Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions

Adult rat hepatocytes were maintained in culture for at least 1 month without losing the expression of their differentiated functions; they were cultured on lethally treated 3T3 fibroblasts inoculated at 35,000 cells/cm2 with medium containing 10-25 micrograms/ml hydrocortisone. Hepatocytes showed their typical morphology; they formed bile canaliculi, microvilli, and intercellular junctions with desmosomes and nexus; some formed structures that may resemble the perisinusoidal space of Disse. In addition, they showed DNA synthesis and expressed some liver-specific functions. They synthesized albumin and other proteins, which were exported to the culture medium. Like parenchymal liver cells in vivo, de novo fatty acid synthesis and esterification took place, and more than 80% of the lipids synthesized by the hepatocytes were secreted into the medium as triglycerides; they also showed cytochrome-P450 activity that was inducible with phenobarbital, suggesting that the hepatocytes have the capacity to metabolize drugs. These culture conditions allow the study of various hepatocyte differentiated functions, and they may provide the means to analyze the effect on liver of hormones, viruses and hepatotoxic chemicals and drugs; they may also indicate conditions adequate for serial growth of hepatocytes.     

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes.

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles' medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, alpha 1-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was alpha-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. gamma-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.     

The influence of medium composition and matrix on long-term cultivation of primary porcine and human hepatocytes

The differentiated hepatocyte phenotype remains difficult to maintain in culture. The duration over which phenotypically stable hepatocytes can be cultured ranges from a couple of days to a few weeks. Shortcomings in medium formulation may be a factor in this lack of success. We have investigated effects of medium formulation on primary porcine and human hepatocyte cultures. We tested seven culture medium compositions (DMEM, ExCell 400, HepatoZYME-SFM, L-15 Leibovitz, SF-3, Waymouth, and Williams' E) and the effects of serum, fibronectin and biomatrix in a sandwich culture configuration. Albumin, urea, cholesterol, GOT, GPT, LDH and triglyceride concentrations were measured over 14 days. For both human and porcine cultures, the best results were obtained with SF-3 medium. Cells cultivated with Williams' E medium and FCS had good morphology and synthetic function during the first days of culture. However, continued addition of serum, was associated with a subsequent loss of differentiated phenotype. Addition of fibronectin was associated with improved function in cultures maintained in SF-3 medium whilst biomatrix had no effect. In contrast, addition of fibronectin did not influence cultures maintained in Williams' E medium, but cultures with biomatrix were associated with improved function at longer time points.     

Primary culture in chemically defined medium of human fetal mammary epithelial cells within reconstituted matrix]

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

Transitional change of colony stimulating factor requirements for erythroid progenitors.

The course of the differentiation and proliferation of the human erythroid burst-forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL-3 requirement correlate with the erythroid differentiated state.     

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles’ medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, α-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was α-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. γ-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Clonal growth of primary cultures of rabbit ear chondrocytes in a lipid-supplemented defined medium

Clonal growth of primary cultures of rabbit ear chondrocytes in a defined medium without serum or other undefined additives has been achieved. The clonal inoculum is a suspension of fully differentiated chondrocytes prepared by collagenase digestion of rabbit ear cartilage and used with no prior adaptation or selection in culture. When inoculated into medium MCDB 104 supplemented with 100 ng/ml fibroblast growth factor (FGF), 1 microgram/ml insulin, and 5 micrograms/ml of a lipid supplement previously developed for human fibroblasts, the isolated chondrocytes undergo clonal multiplication to form large colonies of epithelial-like cells. Colonies grown in the defined medium for 14 days accumulate at their centers refractile cartilage-like matrix that is stained by acidified Alcian green, although the amount is significantly less than with undefined additives. This system opens the way for detailed studies, in a defined background medium, of factors that regulate phenotypic expression of cartilage-like differentiated properties.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

Human granulopoiesis in vitro: an advantage in the use of Iscove's modified Dulbecco's medium

With the aim of determining whether Iscove's Dulbecco's medium (IMDM) provides a growth advantage in the support of granulopoiesis from cultures of human bone marrow in agar, samples from 20 normal subjects were examined in triplicate after 7, 10 and 14 days in parallel cultures containing IMDM or Dulbecco's medium. From every sample, more granulocyte-macrophage colonies were obtained at each culture interval with IMDM. In particular, the number of colonies with IMDM at 14 days (96 +/- 13 per 2x10(5) bone marrow cells) was almost double that with Dulbecco's medium (50 +/- 10). This increment consisted almost entirely of pure granulocyte colonies (P less than 0.001). No significant change in the proportion of eosinophil colonies was observed. These data indicate that IMDM does provide a growth advantage over Dulbecco's medium in the generation of granulocyte (neutrophil and eosinophil) colonies from agar cultures of normal human bone marrow.     

Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts

In preparation for studies on the growth factor requirements of normal and transformed human fibroblasts, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human fibroblasts and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment factors instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal growth factor or platelet-derived growth factor. In this modified serum-free medium, which we have designated McM+SR1, diploid human fibroblasts grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells growing in the medium containing serum.     

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation.     

A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.     

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.