This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell-cell interactions through 'cellular patterning' or 'micropatterning'. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell-cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.

http://lmrt.mit.edu/sites/default/files/Bhatia1999_FASEBJ_0.pdf

Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.

/pdf/identification-and-purification-of-a-sperm-surface-protein-15hkr2vi4r.pdf

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

https://cancerres.aacrjournals.org/content/canres/42/11/4673.full.pdf

Liver Regeneration Studies with Rat Hepatocytes in Primary Culture

A full understanding of the acrosome reaction is central to understanding sperm function. Acrosomal status can be determined on living, motile sperm in only a few mammalian species. For other species, many light microscopic methods have been developed, including colored stains for bright-field microscopy, and probes for fluorescence microscopy. We review the existing methods and the criteria that should be considered in the choice of an assay.

Methods for evaluating the acrosomal status of mammalian sperm.

Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane.

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Mouse sperm antigens that participate in fertilization. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies.

Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b5 and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b5 and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism.

Cryopreservation of isolated rat hepatocytes.

The addition of diethylnitrosamine or cyclophosphamide in cultures of hepatocytes overlaid on confluent diploid human fibroblasts resulted in a dose-dependent increase in the frequency of hypoxanthine-guanine phosphoribosyl transferase human fibroblast mutants with both chemicals. Different toxicity patterns for the two cell types were seen. Diethylnitrosamine was more toxic to the hepatocytes, whereas cyclophosphamide was more toxic to the fibroblasts. These data open the possibility of using strains of human fibroblasts as in vitro screens for mutagenicity of procarcinogens.

Mutagenesis Induced by Procarcinogens at the Hypoxanthine-Guanine Phosphoribosyl Transferase Locus of Human Fibroblasts Cocultured with Rat Hepatocytes

Grace P. Irons

Papers

Mouse sperm antigens that participate in fertilization. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies.

Cryopreservation of isolated rat hepatocytes.

Mutagenesis Induced by Procarcinogens at the Hypoxanthine-Guanine Phosphoribosyl Transferase Locus of Human Fibroblasts Cocultured with Rat Hepatocytes