Continuous culture of Reuber hepatoma cells in serum-free arginine-, glutamine- and tyrosine-deprived chemically defined medium

The rat hepatoma cell line, H4-II-E, was grown serially over a 1-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's minimum essential medium with no supplements other than biotin. The adapted cell line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic "marker" enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.     

Characterization of alpha-fetoprotein secreted from cultured reuber H-35 hepatoma cells

Summary Reuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro. A part of this study was performed in the Division of Biofunction Research, Biomedical Research Laboratories, The Jikei University School of Medicine, with support by Grant-in-Aid for Scientific Research for 1981 of the Ministry of Education, Science and Culture of Japan.     

Growth of neural cell cultures in a chemically defined,serum-free culture medium

The composition of a serum-free, completely chemically defined culture medium which supports active growth of dissociated neural-cells in culture is described. This serum-free medium can also be used to grow many types of human cell lines without modification. It is the first report which describes the development of a wholly chemically defined, synthetic culture medium for growth of neural cells.     

Optimization of the culture medium composition for the antibody response of mouse spleen cells

Summary The Mishell-Dutton culture system for in vitro primary antibody response of mouse spleen cells was used to optimize the amino acid composition of RPMI 1640 media. Each of the 20 amino acids was tested over a broad range of concentrations always leaving the remaining 19 amino acids unaltered (i.e. at the formula recommended concentration). In several instances, higher plaque-forming cell responses were obtained with an amino acid concentration that was either higher or lower than that recommended: (a) the optimum concentration for valine, glutamine, and lysine lies considerably above the recommended one, (b) the optimum concentration for leucine as well as for several other amino acids lies below the recommended concentration, and (c) the optimum concentration for arginine corresponds exactly to the recommended concentration. The second round of optimization, i.e. combining of two conditions that individually yielded an improved response often caused a decrease of response. The possibility is discussed that for an optimal response a ratio of two or several amino acids rather than the absolute concentration of any one amino acid is of importance. The Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche & Co., Ltd.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

Growth of normal oral keratinocytes and squamous cell carcinoma cells in a novel protein-free defined medium

Summary A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

High-Density culture of mouse-human hybridoma in serum-free defined medium

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.     

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium

Summary The production of albumin, α-fetoprotein (AFP), and α-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 μg/ml reduced the level of total secreted protein.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Primary culture of adrenal medullary chromaffin cells in a chemically defined medium

Primary cultures of bovine adrenal medullary chromaffin cells have been maintained in the absence of serum for up to 3 weeks. Chromaffin cell catecholamine and protein contents were maintained, after an initial loss at the time of plating, as were the functional properties of the cells, including nicotine-evoked, calcium-dependent catecholamine secretion. Important factors in the maintenance of chromaffin cells included the cell plating density, frequency of medium replacement, and extent of medium replacement, suggesting ‘conditioning’ of the culture medium. Initially, serum was used for the first 48 h of culture, but pretreatment of the tissue culture plates with fibronectin allows complete elimination of serum from the plating medium. The establishment of serum-free culture conditions for chromaffin cells should facilitate the study of their cell biology and biochemistry.     

Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium

The embryonal carcinoma line C17-S₁ clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1003 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Growth of distal fetal rat lung epithelial cells in a defined serum-free medium

Summary Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 μg/ml), endothelial cell growth supplement (20 μg/ml), bovine pituitary extract (100 μg/ml), bovine serum albumin (50 μg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 μg/ml), transferrin (5 μg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 μg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E₁ (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung. Supported by grants MT-7867 and PG-42 from the Medical Research Council of Canada, HL-40458 from the National Heart, Lung and Blood Institute, Bethesda, MD, and an equipment grant from the Ontario Thoracic Society. Dr. Caniggia is a recipient of a research fellowship from the Italian Ministry of Education.