Institute for Bioengineering of Catalonia, Baldiri i Reixac 10-12, 08028 Barcelona, Spain.
Biomed Mater. 2022 Jun 21;17(4). doi: 10.1088/1748-605X/ac76fb.
Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.
自 20 世纪 50 年代首次展示以来，用于低温保存（又称冷冻保存）活体组织、细胞系和原代细胞的技术已经在临床医生和研究人员中得到了极大的发展，并广泛应用于存储和运输应用。然而，目前对于生物工程的三维（3D）细胞模型，包括患者样本，仍然缺乏可行的冷冻保存和解冻方法。作为解决这一差距的第一步，我们展示了一种基于 3D 羧甲基纤维素支架的可行的球体冷冻保存和生存方案，以及冷冻和解冻的精确条件。该方案使用肝细胞进行了测试，对于这些细胞，支架提供了 3D 结构，使细胞能够自行排列成球体，并在冷冻过程中支撑细胞，以实现最佳的解冻后活力。与传统的细胞球模型相比，解冻后的细胞活力得到了提高，在传统的细胞球模型中，细胞在重力作用下沉降，在冷冻前形成伪组织。该技术可能会推进低温生物学和其他应用，这些应用需要在设施之间（例如，在医疗实践、研究和测试设施之间）对预先组装的 3D 模型（包括细胞系和未来的患者细胞）进行高完整性的运输。
