Albumin Production Of Hepatocytes Research Articles

Human hepatocytes and endothelial cells in organotypic membrane systems

The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell–cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell–cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices.

Gene Expression and Functional Analyses of Primary Rat Hepatocytes on Nanofiber Matrices

Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) – either uncoated or coated with a biological material such as collagen or laminin – is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 ± 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 ± 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 ± 1.8 vs. 15.9 ± 4.5 µg albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 ± 0.25 µM vs. 0.36 ± 0.24 µM urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4αindicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions.

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.