Department of Regenerative Medicine and Biomedical Engineering, Faculty of Medicine, Saga University, Saga, Japan.
PLoS One. 2020 Apr 2;15(4):e0230428. doi: 10.1371/journal.pone.0230428. eCollection 2020.
Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs such as blood vessels, liver, diaphragm, and a conduit for nerves by using this method. These constructs have the potential to be applied in patients. However, the process of fabricating tissue constructs (harvesting cells, expanding cells, making spheroids using cultured cells, printing constructs, and maturing constructs) is time-consuming. Therefore, cryopreservation methods for spheroids or constructs should be developed to increase the efficiency of this method for clinical use. Here, we developed a method for cryopreserving spheroids, which were then used to fabricate constructs. Fibroblast cell-based spheroids were cryopreserved in phosphate-buffered saline or cryopreservation solution at -80°C for 1 week. After thawing, spheroids in cryopreservation solution began to fuse on day 1. Cryopreserved spheroids were printed onto a needle array to fabricate a scaffold-free tubular construct using a bio-3D printer. After 7 days, the printed spheroids fused and formed scaffold-free constructs. We confirmed the viability of cells in the cryopreserved spheroids and fabricated tubular constructs. Our results indicate that spheroids can be cryopreserved and used to prepare scaffold-free constructs for clinical use.
冷冻保存是一种通过将活细胞冷却至极低温度来保存活细胞的方法。尽管已经开发出用于生殖医学的卵母细胞和胚胎的冷冻保存方法，但在再生医学中还没有建立用于保存细胞聚集体（球体）的方法。我们开发了一种生物三维（3D）打印机，可以通过将球体加载到针阵列上来制造无支架的 3D 结构。我们使用这种方法制造了几种结构，例如血管、肝脏、膈肌和用于神经的导管。这些结构有可能应用于患者。然而，制造组织结构（收获细胞、扩增细胞、使用培养细胞制造球体、打印结构和成熟结构）的过程非常耗时。因此，应该开发用于球体或结构的冷冻保存方法，以提高该方法用于临床应用的效率。在这里，我们开发了一种冷冻保存球体的方法，然后使用该方法制造结构。成纤维细胞基球体在磷酸盐缓冲盐水或冷冻保存溶液中于-80°C下冷冻保存 1 周。解冻后，冷冻保存溶液中的球体在第 1 天开始融合。将冷冻保存的球体打印到针阵列上，使用生物 3D 打印机制造无支架管状结构。7 天后，打印的球体融合并形成无支架结构。我们证实了冷冻保存球体和制造无支架管状结构中细胞的活力。我们的结果表明，球体可以冷冻保存并用于制备用于临床应用的无支架结构。
