A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy
Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epit...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-03-01
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| Series: | Regenerative Therapy |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352320422000050 |
| _version_ | 1818315224904630272 |
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| author | Shota Mitsuboshi Jun Homma Hidekazu Sekine Ryo Takagi Tatsuya Shimizu Masato Kanzaki |
| author_facet | Shota Mitsuboshi Jun Homma Hidekazu Sekine Ryo Takagi Tatsuya Shimizu Masato Kanzaki |
| author_sort | Shota Mitsuboshi |
| collection | DOAJ |
| description | Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy. |
| first_indexed | 2024-12-13T09:02:09Z |
| format | Article |
| id | doaj.art-bdc79fe27345444fa578de52b1cf4056 |
| institution | Directory Open Access Journal |
| issn | 2352-3204 |
| language | English |
| last_indexed | 2024-12-13T09:02:09Z |
| publishDate | 2022-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Regenerative Therapy |
| spelling | doaj.art-bdc79fe27345444fa578de52b1cf40562022-12-21T23:53:09ZengElsevierRegenerative Therapy2352-32042022-03-0119113121A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapyShota Mitsuboshi0Jun Homma1Hidekazu Sekine2Ryo Takagi3Tatsuya Shimizu4Masato Kanzaki5Department of Thoracic Surgery, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanInstitute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanInstitute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan; Corresponding author. Fax: +81 3 5269 7616.Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanInstitute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanDepartment of Thoracic Surgery, 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, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanIntroduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.http://www.sciencedirect.com/science/article/pii/S2352320422000050Alveolar epithelial cellFeeder-freeCell sheetRegenerative therapy |
| spellingShingle | Shota Mitsuboshi Jun Homma Hidekazu Sekine Ryo Takagi Tatsuya Shimizu Masato Kanzaki A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy Regenerative Therapy Alveolar epithelial cell Feeder-free Cell sheet Regenerative therapy |
| title | A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy |
| title_full | A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy |
| title_fullStr | A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy |
| title_full_unstemmed | A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy |
| title_short | A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy |
| title_sort | novel alveolar epithelial cell sheet fabricated under feeder free conditions for potential use in pulmonary regenerative therapy |
| topic | Alveolar epithelial cell Feeder-free Cell sheet Regenerative therapy |
| url | http://www.sciencedirect.com/science/article/pii/S2352320422000050 |
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