Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical College of Nanjing University, Jiangsu Province's Key Medical Center for Hepatobiliary Disease, Nanjing, China.
Liver Int. 2010 Apr;30(4):611-22. doi: 10.1111/j.1478-3231.2009.02197.x. Epub 2010 Jan 11.
The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications.
Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed.
Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious.
The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.
能够冷冻和解冻的特征良好的人肝细胞的可用性对于细胞治疗至关重要。我们研究了微囊化冷冻保存后人类肝细胞是否能够恢复，并探讨了这些冷冻保存的微囊化肝细胞是否可用于临床应用。
采用两步法体外胶原酶灌注技术从 18 位不同供体中分离成年肝细胞。在 HepatoZYME-SFM 中于 4°C 预孵育 12-24 小时后，使用藻酸盐-聚-L-赖氨酸-藻酸盐微胶囊对肝细胞进行微囊化。将微囊化的肝细胞转移到含有 10%二甲亚砜的完全培养基中。它们立即被放入异丙醇渐进冷冻容器中，在-80°C 下过夜，第二天浸入液氮中。在解冻培养期间，分析了白蛋白分泌、尿素合成、细胞周期、mRNA 和蛋白水平以及微囊化前预孵育（PMC）组的形态和病理学结构。
与直接冷冻（IC）组相比，我们发现解冻后附着细胞的 mRNA 和蛋白水平显著提高，白蛋白和尿素水平的分泌也更高。在来自 PMC 组的附着培养的人冷冻/解冻肝细胞中，白蛋白的产生在第 2、3 和 4 天与直接培养组没有显著差异。与 IC 组相比，PMC 组的保存形态明显。
本研究结果表明，在微囊化冷冻保存前于 4°C 预孵育 12-24 小时可恢复人肝细胞的功能和形态完整性。这些研究为药物毒理学、生物人工肝和细胞治疗在人类中的临床应用提供了可能性。
