Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland; Center for Applied Biotechnology and Molecular Medicine, Zurich, Switzerland; La Colline Research Fellow, La Colline, Sion, Switzerland.
Center for Applied Biotechnology and Molecular Medicine, Zurich, Switzerland; Musculoskeletal Research Unit, VetSuisse Faculty, University of Zurich, Zurich, Switzerland.
Cytotherapy. 2021 Jun;23(6):536-547. doi: 10.1016/j.jcyt.2021.01.006. Epub 2021 Mar 6.
Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes.
Keratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development.
The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors.
These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients.
自 20 世纪 70 年代以来，培养的患者特异性角质形成细胞片已用于临床治疗大面积严重烧伤。然而，尽管近年来取得了重大进展，但成功和可持续的治疗仍然是一个挑战。为了改善临床结果，需要可靠、高质量、可用性更高且移植时间窗口更灵活的移植物。
角质形成细胞通常在 37°C 下体外培养。鉴于天然皮肤组织中的温度差异很大，作者的研究旨在探讨角质形成细胞片生产的热调节。因此，研究了 31°C、33°C 和 37°C 对细胞增殖和片形成功效的细胞扩增和分化的影响。此外，根据移植物发育的不同阶段释放与伤口愈合相关的生物因子，评估了热效应对移植物生物状态和质量的影响。
作者证明温度是生产人角质形成细胞片的决定性因素。通过使用特定的温度范围，作者成功地优化了各个制造步骤。在细胞扩增阶段，37°C 的培养最有效。与 33°C 和 31°C 相比，在 37°C 下培养 6 天后，可获得的活细胞数量分别增加了三倍和六倍。然而，在细胞分化和片形成阶段，细胞受益于轻度低温 33°C。角质形成细胞显示出更高的分化潜力，并形成更好的表皮结构，这导致在第 18 天更快地获得生物力学片稳定性。此外，33°C 的培养温度导致所研究的免疫调节、抗炎、血管生成和促炎生物因子的持续时间更长和分泌更高。
这些结果表明，通过使用特定的温度范围，可以在提高质量的同时加速大规模生产培养的角质形成细胞片。培养的角质形成细胞片可在活检后 18 天内随时获得，并且在此后 7 天内任何时间都可用，这增加了外科医生和患者对该过程的灵活性。这些发现将有助于提供更好的临床结果，提高严重烧伤患者的成功率。
