Immortalization of chimpanzee hepatocytes with an amphoteric retrovirus encoding simian virus 40 T antigen.

Primary chimpanzee (Pan troglodytes) hepatocyte cultures were maintained in a serum-free medium containing hormones and growth factors and exhibited the de novo synthesis and secretion of numerous liver-specific plasma proteins for over 3 weeks in vitro. The long-term maintenance of differentiated, primate hepatocytes in this serum-free medium allowed for subsequent immortalization events to occur after infection with the amphoteric retrovirus U19, which encodes the simian virus 40 large T antigen oncogene. Several hepatocyte cell lines were selected and examined for the expression of liver-specific plasma proteins and the capacity to synthesize apolipoproteins. Several cell lines expressed a majority of the plasma proteins investigated, including apolipoproteins A1 and E. These results demonstrate the ability of this serum-free medium to maintain long-term differentiated primate hepatocytes, allowing for the experimental immortalization of this cell type in vitro and the maintenance of differentiated functions in the established cell lines. This methodology should be amenable to the study of the liver and its related diseases.     

Serum-free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts.

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis.     

Indole derivatives sustain embryonic stem cell self-renewal in long-term culture

Embryonic stem cells (ESCs), which have characteristics such as self-renewal, indefinite proliferation, and pluripotency, are thought to hold great promise for regenerative medicine. ESCs are generally cultured on mouse embryonic fibroblast (MEF) or MEF conditioned medium (MEF-CM). However, for therapeutic applications, it is preferable for ESCs to be cultured under chemically defined conditions. Here, we report synthetic compounds that allow expansion of undifferentiated mouse ESCs in the absence of MEF, Leukemia Inhibitory Factor (LIF), and Fetal Bovine Serum (FBS). ESCs cultured for more than 30 d in a serum-free medium supplemented with indole derivertives retained their characteristic morphology and expressed markers such as SSEA-1, OCT3/4, Rex-1, Sox2, and Nanog. They consistently differentiated into many types of cells, including neurons, muscle cells, and hepatocytes. These results indicate that our compounds provide a more efficient and safer large-scale culture system for pluripotent ESCs, and hence might contribute to the use of ESCs in therapeutic applications.     

Establishment of a tightly regulated human cell line for the development of hepatocyte transplantation

Hepatocyte transplantation (HTX) could be an attractive treatment for patients with liver failure and liver-based metabolic disease. Human primary hepatocytes are ideal in this modality, but the shortage of human livers available for hepatocyte isolation severely limits the use of this form of therapy. A tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to the primary human hepatocytes. To test the feasibility, human hepatocytes were immortalized by a retroviral vector expressing simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. A highly differentiated immortal hepatocyte line NKNT-3 was established. NKNT-3 cells grew in chemically defined serum-free medium, retained highly differentiated liver functions, and were sensitivity to ganciclovir as a prodrug. Essentially unlimited availability of NKNT-3 cells may be clinically useful for HTX and bioartificial liver.     

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities,and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium

Summary We have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.     

Control of growth and expression of differentiated functions of mature hepatocytes in primary culture

Since methods to disperse and culture hepatocytes were developed 15 years ago, numerous investigations have shown that primary cultures of mature hepatocytes retain most liver functions and respond as well to various hormones as those in vivo. Thus they are the most suitable system in vitro for studies on the liver. Moreover, recently it was found that differentiated hepatocytes in culture can grow under certain conditions and that this growth is regulated not only by several hormones, such as insulin, epidermal growth factor and serum growth factor, but also by a cell membrane factor and proteins in the environmental matrix through cell contact. This article describes the biochemical characterization of regulatory factors for hepatocyte growth and functions and their reciprocal expression. The mechanisms of liver regeneration, differentiation and carcinogenesis and the importance of the tissue architecture for these events are discussed mainly on the basis of our findings.     

Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products

Human embryonic stem cells (hESCs) can serve as an unlimited cell source for cellular transplantation and tissue engineering due to their prolonged proliferation capacity and their unique ability to differentiate into derivatives of all three-germ layers. In order to reliably and safely produce hESCs, use of reagents that are defined, qualified, and preferably derived from a non-animal source is desirable. Traditionally, mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture undifferentiated hESCs. We recently reported a scalable feeder-free culture system using medium conditioned by MEFs. The base and conditioned medium (CM) still contain unknown bovine and murine-derived components, respectively. In this study, we report the development of a hESC culture system that utilizes a commercially available serum-free medium (SFM) containing human sourced and recombinant proteins supplemented with recombinant growth factor(s) and does not require conditioning with feeder cells. In this system, which employs human laminin coated surface and high concentration of hbFGF, the hESCs maintained undifferentiated hESC morphology and had a twofold increase in expansion compared to hESCs grown in MEF-CM. The hESCs also expressed surface markers SSEA-4 and Tra-1-60 and maintained expression of hTERT, Oct4, and Cripto genes similar to cells cultured in MEF-CM. In addition, hESCs maintained in this culture system were able to differentiate in vitro and in vivo into cells of all three-germ layers. The cells maintained a normal karyotype after prolonged culture in SFM. In summary, this study demonstrates that the hESCs cultured in defined non-conditioned serum-free medium (NC-SFM) supplemented with growth factor(s) retain the characteristics and replicative potential of hESCs. The use of defined culture system with NC-SFM on human laminin simplifies scale-up and allows for reproducible generation of hESCs under defined and controlled conditions that would serve as a starting material for production of hESC derived cells for therapeutic use.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

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.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

A serum-free defined medium for retinal pigment epithelial cells

Human and bovine RPE cells underwent changes in morphology and culture doubling times when passaged in serum-supplemented medium (CM). Furthermore, late passage human RPE cells subcultured in CM medium increased synthesis of three acidic, 43 000–63 000 D proteins. In order to provide a controlled environment for the study of RPE cells in vitro, we have devloped a method for growing human and bovine RPE in a serum-free defined medium (DM). RPE cells grown in DM required a 24 h pretreatment with CM to allow the cells to attach and spread on the substrate. Cells grown in DM retained an epithelioid morphology, a stable culture doubling time, and similar 2-D PAGE patterns through several subculturings.     

Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco’s modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.     

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in co-culture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1–2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.     

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Summary Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Human breast epithelium in organ culture: Effect of hormones on growth and morphology

Summary Normal breast tissue from a 17-year-old girl was grown in organ culture for 3 weeks. A comparison was made between the effects on the epithelium of a defined culture medium containing various combinations of hormones and a serum-supplemented medium that has been used to successfully maintain other human tissues for 4 months routinely, and in some cases for up to 1 year. After culture for 3 weeks the explants were exposed to [³H]thymidine and autoradiographs were prepared and evaluated in order to determine labeling indexes. The only serum-free defined medium that permitted any significant survival or labeling of the cells contained insulin + hydroxycortisone + prolactin. However, serum-supplemented medium alone gave an even higher labeling index, and this was elevated more in media containing either progesterone or other combinations of hormones. Our study indicates that normal human breast (removed at the early postovulatory stage of the menstrual cycle) can be maintained in a differentiated state for 12 days in serum-supplemented media. By 2 weeks the cells had begun to migrate onto the surface of the explant. They then began to accumulate tonofilaments so that after 3 weeks in culture nearly all of the cells contained tonofilaments. The one exception was found in breast tissue cultured in the presence of human chorionic gonadotropin, where the cells maintained differentiated characteristics, despite the fact that they contained many lysosomes. This work was supported by Grant R01 CA20764 and in part by Contract N01-CP-95640 awarded by the National Cancer Institute.