High-Density and Large-Scale Culture of Mammalian Liver Cells

在这个项目的第一年，1991年，我们建立了基本的培养条件，既为球状体培养的聚赖氨酸包被的表面和培养在膜支持的胶原蛋白凝胶三明治的原代成年大鼠肝细胞。1992年第二年，本课题的研究重点是利用球状体培养技术，开发一种混合型生物人工肝支持体，这是高密度、大规模培养正常肝细胞最有前途的应用之一。首先，我们尝试在聚赖氨酸涂覆的表面上形成大量漂浮的球状体。培养基补充物被优化用于快速形成。然而，需要大约5-7天才能使形成的球状体的一半漂浮。此外，接种面密度应降低到单层融合培养的一半。SP ...更多信息 因此，当考虑到构建生物人工肝所需的球状体数量时，在平板上自发的球状体形成被证明是明显无效的。尽管悬浮液中过量培养会导致细胞死亡，但由此形成的球状体与形成完成后接种在平板上时在聚赖氨酸涂层表面自发形成的球状体相比，可以表达大致相同水平的功能。另一个关键问题是选择灌注患者血浆的稳定固定方法。我们比较了各种方法固定的球状体功能，并在含有高血清浓度的培养基中培养。胶原凝胶包埋法是维持人工肝良好功能的最有效方法。1993年最后一年，我们制备了中空纤维型生物人工肝模型。将通过大规模悬浮培养快速形成的球状体用胶原凝胶固定在小型中空纤维模块的毛细血管外空间中，并进行10天的灌注培养。在富含酶的培养基中，该模块表达了与先前报道的生物人工肝模块相同的性能。即使在100%动物血清中，也未观察到模块性能的显著劣化。因此，该模块被认为具有高可靠性，特别是在临床应用中。少

In the first year of this project, 1991, we established basic culture conditions both for spheroid culture on polylysine-coated surfaces and culture in membrane-supported collagen gel sandwich of primary adult rat liver cells. The cells can be cultured with high level functional expressions and with extended culture time by using those culture techniques.In the second year 1992, the objectives of this project, by using the spheroid culture, were focused on the development of a hybrid-type bioartificial liver support, which is one of the most promising applications of high-density and large-scale culture of normal liver cells. First, we tried to form a large number of floating spheroids on polylysine-coated surfaces. The medium supplements were optimized for rapid formation. However, approximately 5-7 days were needed to cause the floating of a half number of spheroids formed. Moreover, the inoculation surface density should be lowered to a half of that in confluent monolayr culture. Sp … More ontaneous spheroid formation on flat plates, thus, proved to be markedly inefficient when considering the number of spheroids needed for construction of a bioartificial liver.Second, spheroid formation by suspension culture was examined. Though excess culture in suspension caused cell death, the spheroids thereby formed could express approximately the same level functions as compared to the spheroids spontaneously formed on polylysine-coated surfaces when inoculated on flat plates after the formation was completed.Another crucial subject is the selection of stable immobilization methods in perfused patient's plasma. We compared the spheroid functions immobilized by various methods and cultured in media containing high serum concentration. Collagen gel entrapment was most effective in sustaining the good functions in the media.In the final year 1993, we prepared a hollow-fiber type bioartificial liver model. Spheroids rapidly formed by large-scale suspension culture were immobilized with collagen gel in the extra-capillary space of a small hollow fiber module, and 10 days of perfusion culture was performed. In hormone-enriched media, the module expressed about the same performance as compared to the bioartificial liver modules reported previously. Even in 100% animal sera, no significant deterioration was observed in the module performance. Thus, this module presented in considered to have a high reliability particularly in clinical application. Less