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.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Use of hepatocytes cultured in microcarriers for correction of acute hepatic insufficiency

The ability of hepatocytes cultured on Biosilon microcarriers to secrete albumin and to conjugate bilirubin were examined for the purpose of developing an artificial liver support system. Cultured hepatocytes were able to synthesize 100-120 micrograms albumin per 10(5) cells in 24 hours and to conjugate about 20 micrograms/hr of bilirubin for at least 5 days. Rats with liver failure caused by i.p. injection of CCL4 or D-galactosamine were subjected to hemosorption via minicolumns containing 2 ml of Biosilon microcarriers with 40 X 10(6) cultured hepatocytes. The procedure was performed 20-24 h after hepatotoxins injection and lasted for 3 h at a flow rate of 60 ml/h. This reduced mortality from 100% to 20% after 48% and to 40% after 7 days in case of CCl4 and from 100% to 40% after 48 h and 7 days in case of D-galactosamine. Our results suggest that hepatocytes cultured on microcarriers may be efficiently applied to correction of fulminant hepatic failure caused by different hepatotoxins.     

Acute phase response to recombinant interleukin-6 and macrophage- and fibroblast-derived crude cytokine preparations in primary cultures of mouse hepatocytes

A number of acute phase proteins were determined by electroimmunoassay in media from CBA mouse hepatocytes cultured for 2 days with human recombinant IFN beta 2/IL-6, as well as with conditioned media from LPS-stimulated rat macrophages, and of murine L fibroblasts. It was found that human recombinant IL-6 caused three-fold increase in secretion of fibrinogen, while haptoglobin, complement C3 and transferrin were increased respectively, to 168 per cent, 151 per cent, and 145 per cent of the control. DEX(10(-7) M) in DMEM supplemented with 5 per cent FCS, enhanced the IL-6 effect on the three positive acute phase proteins. IL-6 elevated haptoglobin mRNA in mouse hepatocytes to a degree comparable with the concentration of the protein in the culture medium. The effect of conditioned media from murine fibroblasts and peritoneal rat macrophages was generally similar to that of recombinant IL-6. However, both natural preparations of the cytokines caused decrease in albumin and alpha-1-proteinase inhibitor secretion.     

Fatty acids alter time dependent loss of apolipoprotein E expression by primary cultures of rat hepatocytes

Although the phenomenon of intracellular apolipoprotein E (apoE) degradation has been reported in other cell types, the fate of newly synthesized apoE in the liver is not well understood. In the present study, we examined the expression (the balance of synthesis, secretion, and degradation) of apoE in primary cultures of rat hepatocytes and compared it with albumin, a typical secretory protein. Synthesis and secretion of [(35)S]apoE was diminished in primary hepatocytes cultured for more than 2 days, in agreement with an observed decrease in apoE mRNA. Cells cultured for 1 day and labeled for up to 4 hours secreted total protein, apoE, and albumin, linearly. The apparent rates of synthesis for apoE and albumin were similar (1,158 vs. 1,334 dpm/mg/min) but rates of their secretion differed significantly (225 vs. 1,159 dpm/mg/min). Pulse-chase experiments indicated that cell-associated [(35)S]albumin was secreted without degradation, whereas significant quantities of newly synthesized apoE were degraded. The overall synthesis and secretion of total proteins, including secretion of apoE, was enhanced by oleic acid (1 mmol/L). However, this effect may not be limited to oleic acid because other fatty acids showed a similar effect on apoE mRNA abundance. In control cells, apoE was found to associate with high density lipoproteins predominantly, although the fraction associated with very low density lipoprotein was increased in hepatocytes incubated with oleic acid. Overall, the findings from this study suggest that the level of apoE expression by primary hepatocytes is dependent on the age of the culture. The study also indicates that the phenomenon of apoE degradation occurs in primary hepatocytes.     

Postthaw viability of precultured hepatocytes

Hepatocytes are being studied for a wide variety of applications, including drug metabolism studies, gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordination of the cell supply with patient care regimes and the completion of safety and quality-control testing. The objective of this investigation was to develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different applications. Freshly isolated hepatocytes were cultured in a spinner flask for different periods of time, up to 48 h. The cells were cryopreserved by use of a range of solution concentrations and cooling rates. For fresh, nonfrozen hepatocytes precultured for 24 h prior to being plated on collagen, the albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were precultured for 24 h, frozen in a solution containing 10% Me2SO with a cooling rate of 1 degrees C/min, thawed, plated on collagen, and cultured, the albumin secretion rate was 0.21 +/- 0.24 microg/ml/h. In contrast, freshly isolated hepatocytes cryopreserved without preculture and cultured on collagen had an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of different solution compositions and cooling rates on postthaw function of precultured hepatocytes were also determined. These results indicate that the use of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes.     

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A domain of methylation change at the albumin locus in rat hepatoma cell variants.

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.     

Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes.

Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF-beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response.     

Enhancement of albumin secretion by short-term hypothermic incubation of primary rat hepatocytes

A feasibility of hypothermic incubation of hepatocytes as a means of enhancing liver-specific activity was investigated to obtain preferable hepatocytes for a bioartificial liver (BAL) system. Freshly isolated rat hepatocytes were incubated at hypothermic temperatures from 10 to 33 °C for several days, and subsequently cultured at normothermic temperature of 37 °C to evaluate cell viability and albumin secretion activity. The cell viability was decreased by 3-day hypothermic incubations at 10 and 20 °C, while it was maintained even after 3-day hypothermic incubations between 25 and 33 °C. The activity of albumin secretion gradually decreased with prolonging the period of hypothermic incubation at 25 °C. Enhancement of albumin secretion activity was observed in the hypothermic incubations at 30 and 33 °C. The maximum activation of albumin secretion was obtained when hypothermic incubation was performed for 3 days at 30 °C, where the activity increased to 145% of the original activity. The hypothermic incubation at 30 °C also reduced the required time to be the peak of the activity of albumin secretion in the normothermic culture. It was considered that the hypothermic incubation at 30 °C would be effective as a method for pretreatment of isolated hepatocytes for a BAL system.     

The effect of dexamethasone on albumin production by fetal rat hepatocytes in culture

Hepatocytes derived from 15 and 19-day gestation rats synthesize and secrete albumin during culture. Albumin secretion is maintained when the culture medium is supplemented with dexamethasone but declines in its absence. The fall in secretion rate correlates with the level of albumin messenger RNA in the respective cultures. Even when dexamethasone is present, the level of albumin production in 19-day gestation hepatocytes is 6 to 7 times greater than that observed in hepatocytes derived from 15-day gestation rats. Immunocytochemical studies were undertaken to establish whether the difference in secretion rate was due to a difference in the amount of albumin produced by all the hepatocytes of the respective cultures or whether there were fewer hepatocytes which were capable of synthesizing albumin in the less mature liver. The results indicate that albumin production is reduced in all hepatocytes when cultured in the absence of dexamethasone.     

Characteristics and significance of albumin-positive hepatocytes in analbuminemic rats

Analbuminemic rats (NAR) are a mutant strain in which splicing of the albumin mRNA is blocked due to a seven-base-pair deletion in an intron of the albumin gene. NAR liver contains a few hepatocytes that react with anti-rat albumin antibody (Alb+ hepatocytes), and these cells increase in number during aging and on treatment with hepatocarcinogens. To characterize these Alb+ hepatocytes, we examine their albumin mRNA, the biochemical specificity of their albumin, and its intracellular distribution. Signals of albumin mRNA were observed in a few hepatocytes by in situ hybridization. Moreover, a small amount of cytoplasmic albumin mRNA was detected by RNA blot analysis in the liver of aged NAR and NAR treated with 3'-methyl-4-diaminoazobenzene (DAB). Immunoelectron microscopic examination revealed the cisternae of the rough and smooth endoplasmic reticula, Golgi complexes, and secretory vesicles of the Alb+ hepatocyte of NAR being filled with material that reacted with anti-rat albumin antibody. These facts suggested that albumin was gradually synthesized in Alb+ hepatocytes but that its secretion was disturbed. The albumin-like proteins of NAR were shown by Western blot analysis to consist of three species of 68 kDa, 50 kDa, and 25 kDa proteins. The 50 kDa albumin was thought to be formed by exon-skipping splicing of the albumin mRNA precursor, which was recently reported by Shalaby and Shafritz (Proc. Natl. Acad. Sci. USA 87, 2652-2656 (1990)). The 25 kDa protein was suspected to be formed by fragmentation of the 50 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.     

A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions. A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J. C. Y. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J. 3, 174, 1989). Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C. Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing. Comparison was made with cryopreservation of liver cells in single cell suspensions. Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes. Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively. Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture. These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.     

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.     

The importance of proline on long-term hepatocyte function in a collagen gel sandwich configuration: regulation of protein secretion

A serum-free culture system for primary hepatocytes which maintains stabel high-level hepatocyte function for prolonged periods in culture has been developed. Isolated rat primary hepatocytes were cultured in serum-free media between two layer of gelled collagen in a sandwich configuration which reinstates the cellular polarity necessary for long-term function in vitro. Thsee serum-free hepatocyte cultures maintained near physiological rates of albumin and transferrin secretion for a minimum of 26 days in culture. L-Proline was shown to be critical for both the approach to steady state and maximal level of protein secretion. Analysis of does-response data gave K(m) values of 2.9 and 1.7 mug/mL for albumin and transferrin secretion, respectively.