A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers

Abstract

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

Transplantation of multiciliated airway cells derived from human iPS cells using an artificial tracheal patch into rat trachea

“…It exhibits excellent transparency and permeability for high‐molecular weight proteins and has, therefore, been successfully used as a cell culture scaffold in various studies. Human corneal models (Ko et al, ; McIntosh Ambrose et al, ; Takezawa, Nishikawa, & Wang, ; Chae et al, ; Yamaguchi et al, 2016; Yoshida et al, ; Yamaguchi & Takezawa, ) and human hepatocytes models (Oshikata‐Miyazaki & Takezawa, ; Watari et al, ) have been successfully reconstructed using the collagen vitrigel membrane. An artificial skin based on collagen vitrigel was shown to promote the epithelialization of regenerative skin and to inhibit scar formation and inflammation, whereas the existing products such as DuoACTIVEV ET of a hydrocolloid dressing and Pelnac of a collagen sponge was not able to sufficiently inhibit scar formation (Aoki et al, ).…”

Prediction of Human Hepatic Clearance for Cytochrome P450 Substrates <i>via</i> a New Culture Method Using the Collagen Vitrigel Membrane Chamber and Fresh Hepatocytes Isolated from Liver Humanized Mice

“…Regarding the concern about the contamination of mouse hepatocytes, PXB-cells have been reported to decrease the mRNA expression level of mouse CYP to 1/1000 or less upon culturing for 1 week or more after cell seeding. 12) Since our vitrigel culture method employs a 2-week culture period to maximize CYP activity, the influence of mouse hepatocytes may be as small as possible. In fact, the prediction accuracy of diazepam was improved within a 3-fold difference in this study, even though diazepam is easily metabolized by mouse hepatocytes, supporting our contention that PXB-cells cultured using CVM chambers for 2 weeks would minimize or diminish the contribution of mouse hepatocytes.…”

“…On the other hand, PXB-cells have been reported to decrease the mRNA expression level of mouse CYP to 1/1000 or less upon culturing for 1 week or more after cell seeding. 12) It is * To whom correspondence should be addressed. e-mail: r-watari@hhc.eisai.co.jp…”

Art of Making Artificial Liver: Depicting Human Liver Biology and Diseases in Mice

“…This is extremely important because hepatocytes cultured at optimal confluency appropriately polarize through the formation of tight junctions and bile canaliculi with neighboring cells, thereby preventing the cells from undergoing epithelial to mesenchymal transition (EMT) and thus sustaining precise morphology and functionality over a longterm culture. [106][107][108] In contrast, these factors, especially the platability and the degree of hepatocyte function preservation of commercially available cryopreserved PHH, are highly variable.…”

“…HLCM-derived human hepatocyte has been heavily utilized in a broad spectrum of in vitro experimental applications such as, but not limited to the studies of hepatitis virus infection, DMPK, and regenerative biology. 106,107,[109][110][111] In addition, HLCM-derived human hepatocyte could be applied to the development of microfabricated in vitro culture systems that mimic the cellular environment of tissue and organ. These include co-culture nonparenchymal cells of the liver, 3D organoid culture, and organ-on a-chip system in conjunction with microfluid circulatory system.…”