Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
PLoS One. 2020 May 20;15(5):e0232270. doi: 10.1371/journal.pone.0232270. eCollection 2020.
Transplantation of cultured epidermal cell sheets (CES) can be life-saving for patients with large area burns. CES have also been successfully used to regenerate eye and urethral epithelia in animal models. Short-term storage aims to extend the transplantation window, offers flexibility in timing surgery and allows testing of CES quality, phenotype and sterility. This study investigated extended CES storage and explored the effect of additional re-incubation recovery time following storage. The proliferative quality of stored confluent versus pre-confluent CES was also investigated using functional testing. CES were stored at 12°C and results compared to non-stored control CES. Investigation of timepoints during 15 days storage revealed that viability began to deteriorate by day 11 and was associated with increased lactate in the storage medium. The percentage of apoptotic cells also significantly increased by day 11. Flow cytometry analysis of integrin β1 expression and cell size indicated best retention of stem cells at 7 days of storage. Functional testing of pre-confluent and confluent cells following 7 days storage showed that pre-confluent cells responded well to 1-day re-incubation after storage; they became highly prolific, increasing in number by ~67%. Conversely, proliferation in stored confluent cells declined by ~50% with 1-day re-incubation. Pre-confluent stored CES also had far superior stem cell colony forming efficiency (CFE) performance compared to the confluent group. Re-incubation improved CFE in both groups, but the pre-confluent group again out-performed the confluent group with significantly more colonies. In conclusion, a maximum storage period of 7 days is recommended. Use of pre-confluent cells and one day recovery incubation greatly improves viability, colony-forming ability and proliferation of cells stored for 7 days at 12°C. Thus, these recommendations should be considered under culture and storage of high-quality CES for clinical use.
培养表皮细胞片（CES）的移植对于大面积烧伤的患者来说可以救命。CES 已成功用于在动物模型中再生眼和尿道上皮。短期储存旨在延长移植窗口，提供手术时间的灵活性，并允许测试 CES 的质量、表型和无菌性。本研究调查了 CES 的延长储存，并探讨了储存后额外再孵育恢复时间的影响。还使用功能测试研究了储存的致密与预致密 CES 的增殖质量。CES 在 12°C 下储存，并将结果与未储存的对照 CES 进行比较。在 15 天储存期间的时间点研究表明，活力在第 11 天开始恶化，与储存介质中乳酸的增加有关。第 11 天，凋亡细胞的比例也显著增加。通过流式细胞术分析整合素 β1 表达和细胞大小表明，在储存 7 天时干细胞的保留最好。在储存 7 天后对预致密和致密细胞进行功能测试表明，预致密细胞在储存后 1 天再孵育反应良好；它们的数量增加了约 67%。相反，储存的致密细胞在 1 天再孵育时增殖下降了约 50%。预致密储存的 CES 还具有比致密组更高的干细胞集落形成效率（CFE）性能。再孵育提高了两组的 CFE，但预致密组再次表现优于致密组，集落数明显更多。总之，建议最大储存时间为 7 天。使用预致密细胞和 1 天恢复孵育可显著提高在 12°C 下储存 7 天的细胞活力、集落形成能力和增殖能力。因此，在培养和储存用于临床应用的高质量 CES 时，应考虑这些建议。
