Krause P, Markus P M, Schwartz P, Unthan-Fechner K, Pestel S, Fandrey J, Probst I
Zentrum Chirurgie, Georg-August-Universität Göttingen, Germany.
J Hepatol. 2000 May;32(5):718-26. doi: 10.1016/s0168-8278(00)80239-1.
BACKGROUND/AIMS: A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h. This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days.
Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen.
At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v). Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h. The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein.
The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.
背景/目的：大鼠肝内皮细胞培养中的一个主要问题是48小时后细胞迅速丢失。本研究旨在开发一种方案，使肝血窦内皮细胞在无血清培养中易于维持并增殖5 - 6天。
通过胶原酶消化从成年大鼠肝脏分离的细胞经离心淘析纯化，并在戊二醛交联的胶原上培养。
在高接种密度下，细胞在氢化可的松和碱性成纤维细胞生长因子存在的情况下可无血清维持6天；在较低接种密度下，培养基必须补充额外的促生长因子。成年大鼠肝细胞的条件培养基被证明是最有效的生长刺激物；它增加了胸苷掺入、DNA含量和每皿细胞数量，在20%（v/v）时具有半数最大效应。当培养基中额外添加1%新生牛血清时，血管内皮生长因子、佛波酯或来自FAO或HEPG2肝癌细胞系的条件培养基也能观察到细胞增殖。在所有条件培养基中均检测到血管内皮生长因子。在没有肝细胞条件培养基的情况下，1%血清有助于维持培养；其本身具有较低的增殖作用。然而，更高的血清浓度（>5%）会导致48小时后细胞丢失。培养过程中，6小时龄细胞的众多筛板逐渐消失，被随机分布的小孔取代，这些小孔随后在细胞 - 细胞边界聚集在一起。超过90%的细胞摄取乙酰化低密度脂蛋白。
该研究表明，培养的肝细胞分泌促进生长物质，在无血清条件下刺激体外内皮细胞增殖；血管内皮生长因子和1%血清联合添加可模拟这种效应。新的培养基配方应有助于未来对肝内皮细胞单培养或共培养的研究。
