Low Temperature Preservation Unit, National University Medical Institutes Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
Biomaterials. 2012 Jan;33(3):829-36. doi: 10.1016/j.biomaterials.2011.10.015. Epub 2011 Nov 9.
Cryopreservation has been identified as a necessary barrier to overcome in the production of tissue engineered products for clinical application. Liver engineering and bioartificial liver assisting devices are on the forefront of tissue engineering research due to its high demand and clinical potential. In this study we propose that the cryopreservation of primary mammalian hepatocytes yields better results when these cells are in a tissue-like culture configuration since cell attachment is essential for cell survival in this cell type. We used two different tissue-engineered culture configurations: monolayers and spheroid culture; and two different concepts of cryopreservation, namely vitrification and freezing. Cell suspensions were also cryopreserved using both approaches and results were compared to the engineered cultures. Both engineered configurations and suspension were cryopreserved using both conventional freezing (cooling at 1 °C/minute using 10% DMSO in foetal calf serum) and vitrification (using 40% ethylene glycol 0.6 m sucrose supplemented with 9% Ficoll). These two approaches differ on the degree of mechanical stress they inflict on the material to be cryopreserved. The maintenance of cell-to-cell and the integrity of the actin cytoskeleton were assessed using scanning electron microscopy and immunohistochemistry respectively. Results showed that while there was no significant difference between the degree of integrity shown between vitrified and control engineered cultures, the same did not happen to the frozen engineered constructs. The disruption of the cytoskeletal structure correlated with increased levels of apoptotic markers. With cryopreserved suspensions there was evidence of disruption of the cytoskeletal structure. This study concluded that cell-to-cell contact is beneficial in the maintenance of viability post-cryopreservation and that the vitrification approach was far superior to those of conventional freezing when applied to 2D and 3D hepatocyte based engineered cultures.
冷冻保存已被确定为在生产用于临床应用的组织工程产品时必须克服的障碍。由于肝脏工程和生物人工肝辅助设备具有高需求和临床潜力，因此它们处于组织工程研究的前沿。在这项研究中，我们提出当这些细胞处于组织样培养状态时，对原代哺乳动物肝细胞进行冷冻保存会产生更好的结果，因为细胞附着对于这种细胞类型的细胞存活至关重要。我们使用了两种不同的组织工程培养配置：单层和球体培养；以及两种不同的冷冻保存概念，即玻璃化和冷冻。使用这两种方法分别对细胞悬浮液进行冷冻保存，并将结果与工程培养物进行比较。使用常规冷冻（使用 10％二甲基亚砜在胎牛血清中以 1°C/分钟的速度冷却）和玻璃化（使用 40％乙二醇 0.6 m 蔗糖补充 9％ Ficoll）对这两种工程配置和悬浮液进行了冷冻保存。这两种方法在对要冷冻保存的材料施加的机械应力程度上有所不同。使用扫描电子显微镜和免疫组织化学分别评估细胞间连接和肌动蛋白细胞骨架的完整性。结果表明，虽然玻璃化和对照工程培养物之间显示的完整性程度没有差异，但冷冻工程构建体却没有。细胞骨架结构的破坏与凋亡标志物水平的增加相关。对于冷冻保存的悬浮液，有证据表明细胞骨架结构遭到破坏。这项研究得出的结论是，细胞间接触有益于冷冻保存后的细胞活力维持，并且与传统冷冻相比，玻璃化方法在应用于基于 2D 和 3D 肝细胞的工程培养物时具有明显优势。
