laval Research Centre, Québec, QC G1J 1Z4, Canada.
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada.
| | - Danielle Larouche
- Université Laval Research Center on Experimental Organogenesis/LOEX, Québec, QC G1J 1Z4, Canada.
- Regenerative Medicine Division, CHU de Québec-Université Laval Research Centre, Québec, QC G1J 1Z4, Canada.
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada.
| | - Lucie Germain
- Université Laval Research Center on Experimental Organogenesis/LOEX, Québec, QC G1J 1Z4, Canada.
- Regenerative Medicine Division, CHU de Québec-Université Laval Research Centre, Québec, QC G1J 1Z4, Canada.
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada.
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López-Paniagua M, Nieto-Miguel T, de la Mata A, Galindo S, Herreras JM, Corrales RM, Calonge M. Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions. Curr Eye Res 2016; 42:685-695. [PMID: 27911610 DOI: 10.1080/02713683.2016.1250278] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.
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Affiliation(s)
- Marina López-Paniagua
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - Teresa Nieto-Miguel
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - Ana de la Mata
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - Sara Galindo
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - José M Herreras
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - Rosa M Corrales
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
| | - Margarita Calonge
- a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.,b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain
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10
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Engineered three-dimensional rabbit oral epithelial-mesenchymal-muscular hybrid sheets. Int J Oral Sci 2016; 8:145-54. [PMID: 27341388 PMCID: PMC5113088 DOI: 10.1038/ijos.2016.16] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/25/2016] [Indexed: 11/09/2022] Open
Abstract
Regenerative muscles are required for swallowing and mastication, and are important for functional recovery from diseases involving oral muscular defects. Therefore, we generated three-layer hybrid sheets, similar to oral mucosal structures containing submucosal muscles, using rabbit oral mucosa epithelial, mesenchymal, and myoblastic progenitor cells, and examined the structural proteins. Each cell type was obtained from rabbit oral mucosa using enzymatic digestion. Isolated mesenchymal and myoblastic cells were multi-differentiated into osteoblasts, adipocytes, and chondrocytes or myotubes. Isolated epithelial cells were cultured on collagen gels containing isolated mesenchymal cells for 2 weeks, and these epithelial-mesenchymal cell sheets were laminated onto myoblastic cell sheets. The engineered hybrid sheets were multi-stratified in the epithelial and myoblastic layers in a time-dependent manner, expressing intermediate cytoskeletal filament proteins of epithelium and muscle. Hybrid sheets also expressed extracellular matrix basement membrane proteins. Immature cell markers for epithelial and myoblastic cells were observed continuously in hybrid sheet cultures. We established engineered three-dimensional rabbit oral mucosa hybrid sheets containing each immature cell type in vitro.
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11
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Preparation of hyperbranched polystyrene-g-poly(N-isopropylacrylamide) copolymers and its application to novel thermo-responsive cell culture dishes. POLYMER 2015. [DOI: 10.1016/j.polymer.2015.06.035] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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12
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EXP CLIN TRANSPLANTExp Clin Transplant 2015; 13. [DOI: 10.6002/ect.mesot2014.p216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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13
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Ohki T, Yamato M, Ota M, Takagi R, Kondo M, Kanai N, Okano T, Yamamoto M. Application of regenerative medical technology using tissue-engineered cell sheets for endoscopic submucosal dissection of esophageal neoplasms. Dig Endosc 2015; 27:182-8. [PMID: 25181559 DOI: 10.1111/den.12354] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2014] [Accepted: 08/22/2014] [Indexed: 12/24/2022]
Abstract
We have developed a technique for endoscopic transplantation of cultured autologous oral mucosal epithelial cell sheets to an esophageal ulcer following endoscopic submucosal dissection (ESD). The epithelial cell sheets successfully prevented esophageal stricture after ESD. Key technology is that epithelial cell sheets cultured from oral mucosal tissue and attached proteins can be harvested using cell sheet technology and can be transplanted to a wound site without the use of adhesive material. This regenerative procedure can promote the epithelialization of ulceration safely and effectively. In the near future, the development of advanced endoscopic treatment of regenerative medicine shows promise.
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Affiliation(s)
- Takeshi Ohki
- Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Tokyo, Japan; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
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14
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Kobayashi S, Kanai N, Ohki T, Takagi R, Yamaguchi N, Isomoto H, Kasai Y, Hosoi T, Nakao K, Eguchi S, Yamamoto M, Yamato M, Okano T. Prevention of esophageal strictures after endoscopic submucosal dissection. World J Gastroenterol 2014; 20:15098-15109. [PMID: 25386058 PMCID: PMC4223243 DOI: 10.3748/wjg.v20.i41.15098] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Revised: 02/14/2014] [Accepted: 05/19/2014] [Indexed: 02/06/2023] Open
Abstract
Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have recently been accepted as less invasive methods for treating patients with early esophageal cancers such as squamous cell carcinoma and dysplasia of Barrett’s esophagus. However, the large defects in the esophageal mucosa often cause severe esophageal strictures, which dramatically reduce the patient’s quality of life. Although preventive endoscopic balloon dilatation can reduce dysphagia and the frequency of dilatation, other approaches are necessary to prevent esophageal strictures after ESD. This review describes several strategies for preventing esophageal strictures after ESD, with a particular focus on anti-inflammatory and tissue engineering approaches. The local injection of triamcinolone acetonide and other systemic steroid therapies are frequently used to prevent esophageal strictures after ESD. Tissue engineering approaches for preventing esophageal strictures have recently been applied in basic research studies. Scaffolds with temporary stents have been applied in five cases, and this technique has been shown to be safe and is anticipated to prevent esophageal strictures. Fabricated autologous oral mucosal epithelial cell sheets to cover the defective mucosa similarly to how commercially available skin products fabricated from epidermal cells are used for skin defects or in cases of intractable ulcers. Fabricated autologous oral-mucosal-epithelial cell sheets have already been shown to be safe.
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15
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Kanai N, Yamato M, Okano T. Cell sheets engineering for esophageal regenerative medicine. ANNALS OF TRANSLATIONAL MEDICINE 2014; 2:28. [PMID: 25333004 DOI: 10.3978/j.issn.2305-5839.2014.03.06] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 03/20/2014] [Indexed: 12/11/2022]
Abstract
Recently, cell-based therapies, regenerative medicine, and tissue engineering have been progressing rapidly. We have developed a novel strategy for regenerative medicine to recover tissue functions using temperature-responsive cell culture surfaces. To overcome of conventional methods such as the usage of single-cell suspension injection, we have applied transplantable cell sheets fabricated with temperature-responsive culture surfaces for cell delivery. In the field of gastroenterology, transplantable cell sheets from autologous oral mucosal epithelial cells can prevent esophageal stricture following extensive endoscopic mucosal resection.
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Affiliation(s)
- Nobuo Kanai
- Institute of Advanced Biomedical Engineering and Science (TWIns), Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and Science (TWIns), Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Teruo Okano
- Institute of Advanced Biomedical Engineering and Science (TWIns), Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
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16
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The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts. Biomaterials 2014; 35:9591-8. [PMID: 25176070 DOI: 10.1016/j.biomaterials.2014.07.048] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Accepted: 07/23/2014] [Indexed: 11/23/2022]
Abstract
Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing.
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17
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Mainzer C, Barrichello C, Debret R, Remoué N, Sigaudo-Roussel D, Sommer P. Insulin-transferrin-selenium as an alternative to foetal serum for epidermal equivalents. Int J Cosmet Sci 2014; 36:427-35. [PMID: 24847782 DOI: 10.1111/ics.12141] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2014] [Accepted: 04/26/2014] [Indexed: 12/27/2022]
Abstract
OBJECTIVE Organotypic skin models are powerful tools for research in development, ageing and diseases. They have become more and more complex with the use of multiple cell types. This requires a culture medium adapted to optimize the development of such in vitro skin. Foetal bovine serum (FBS) is the most complete supplement in existence at the moment, providing at once growth factors, vitamins, hormones and other circulating compounds. However, this cocktail suffers from batch variability and its animal origin is ethically questionable. More importantly, its biological activities may interfere with the study of certain signalling pathways. Here, we present a strategy for constructing an epidermal equivalent using a defined culture medium without serum. METHODS An epidermal equivalent was constructed with primary human keratinocytes cultured using an insulin-transferrin-selenium (ITS) medium. Determination of steady-state gene expression levels and the immunohistological characterization of keratinocyte markers were performed to compare the ITS medium condition with a reference model, where keratinocytes were co-cultured with fibroblasts in the presence of FBS. RESULTS The data show that the ITS medium promoted the expression of keratinocyte proliferation and differentiation markers at the protein and transcript levels in a similar way to that of the reference model. CONCLUSION We show that culture using the ITS medium appears as a viable replacement for FBS in the construction of epidermal equivalents, opening the way to signal transduction studies.
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Affiliation(s)
- C Mainzer
- Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, CNRS, UMR5305, Université Lyon 1, 7 passage du Vercors, 69367, Lyon, France
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18
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Ohki T, Yamato M, Okano T, Yamamoto M. Regenerative medicine: tissue-engineered cell sheet for the prevention of post-esophageal ESD stricture. Gastrointest Endosc Clin N Am 2014; 24:273-81. [PMID: 24679238 DOI: 10.1016/j.giec.2013.11.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Induced pluripotent stem (iPS) cells have captured the world's attention and directed an unprecedented focus on regenerative medicine. The potential of iPS cells to aid in the development of new treatments for various diseases is exciting, and researchers are only beginning to discover their potential benefits for humans. iPS cells are more effective if they are interconnected with tissues; however, new technologies are needed to create and transplant these tissues. This study introduces a new connection between endoscopy and regenerative medicine in gastroenterology through specifically addressing how cell sheet technology can be a viable method of tissue creation and transplantation.
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Affiliation(s)
- Takeshi Ohki
- Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan; Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Teruo Okano
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Masakazu Yamamoto
- Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
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19
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Suzuki M, Yamane S, Higa K, Umezawa T, Serikawa M, Shimazaki J, Abe S. Expression of Cross-Linked Protein on Tissue-Engineered Epithelial Cell Sheets from Rabbit Oral Mucosa. J HARD TISSUE BIOL 2014. [DOI: 10.2485/jhtb.23.275] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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20
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Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces. Biomaterials 2013; 34:9082-8. [DOI: 10.1016/j.biomaterials.2013.08.029] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2013] [Accepted: 08/12/2013] [Indexed: 11/18/2022]
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21
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Ilmarinen T, Laine J, Juuti-Uusitalo K, Numminen J, Seppänen-Suuronen R, Uusitalo H, Skottman H. Towards a defined, serum- and feeder-free culture of stratified human oral mucosal epithelium for ocular surface reconstruction. Acta Ophthalmol 2013; 91:744-50. [PMID: 22963401 DOI: 10.1111/j.1755-3768.2012.02523.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
PURPOSE Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. METHODS In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration. RESULTS In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. CONCLUSION In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.
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Affiliation(s)
- Tanja Ilmarinen
- Institute of Biomedical Technology, University of Tampere, Tampere, FinlandBioMediTech, Tampere, FinlandDepartment of Eye, Ear, and Oral Diseases, Tampere University Hospital, Tampere, FinlandSILK, Department of Ophthalmology, University of Tampere, Tampere, FinlandEye Center, Tampere University Hospital, Tampere, Finland
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22
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Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model. Biomaterials 2013; 34:9010-7. [DOI: 10.1016/j.biomaterials.2013.07.065] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Accepted: 07/19/2013] [Indexed: 11/23/2022]
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Varkey M, Ding J, Tredget EE. Fibrotic remodeling of tissue-engineered skin with deep dermal fibroblasts is reduced by keratinocytes. Tissue Eng Part A 2013; 20:716-27. [PMID: 24090416 DOI: 10.1089/ten.tea.2013.0434] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Two-thirds of burn patients with deep dermal injuries are affected by hypertrophic scars, and currently, there are no clinically effective therapies. Tissue-engineered skin is a very promising model for the elucidation of the role of matrix microenvironment and biomechanical characteristics and could help in the identification of new therapeutic targets for hypertrophic scars. Conventionally, tissue-engineered skin is made of heterogeneous dermal fibroblasts and keratinocytes; however, recent work has shown that superficial and deep dermal fibroblasts are antifibrotic and profibrotic, respectively. Furthermore, keratinocytes are believed to regulate the development and remodeling of fibrosis in skin. This study aimed to assess the influence of keratinocytes and layered fibroblasts on the characteristics of tissue-engineered skin. Layered fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of the lower abdominal tissue were independently co-cultured on collagen-glycosaminoglycan scaffolds, and the resulting tissue-engineered skin was assessed for differences in tissue remodeling based on the underlying specific dermal fibroblast subpopulation. Collagen production by deep fibroblasts but not by superficial fibroblasts was significantly reduced upon co-culture with keratinocytes. Also, keratinocytes in the tissue-engineered skin resulted in significantly reduced expression of profibrotic connective tissue growth factor and fibronectin, and increased expression of the antifibrotic matrix metalloproteinase-1 by deep fibroblasts but not by superficial fibroblasts. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower levels of small proteoglycans, decorin, and fibromodulin, and higher levels of large proteoglycan, versican, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Tissue-engineered skin made of deep fibroblasts and keratinocytes had lower expression of transforming growth factor (TGF)-α, interleukin (IL)-1, and keratinocyte growth factor but higher expression of platelet-derived growth factor and IL-6, compared to tissue-engineered skin made of superficial fibroblasts and keratinocytes. Furthermore, co-culture with keratinocytes reduced TGF-β1 production of deep but not superficial fibroblasts. Additionally, keratinocytes reduced the differentiation of deep fibroblasts to myofibroblasts in tissue-engineered skin constructs, but not that of superficial fibroblasts. Taken together, keratinocytes reduce fibrotic remodeling of the scaffolds by deep dermal fibroblasts. Our results therefore demonstrate that tissue-engineered skin made specifically with a homogeneous population of superficial fibroblasts and keratinocytes is less fibrotic than that with a heterogeneous population of fibroblasts and keratinocytes.
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Affiliation(s)
- Mathew Varkey
- 1 Wound Healing Research Group, Division of Plastic and Reconstructive Surgery, University of Alberta , Edmonton, Canada
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Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method. Exp Eye Res 2013; 118:53-60. [PMID: 24184720 DOI: 10.1016/j.exer.2013.10.019] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2013] [Revised: 09/11/2013] [Accepted: 10/19/2013] [Indexed: 01/28/2023]
Abstract
The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration.
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25
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Varkey M, Ding J, Tredget EE. Superficial dermal fibroblasts enhance basement membrane and epidermal barrier formation in tissue-engineered skin: implications for treatment of skin basement membrane disorders. Tissue Eng Part A 2013; 20:540-52. [PMID: 24004160 DOI: 10.1089/ten.tea.2013.0160] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Basement membrane is a highly specialized structure that binds the dermis and the epidermis of the skin, and is mainly composed of laminins, nidogen, collagen types IV and VII, and the proteoglycans, collagen type XVIII and perlecan, all of which play critical roles in the function and resilience of skin. Both dermal fibroblasts and epidermal keratinocytes contribute to the development of the basement membrane, and in turn the basement membrane and underlying dermis influence the development and function of the epidermal barrier. Disruption of the basement membrane results in skin fragility, extensive painful blistering, and severe recurring wounds as seen in skin basement membrane disorders such as epidermolysis bullosa, a family of life-threatening congenital skin disorders. Currently, there are no successful strategies for treatment of these disorders; we propose the use of tissue-engineered skin as a promising approach for effective wound coverage and to enhance healing. Fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of tissue from abdominoplasty patients were independently cocultured on collagen-glycosaminoglycan matrices, and the resulting tissue-engineered skin was assessed for functional differences based on the underlying specific dermal fibroblast subpopulation. Tissue-engineered skin with superficial fibroblasts and keratinocytes formed a continuous epidermis with increased epidermal barrier function and expressed higher levels of epidermal proteins, keratin-5, and E-cadherin, compared to that with deep fibroblasts and keratinocytes, which had an intermittent epidermis. Further, tissue-engineered skin with superficial fibroblasts and keratinocytes formed better basement membrane, and produced more laminin-5, nidogen, collagen type VII, compared to that with deep fibroblasts and keratinocytes. Overall, our results demonstrate that tissue-engineered skin with superficial fibroblasts and keratinocytes forms significantly better basement membrane with higher expression of dermo-epidermal adhesive and anchoring proteins, and superior epidermis with enhanced barrier function compared to that with deep fibroblasts and keratinocytes, or with superficial fibroblasts, deep fibroblasts, and keratinocytes. The specific use of superficial fibroblasts in tissue-engineered skin may thus be more beneficial to promote adhesion of newly formed skin and wound healing, and is therefore promising for the treatment of patients with basement membrane disorders and other skin blistering diseases.
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Affiliation(s)
- Mathew Varkey
- 1 Wound Healing Research Group, Division of Plastic and Reconstructive Surgery, University of Alberta , Edmonton, Canada
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Iwata T, Washio K, Yoshida T, Ishikawa I, Ando T, Yamato M, Okano T. Cell sheet engineering and its application for periodontal regeneration. J Tissue Eng Regen Med 2013; 9:343-56. [DOI: 10.1002/term.1785] [Citation(s) in RCA: 111] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2012] [Revised: 05/01/2013] [Accepted: 05/06/2013] [Indexed: 01/01/2023]
Affiliation(s)
- Takanori Iwata
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
- Department of Oral and Maxillofacial Surgery; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Kaoru Washio
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Toshiyuki Yoshida
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Isao Ishikawa
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Tomohiro Ando
- Department of Oral and Maxillofacial Surgery; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
| | - Teruo Okano
- Institute of Advanced Biomedical Engineering and Science; Tokyo Women's Medical University; Shinjuku-ku Tokyo Japan
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Kondo M, Yamato M, Takagi R, Murakami D, Namiki H, Okano T. Significantly different proliferative potential of oral mucosal epithelial cells between six animal species. J Biomed Mater Res A 2013; 102:1829-37. [DOI: 10.1002/jbm.a.34849] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2013] [Revised: 05/18/2013] [Accepted: 06/10/2013] [Indexed: 11/09/2022]
Affiliation(s)
- Makoto Kondo
- Graduate School of Advanced Science and EngineeringWaseda UniversityShinjuku‐ku Tokyo162‐8480 Japan
- Institute of Advanced Biomedical Engineering and ScienceTokyo Women's Medical University, TWInsShinjuku‐ku Tokyo162‐8666 Japan
| | - Masayuki Yamato
- Institute of Advanced Biomedical Engineering and ScienceTokyo Women's Medical University, TWInsShinjuku‐ku Tokyo162‐8666 Japan
| | - Ryo Takagi
- Institute of Advanced Biomedical Engineering and ScienceTokyo Women's Medical University, TWInsShinjuku‐ku Tokyo162‐8666 Japan
| | - Daisuke Murakami
- Graduate School of Advanced Science and EngineeringWaseda UniversityShinjuku‐ku Tokyo162‐8480 Japan
- Institute of Advanced Biomedical Engineering and ScienceTokyo Women's Medical University, TWInsShinjuku‐ku Tokyo162‐8666 Japan
| | - Hideo Namiki
- Graduate School of Advanced Science and EngineeringWaseda UniversityShinjuku‐ku Tokyo162‐8480 Japan
| | - Teruo Okano
- Institute of Advanced Biomedical Engineering and ScienceTokyo Women's Medical University, TWInsShinjuku‐ku Tokyo162‐8666 Japan
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Umemoto T, Yamato M, Nishida K, Okano T. Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces. ACTA ACUST UNITED AC 2013. [DOI: 10.1007/s11434-013-5742-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
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Abstract
The ultimate goal of the treatment of cutaneous burns and wounds is to restore the damaged skin both structurally and functionally to its original state. Recent research advances have shown the great potential of stem cells in improving the rate and quality of wound healing and regenerating the skin and its appendages. Stem cell-based therapeutic strategies offer new prospects in the medical technology for burns and wounds care. This review seeks to give an updated overview of the applications of stem cell therapy in burns and wound management since our previous review of the “stem cell strategies in burns care”.
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Affiliation(s)
- Lin Huang
- Department of Surgery, Division of Plastic, Reconstructive and Aesthetic Surgery, The Chinese University of Hong Kong, Hong Kong
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Kondo M, Yamato M, Takagi R, Namiki H, Okano T. The regulation of epithelial cell proliferation and growth by IL-1 receptor antagonist. Biomaterials 2012; 34:121-9. [PMID: 23059003 DOI: 10.1016/j.biomaterials.2012.09.036] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2012] [Accepted: 09/17/2012] [Indexed: 01/21/2023]
Abstract
We have performed clinical translation of epithelial cell sheets fabricated on temperature-responsive culture surfaces to treat cornea and esophagus. In the preclinical study using animal models, we found epithelial cell growth potential varied among species. Canine epithelial cell growth was prominent, while rat one was poor under 3T3 feeder layer-free condition. The aim of the present study was to identify growth-promoting factors for epithelial cells. Conditioned medium of canine cell culture harvested at different time points showed different growth promotive activity for rat epithelial cells. Time-dependent gene expression was quantitatively evaluated for forty growth factors, and compared with conditioned medium results. Statistically significant promotive activity was observed with IL-1RA, and significant inhibitory activity was observed with IL-1α. Furthermore, neutralizing anti-IL-1α antibody also showed significant promotive activity. Human epidermal keratinocytes were promoted to proliferate by IL-1RA and neutralizing anti-IL-1α antibody, and showed well differentiation to form transplantable, squamous stratified epithelial cell sheets. These findings would be useful to fabricate reproducible, transplantable epithelial cell sheets for regenerative medicine.
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Affiliation(s)
- Makoto Kondo
- Graduate School of Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan
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Takagi R, Yamato M, Kanai N, Murakami D, Kondo M, Ishii T, Ohki T, Namiki H, Yamamoto M, Okano T. Cell sheet technology for regeneration of esophageal mucosa. World J Gastroenterol 2012; 18:5145-50. [PMID: 23066307 PMCID: PMC3468845 DOI: 10.3748/wjg.v18.i37.5145] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2011] [Revised: 04/18/2012] [Accepted: 04/27/2012] [Indexed: 02/06/2023] Open
Abstract
The progress of tissue-engineering technology has realized development of new therapies to treat various disorders by using cultured cells. Cell- and tissue-based therapies have been successfully applied to human patients, and several tissue-engineered products have been approved by the regulatory agencies and are commercially available. In the review article, we describe our experience of development and clinical application of cell sheet-based regenerative medicine. Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have been shown to be useful for removal of gastrointestinal neoplasms with less invasiveness compared with open surgery, especially in esophageal surgery. However, postoperative inflammation and stenosis are major complications observed after intensive mucosal resection. Therefore, we have developed novel regenerative medicine to prevent such complications and promote wound healing of esophageal mucosa after EMR or ESD. Transplantable oral mucosal epithelial cell sheets were fabricated from patients’ own oral mucosa. Immediately after EMR or ESD, fabricated autologous cell sheets were endoscopically transplanted to the ulcer sites. We performed a preclinical study with a canine model. In human clinical settings, cell culture and cell sheet fabrication were performed in clean rooms according to good manufacturing practice guidelines, and pharmaceutical drugs were used as supplements to culture medium in place of research regents used in animal study. We believe that cell-based regenerative medicine would be useful to improve quality of life of patients after EMR or ESD.
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PENFIELD JOSHUAD, GOROSPE EMMANUELC, WANG KENNETHK. Tissue-engineered cell sheets for stricture prevention: a new connection between endoscopy and regenerative medicine. Gastroenterology 2012; 143:526-529. [PMID: 22842059 PMCID: PMC3815668 DOI: 10.1053/j.gastro.2012.07.030] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
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Takagi R, Yamato M, Murakami D, Kondo M, Ohki T, Sasaki R, Nishida K, Namiki H, Yamamoto M, Okano T. Fabrication and validation of autologous human oral mucosal epithelial cell sheets to prevent stenosis after esophageal endoscopic submucosal dissection. Pathobiology 2011; 78:311-9. [PMID: 22104202 DOI: 10.1159/000322575] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
OBJECTIVES Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. METHODS The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. RESULTS Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. CONCLUSIONS The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.
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Affiliation(s)
- Ryo Takagi
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University and Global Center of Excellence, Japan
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