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

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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