Khaoustov V I, Darlington G J, Soriano H E, Krishnan B, Risin D, Pellis N R, Yoffe B
Department of Medicine, Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030, USA.
In Vitro Cell Dev Biol Anim. 1999 Oct;35(9):501-9. doi: 10.1007/s11626-999-0060-2.
The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.
建立功能性原代人肝细胞（PHLC）的长期培养体系颇具难度。旋转细胞培养系统（RCCS）由美国国家航空航天局研发，可创造独特的微重力环境，具有低剪切力、高传质以及对不同细胞类型进行三维细胞培养的特点。我们研究的目的是在模拟微重力条件下建立肝细胞长期培养体系。通过胶原酶灌注从人肝脏获取PHLC，并在RCCS中进行培养。PHLC聚集体易于形成，长度增加至1厘米。使用微载体和可生物降解支架进一步评估了生物反应器中PHLC的扩增情况。虽然微载体不利于球体形成，但与可生物降解支架一起培养的PHLC形成了长达3厘米的聚集体。对PHLC球体的分析显示，其组织结构类似由肝细胞、胆管上皮细胞和/或肝祖细胞组成，这些细胞沿新生血管芽排列成胆管样结构。电子显微镜观察显示，成簇的紧密相连的肝细胞被复杂的基质结构和网状纤维包围，有带有多个微绒毛的胆小管以及紧密的细胞连接。在整个60天的培养过程中均表达白蛋白mRNA。模拟微重力环境有利于维持功能性肝细胞的长期培养。该模型系统将有助于制定改进的自体肝细胞移植、基因治疗和肝辅助装置方案，并促进对体内肝再生和细胞间相互作用的研究。
