Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions.

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.

Liver regeneration.

Liver regeneration after the loss of hepatic tissue is a fundamental parameter of liver response to injury. Recognized as a phenomenon from mythological times, it is now defined as an orchestrated response induced by specific external stimuli and involving sequential changes in gene expression, growth factor production, and morphologic structure. Many growth factors and cytokines, most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-alpha, interleukin-6, tumor necrosis factor-alpha, insulin, and norepinephrine, appear to play important roles in this process. This review attempts to integrate the findings of the last three decades and looks toward clues as to the nature of the causes that trigger this fascinating organ and cellular response.

Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha 1 adrenoreceptor by norepinephrine.

Addition of norepinephrine to primary cultures of adult rat hepatocytes stimulates the incorporation of [3H]thymidine in a dose-dependent manner. This effect has been observed in serum-free medium containing epidermal growth factor and insulin. Stimulation of DNA synthesis by norepinephrine was strongly antagonized by the alpha 1-adrenergic antagonist prazosin but not by an alpha 2 antagonist or by a beta-adrenergic blocker. The beta agonist isoproterenol did not stimulate significant DNA synthesis. These results indicate that catecholamines interact with the alpha 1 adrenoreceptor to stimulate DNA synthesis in hepatocytes. Since alpha 1 receptors are present in most cells, this receptor may be important in cell growth regulation.

Cytochrome P-450 induction by phenobarbital and 3-methylcholanthrene in primary cultures of hepatocytes.

The characteristic hepatocellular changes resulting from phenobarbital administration in vivo, namely an increase in the levels of cytochrome P-450 and proliferation of membranes of the smooth endoplasmic reticulum, have been demonstrated in primary cultures of nonreplicating hepatocytes on floating collagen membranes. Addition of methylcholanthrene to the medium resulted in an increase in cytochrome P-448 within 48 hours, whereas the phenobarbital induction of P-450 required 5 days. These results demonstrate that responses induced in adult liver cells in vivo by phenobarbital can be reporoduced in cultured hepatocytes, contrary to previous reports.

Novel potential biomarkers for the diagnosis and monitoring of patients with ulcerative colitis.

Unambiguously, great progress has been achieved in the unraveling of more pathological pathways implicated in the development and progression of ulcerative colitis during the last decades. Novel effective drugs that have augmented the management armamentarium have been developed alongside this growing comprehension of the disease, rendering mucosal healing not only a feasible but the optimal goal of every therapy. Clinical evaluation, colonoscopy and biomarkers are the tools used by practitioners for the diagnosis and assessment of the status of the disease in order to achieve clinical remission and mucosal healing for their patients. Among these tools, colonoscopy is the gold method for the cause but is still an invasive, high-cost procedure with possible adverse events such as perforation. While clinical evaluation entails much subjectivity, biomarkers are objective, easily reproducible, non-invasive, cheap and potent surrogate tools of mucosal inflammation. Unfortunately, the well-established, currently in use serum biomarkers, such as C-reactive protein, erythrocyte sedimentation rate and others, do not display sufficiently acceptable sensitivity and specificity rates for the diagnosis of ulcerative colitis and, most importantly, do not represent precisely the mucosal inflammation status of the disease. Therefore, the discovery of new serum biomarkers has been the cause of several studies attempting to discover an "optimal" serum biomarker during the recent years. After thorough research, collection and examination of current data, this review focuses on and selectively presents promising, potential, novel serum biomarkers of ulcerative colitis as they are indicated by studies on the patient over the last years.

MCM7 amplification and overexpression are associated with prostate cancer progression.

The genomic DNA profiles of prostate cancers with aggressive features were compared to the profiles of matched normal DNA to identify genes that are selectively amplified in the cancer cells. One of the identified genes, MCM7, which is a component of the DNA replication licensing complex, has been studied extensively both at the DNA and protein levels in human prostate tissues. Approximately half of the prostate cancer specimens studied showed MCM7 gene amplification, and 60% of the aggressive prostate cancer specimens had increased MCM7 protein expression. Amplification or overexpression of MCM7 was significantly associated with relapse, local invasion and a worse tumor grade. Constitutive expression of MCM7 in a human prostate cancer cell line, DU145, resulted in markedly increased DNA synthesis and cell proliferation compared to vector-only controls, and an increased cell invasion in vitro. Indeed, MCM7 overexpression produced primary tumors 12 times larger than vector-only controls and resulted in a rapid demise of mice bearing those tumors. These studies implicate MCM7, and the DNA replication licensing gene family, in prostate cancer progression, growth and invasion.

Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain barrier and impairs iron-dependent hippocampal neuron dendrite development.

Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood-brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use. SUMMARY: Background Thrombocytopenia is common in sick neonates. Thrombopoietin mimetics (e.g. eltrombopag [ELT]) might provide an alternative therapy for selected neonates with severe and prolonged thrombocytopenia, and for infants and young children with different varieties of thrombocytopenia. However, ELT chelates intracellular iron, which may adversely affect developing organs with high metabolic requirements. Iron deficiency (ID) is particularly deleterious during brain development, impairing neuronal myelination, dopamine signaling and dendritic maturation and ultimately impairing long-term neurological function (e.g. hippocampal-dependent learning and memory). Objective To determine whether ELT crosses the blood-brain barrier (BBB), causes neuronal ID and impairs hippocampal neuron dendrite maturation. Methods ELT transport across the BBB was assessed using primary bovine brain microvascular endothelial cells. Embryonic mouse primary hippocampal neuron cultures were treated with ELT or deferoxamine (DFO, an iron chelator) from 7 days in vitro (DIV) through 14 DIV and assessed for gene expression and neuronal dendrite complexity. Results ELT crossed the BBB in a time-dependent manner. 2 and 6 µm ELT increased Tfr1 and Slc11a2 (iron-responsive genes involved in neuronal iron uptake) mRNA levels, indicating neuronal ID. 6 µm ELT, but not 2 µm ELT, decreased BdnfVI, Camk2a and Vamp1 mRNA levels, suggesting impaired neuronal development and synaptic function. Dendrite branch number and length were reduced in 6 µm ELT-treated neurons, resulting in blunted dendritic arbor complexity that was similar to DFO-treated neurons. Conclusions Eltrombopag treatment during development may impair neuronal structure as a result of neuronal ID. Preclinical in vivo studies are warranted to assess ELT safety during periods of rapid brain development.

Isolation, culture, and transplantation of human hepatocytes.

The in situ two-step collagenase perfusion technique used for the isolation of hepatocytes from rat liver was adapted into a procedure applicable to pieces of human liver obtainable from surgical procedures. Human hepatocytes obtained by this method were maintained in primary culture for 10 days. The cellular changes observed at the light microscopic and electron microscopic levels are described. The changes in microsomal enzymes as a function of the age of the cultures were also measured. Exposure of the human hepatocytes to procarcinogens known to be metabolized by rodent liver resulted in unscheduled DNA synthesis. The isolated hepatocytes were also transplanted into two-thirds partially hepatectomized athymic nude mice. The transplanted cells formed nodules with characteristic hepatic architecture. These studies demonstrate that hepatocytes obtained from human liver by the described modified collagenase technique can be used for in vitro studies in chemical carcinogenesis.

Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes.

We have previously reported the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. We referred to this activity as hepatopoietin A (HPTA) (Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. Control of hepatocytes replication by two serum factors. Cancer Res., 44: 4414-4419, 1984; Thaler, J., and Michalopoulos, G. Hepatopoietin A. Partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res., 45: 2545-2549, 1985). At that time, however, complete purification of this growth factor had not been achieved. In the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. The purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and reversed phase HPLC. The final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, however, the purified HPTA migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 69,000. The mitogenic activity of HPTA was associated with this band when it was eluted from unstained sodium dodecyl sulfate-polyacrylamide gels. Gel filtration HPLC under neutral isotonic conditions indicated that HPTA tends to form aggregates with molecular weights of greater than 300,000. Chromatofocusing indicated that HPTA is an acidic protein with an isoelectric point value of about 5.5. The mitogenic activity of HPTA was sensitive to heat, trypsin, and 2-mercaptoethanol, but relatively resistant to exposure to 1 N acetic acid, 2 M guanidine-HCl, and 0.1% sodium dodecyl sulfate. The stimulation of DNA synthesis induced by HPTA was totally abrogated by transforming growth factor-beta and markedly reduced in the presence of heparin. We present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, fibroblast growth factor, and thrombin.

Mutagenesis and DNA binding of benzo(a)pyrene in cocultures of rat hepatocytes and human fibroblasts.

The genotoxicity of benzo(a)pyrene (BP) was investigated in combined cultures of rat hepatocytes and human diploid fibroblasts. Freshly isolated rat hepatocytes were shown to activate BP to a species which bound to and damaged hepatocyte and fibroblast DNA. A significant increase in the hypoxanthine-guanine phosphoribosyltransferase mutation frequency was induced when 10 to 100 microM BP was added to the cocultures. A comparative analysis of the binding of BP metabolites to hepatocyte and fibroblast DNA revealed that approximately 4 times more [3H]BP metabolites were bound to the fibroblast DNA than were bound to the hepatocyte DNA (per microgram DNA). Activation of BP by the fibroblasts themselves was shown not to be the cause of the relatively greater binding of BP to fibroblast DNA than to the hepatocyte DNA. These results suggest that proximate and/or ultimately carcinogenic metabolites of BP are readily released from isolated hepatocytes and that the metabolites are sufficiently stable and long lived so as to bind to the DNA of an adjacent cell. The relative protection of the hepatocytic DNA from BP metabolites that generated in the cytoplasm of the hepatocyte may be significant in view of the observations that the liver is not under normal conditions a target of BP carcinogenicity in vivo.

Effects of partial hepatectomy on transplanted hepatocytes.

When hepatocytes are injected into the fat pads of syngeneic rats, they behave like clonogenic units and proliferate to form nodules at the site of transplantation. The probability of formation of nodules was quantitated by use of the serial dilution assay. When hepatocytes are injected into hepatectomized hosts, the probability of formation of nodules is enhanced 8-fold. There is slight decrease in the initial rate of disappearance of hepatocytes transplanted into hepatectomized hosts, but the percentage of hepatocytes that remain at the site of injection after 6 days is the same for both recipient types. Hepatectomy in the recipient animals is accompanied by a parallel increase in DNA synthesis and mitosis in the hepatocytes at the transplantation site. The probability of clone formation was found to be inversely proportional to the time between host hepatectomy and hepatocyte transplantation. The implication of these findings for the mode of action and the nature of the hepatectomy stimulus is discussed.

Hepatopoietin A: partial characterization and trypsin activation of a hepatocyte growth factor.

Rat hepatocytes in primary culture are stimulated to synthesize DNA by high-molecular-weight fractions from rat serum. This activity has been previously given the name hepatopoietin A (HPTA). HPTA, with an apparent molecular weight of 150,000 to 250,000, stimulated the incorporation of [3H]thymidine into DNA, quantitated by autoradiography as percentage of nuclear labeling. Properties of HPTA include: sensitivity to heat; stability in minimal essential media at 4 degrees C; in lyophilized form; or in 25% glycerol at -20 degrees C; and instability at 4 degrees C in isotonic buffer. Trypsin digestion of HPTA resulted in an increase in biological activity. Both the trypsinized and native forms of this activity were not inhibited by antiserum against mouse epidermal growth factor in this bioassay. Treatment of HPTA with trypsin resulted in a shift of its apparent molecular weight on a Sephadex G-50 column to less than 6000. This trypsinized HPTA activity did not comigrate with 125I-labeled epidermal growth factor on the same column. These results demonstrate that HPTA exists in normal serum as a large precursor to a more active moiety, generated by proteolytic cleavage, which is not identical to epidermal growth factor. Fractions from human serum and plasma of molecular weight similar to that of HPTA have also been shown to stimulate DNA synthesis in rat hepatocytes.

Partial purification and characterization of a hepatocyte growth factor produced by rat hepatocellular carcinoma cells.

Serum-free medium conditioned by confluent cultures of JM1 of JM2 rat hepatocellular carcinoma cells stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. The hepatotrophic activity was nondialyzable in Mr 50,000 cutoff membranes, heat (60 degrees C) and acid stable, and sensitive to trypsin and dithiothreitol treatment. Gel filtration of concentrated JM1 or JM2 conditioned medium on Sephadex G-100 separated the activity into two regions, the major broad peak migrating with apparent Mr 25,000. Chromatography fractions active in the hepatocyte proliferation bioassay also inhibited specific binding of iodinated epidermal growth factor to cultures of A431 carcinoma cells and rat hepatocytes. These results suggest that neoplastic liver cells synthesize and secrete polypeptide growth factors which can bind to epidermal growth factor receptors and stimulate proliferation of normal adult rat hepatocytes in primary culture.

Unscheduled DNA synthesis in cells from N-2-fluorenylacetamide-induced hyperplastic nodules of rat liver maintained in a primary culture system.

The level of unscheduled DNA synthesis in the parenchymal cells from hyperplastic nodules and from the entire liver of rats fed N-2-fluorenylacetamide was studied and compared with that of normal liver cells. Measurements of unscheduled DNA synthesis were carried out by the use of a primary liver cell culture system. Livers were perfused with collagenase, the cells from individual hyperplastic nodules, and/or from the whole liver aspirated and plated onto plastic Petri dishes. Simultaneous histochemical measurements of beta-glucuronidase were carried out in the cultured cells as an aid in distinguishing functional cell types. The cells from hyperplastic nodules obtained from the liver during carcinogen feeding survived much longer than normal liver cells in culture. The level of unscheduled DNA synthesis was determined radioautographically after exposing cells to ultraviolet light and incubating with [3H]thymidine. [3H]Thymidine labeling was variable among individual nodules or animals and fluctuated as a function of the number of days in culture. In general, however, the level of unscheduled DNA synthesis in the cells from hyperplastic nodules was always higher than or similar to that of normal liver cells. Thus, the cells of hyperplastic nodules are not more readily transformed into the malignant state than normal cells as a result of their lowered DNA repair mechanisms.

Ability of partial hepatectomy to induce gamma-glutamyltranspeptidase in regenerated and transplanted hepatocytes of Fischer 344 and Wistar-Furth rats.

The purpose of this study was to investigate the effect of a two-thirds partial hepatectomy on the expression of gamma-glutamyltranspeptidase (GGT) in parenchymal hepatocytes. Two to 3-month-old Fischer 344 (F344), Wistar-Furth (WF), and WF X F344 F1 rats of both sexes were used in this investigation. Partial hepatectomy in the F344 female rat significantly increased the percent of liver area positive for GGT from 1.3 to 20.4%, a 15-fold increase. In contrast, GGT was not induced by partial hepatectomy in either the female WF or WF X F344 F1 hybrid rat or in the male animals of these three strains of rats. This phenomenon, which was only observed in the F344 female rat, was blocked by oophorectomy 2 weeks prior to partial hepatectomy, indicating that the induction of GGT is in part dependent upon female sex hormones. By transplanting both F344 and WF hepatocytes into the axillary fat pads of isogeneic and WF X F344 F1 rats, we showed that the observed strain difference did not result from a variation in the animal hormonal environment but rather was due to the hepatocyte from the F344 rat being intrinsically more susceptible to GGT induction than that from the WF rat. Further, the extent of the enzyme induction was significantly greater when the F344 liver cells were transplanted into female recipient animals. Since hepatectomy is often used in hepatocarcinogenesis studies, it is important to note that in the F344 female animal, the surgical procedure itself increases hepatic GGT levels in both transplanted hepatocytes and the liver in situ. Thus, the F344 female rat should be used with caution in investigations where the carcinogenic potential of chemicals is based upon the formation of GGT-positive hyperplastic nodules in the liver.

Hormonal regulation and the effects of glucose on tyrosine aminotransferase activity in adult rat hepatocytes cultured on floating collagen membranes.

Adult rat parenchymal hepatocytes can be maintained in primary culture on floating collagen membranes of prolonged periods of time. In this system the enzyme tyrosine aminotransferase is induced by glucagon, (10(-6) to 10(-8) M) hydrocortisone (10(-5) to 10(-8) M), and cyclic adenosine 3':5'-monophosphate (cAMP) (10(-4) to 10(-5) M). Epinephrine (10(-4) M) induces the enzyme only in the presence of hydrocortisone. Addition of actinomycin D inhibited the induction of tyrosine aminotransferase by hydrocortisone and cAMP. Maintenance of the cultured hepatocytes in the presence of glucose (3g/liter) results in partial suppression of the inducing effects of glucagon and cAMP. Cyclic quanosine 3':5'-monophosphate does not mimic the effects of glucose. These results demonstrate that the phenomenon of glucose repression of enzyme induction, demonstrated in vivo in mammalian liver, is independent of changes in levels of serum hormones, which occur in vivo as a result of glucose administration. This study also demonstrates that glucose repression is not mediated by changes in intracellular levels of cAMP and cyclic quanosine 3':5'-monophosphate.

Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes.

Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats (Michalopoulos, G., Cianciulli, H. D., Novotny, A. R., Kligerman, A. D., Strom, S. C., and Jirtle, R. L. Cancer Res., 42: 4673-4682, 1982). The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity. The ability of hepatocytes to activate 2-acetylaminofluorene to reactive intermediates capable of binding to DNA and inhibiting new DNA synthesis decreased as a function of time in culture. gamma-Glutamyl-transferase-positive hepatocytes from diethylnitrosamine-treated rats were observed to be relatively insensitive to carcinogen inhibition of DNA synthesis.

Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes.

Unscheduled DNA synthesis was induced by procarcinogens in freshly isolated suspensions and primary cultures (6 days old) of hepatocytes on collagen membranes. Incorporation of [3H]thymidine in the presence of hydroxyurea was used to measure unscheduled DNA synthesis. When hepatocellular DNA was isolated on cesium chloride gradients, significant levels of unscheduled DNA synthesis were measured. Similar concentrations of procarcinogens elicited higher levels of unscheduled DNA synthesis in hepatocellular suspensions than in primary cultures. The results demonstrate that hepatocytes cultured on collagen membranes can metabolize chemical carcinogens. Suspensions of freshly isolated hepatocytes, however, are more active in procarcinogen metabolism than are those of primary cultures. The selective advantages of the two systems of hepatocytes can be utilized for the establishment of short-term in vitro screening systems of mutagens and carcinogens.

Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes.

The ultrastructure of primary hepatocytes cultured for 2 to 17 days on floating collagen membrane was evaluated. A pellet of the hepatic cell suspension used to inoculate the collagen membranes contained some single cells and many aggregates of two, three, or four cells. Desmosomes were split during the perfusion, but tight junctions and gap junctions remained intact. By 2 days in culture, the hepatocytes had formed a monolayer of cells of polygonal shape with newly synthesized desmosomes between cells. Since the flexible floating collagen membrane decreases in size as the monolayer forms, the hepatocytes do not flatten out, as is characteristic of cells cultured on a rigid substrate. Hepatocytes in culture for 10 days or less exhibited large lamellar arrays of rough endoplasmic reticulum, well developed Golgi complexes, and structures resembling bile canaliculi, which possess tight junctions and desmosomes separating them from the intercellular space. Microfilaments oriented parallel to the plasma membranes of adjoining cells and in an intermeshed network at the edge of the monolayer and beneath the plasma membrane bordering the medium increased in size and number in older cultures. After 17 days in culture, the cells maintained tight junctions, desmosomes, Golgi complexes, and rough endoplasmic reticulum in small lamellar stacks and in small vesicles. Since hepatocytes on the floating collagen membrane retain most of the subcellular structural elements characteristic of normally functioning hepatocytes for 2.5 weeks, this system may be valuable for future experiments involving drug metabolism and carcinogenesis in vitro.