Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration

Adult rat hepatocytes cultured in a collagen sandwich system maintained normal morphology and a physiological rate of albumin secretion for at least 42 days. Hepatocytes cultured on a single layer of collagen gel essentially ceased albumin secretion within 1 wk but could recover function with the overlay of a second layer of collagen gel. This culture configuration more closely mimics the hepatocytes' in vivo environment and provides a simple method for their long-term maintenance.     

An organ culture of postnatal rat liver slices

Summary A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O₂:5% CO₂ atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals. Deceased October 1, 1983. This research was supported in part by a grant from the South African Council for Scientific and Industrial Research.     

Continuous and long-term monoxenic culture of the arbuscular mycorrhizal fungus Gigaspora decipiens in root organ culture

Establishment of arbuscular mycorrhizal (AM) germplasm collections is complex because of the obligate biotrophic nature of AM fungi. Only a few AM species are routinely maintained in monoxenic culture with Ri T-DNA transformed roots as host. Incorporation of new AM species into this culture system is important for molecular, physiological, and taxonomical studies. Here we report for the first time the successful monoxenic culture of Gigaspora decipiens (JA2 strain) with transformed carrot (Daucus carota) roots. In vitro cultures were established from field-collected spores; sub-culture of newly in vitro formed spores was established over five successive generations for a period of 6 y. Although initial culture of field-collected spores was difficult successive sub-cultures appeared to be adapted to the in vitro growing conditions. The JA2 strain of G. decipiens completed its life cycle while maintaining its morphological characteristics, stability, and propagule viability under the monoxenic conditions over several generations. This stable and homogeneous monoxenic material obtained for G. decipiens is part of the Banco de Glomeromycota In Vitro (BGIV, http://www.bgiv.com.ar), and could facilitate morphological, physiological, and molecular analysis of this AM species.     

Dynamic organ culture of precision liver slices for in vitro toxicology

The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro.     

Culture of fetal mouse liver in plastic chambers: a new technique for organ culture.

A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO2-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate that potential of this culture method for the study of physiological and pathological processes.     

Immunohistochemical study of cell phenotypes in organ culture of the rat embryo liver

The organ culture of the liver of a 14 day old rat embryo was used for immunohistochemical recognition of the available cell types. This culture contains the tissue explant, confluent monolayer cells and a zone of single cells (ZSC). At the start of cultivation a monolayer is formed at the expense of cells slipping down together from the explant, and later these cells were seen to migrate actively from the explant and to proliferate. ZSC is formed by cells migrating from the monolayer. Desmin-containing cells (hepatoblasts, Ito cells and myofibroblasts) migrate from the explant to the monolayer and ZSC. In the monolayer hepatoblasts lose gradually desmin and are converted into cytokeratin 18 (CK-18) expressing hepatocytes. In ZSC, hepatoblasts lose desmin, but no CK-18 synthesis occurs in them. The Ito cells (hypothetical progenitor cells) are spreading over the whole culture, and desmin expression in them does not change. The embryonal Ito cells may transform presumably into myofibrils. Myofibroblasts lie flat on the periphery of ZSC. Besides, desmin myofibroblasts express alpha-actin from smooth muscles. Expression of CK-18 in cells depends on the pattern of intercellular interactions. In the monolayer, CK-18 expression extends successively via the adjacent cells towards the explant. In hepatocytes, migrating into ZSC, CK-18 expression stops. In ZSC, CK-18 expression reappears in compact clusters of stopped cells. It supposed that during formation of close contacts in the monolayer the cells-predecessors may be differentiated into hepatocytes, whereas in the case of disturbance of intercellular connections in ZSC the cells-progenitors may be converted into myofibroblasts. However, on the reconstruction of cell contacts in motionless dense clusters the cells in ZSC are differentiated into hepatocytes.     

Beta-cell function in isolated human pancreatic islets in long-term tissue culture

Human pancreatic islets were isolated by collagenase treatment of pancreatic tissue obtained from 27 individuals aged 12 to 69 years. The islets were maintained free floating in tissue culture medium RPMI 1640 supplemented with calf or human serum. In two cases the insulin production was followed up to nearly two years. The insulin production rate of the individual islet preparations varied between 0.2 and 8 ng per islet per day. No significant correlation with donor age or sex was found. The glucose concentration in the medium influenced the insulin release in a dose dependent manner. The acute response of the cultured islets to glucose was evaluated both by batch incubation and by perifusion. Both in the acute and the chronic experiments maximal insulin release was found at 10 mM glucose. In conclusion, these experiments indicate that viable islets of Langerhans can be obtained from adult human pancreatic tissue and that their beta-cell function can be maintained for up to two years. The variation in insulin production rate could not be ascribed to age or sex and may reflect both physiological and methodological factors.     

Putative liver progenitor cells: conditions for long-term survival in culture

Oval cells proliferate extensively in the livers of animals exposed to oncogenic insults, are bipotent and are believed to be related to the so far unidentified liver stem cell. In normal liver, cells antigenica lly related to oval cells and expressing liver and epithelial markers are considered to be liver progenitor cells. We isolated, by fluorescence-activated cell sorting or magnetic bead sorting, cells expressing the oval cell antigens OC.2 or OC.3 from the liver of normal newborn or day 12 embryonal age rats. Magnetic bead sorting of positive cells was as efficient as fluorescence-activated cell sorting. A two-chamber culture system was devised in which cells were plated onto transwell filters coated with type IV collagen and cultured in a serum-free Ham's F12 medium supplemented with free fatty acids and bovine serum albumin. Under these conditions, cells remained viable for up to 6 weeks and their antigenic phenotype was unchanged throughout. Approximately 30% of sorted cells expressed epithelial and/or liver-specific markers. Growth factors mitogenic for epithelial cells and hepatocytes did not elicit cell proliferation. These results provide an important background for further studies designed to determine the biological significance of OC.2⁺ and OC.3⁺ cells in normal liver, to test the liver stem cell hypothesis and to develop protocols for the expansion in vitro of normal liver progenitors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Rat liver cells in culture: effect of storage, long-term culture, and transformation on some enzyme levels.

Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.