Low-serum Medium Research Articles

Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium

Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10 −7 M and 10 −8 M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10 −7 M, MMC is cytotoxic to cells in both the media; at 10 −8 M, MMC is non-cytotoxic to cells in both media.

Production of recombinant protein C in serum-containing and serum-free perfusion culture.

For the development of a perfusion culture producing recombinant human protein C, the effects of fetal calf serum and growth factors on cell growth and recombinant protein production were investigated. Although the growth of recombinant cells was stimulated by serum in a dose-dependent manner, a lower concentration of serum (2%) could support both synthesis and post-translational modification of protein C as efficiently as 10% serum. Among the growth factors tested, transferrin enhanced protein C production to the level comparable with 10% serum, while insulin was effective in maintaining cellular metabolism. Based on these results, a perfusion culture for a scale-up production of recombinant protein C was done using an Opticell culture system. A good productivity of the recombinant protein was obtained in low serum or serum-free medium for more than one month.

Overexpression of c-jun, junB, or junD affects cell growth differently.

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.

“Spontaneous” differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II

Differentiation of muscle cells to form postmitotic myotubes is usually viewed as being negatively controlled by medium components, sometimes designated "mitogens." However, we have found that a family of mitogenic agents, the insulin-like growth factors (IGFs), are potent stimulators of differentiation in myoblasts which act by inducing expression of the myogenin gene. We show here that this action of the IGFs occurs even when these growth factors are not added to the cell medium; upon transfer to low-serum "differentiation medium," myoblasts begin active expression of the IGF-II gene, at both the mRNA and protein levels. Furthermore, autocrine secretion of IGF-II is essential for the process of terminal differentiation of the cells. These conclusions are based upon four lines of evidence. (1) The rate of spontaneous differentiation in several sublines of myogenic cells correlates with their level of expression of IGF-II. (2) C2 and Sol 8 cells, which secrete high levels of IGF-II, are relatively insensitive to exogenous IGFs, in contrast to L6 lines, which exhibit lower levels of IGF-II gene expression. (3) An antisense oligodeoxyribonucleotide complementary to the first five codons of IGF-II inhibits myogenic differentiation in the absence but not in the presence of exogenous IGF-II. (4) Spontaneous differentiation in response to autocrine IGF-II involves the same mechanism that occurs in cells stimulated by the IGFs, i.e. elevation of expression of the myogenin gene.

V-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

Development of Low- and High-serum Culture Conditions for Use of Human Oral Fibroblasts in Toxicity Testing of Dental Materials

With the aim of establishing conditions applicable to the testing of dental materials in human target cells, fibroblastic cell lines have been derived and grown from explants of human oral mucosa. Both a high-serum medium (termed "HSM") (CMRL 1066 supplemented with 10% fetal bovine serum) and a low-serum medium (termed "LSM") (a 1:1 mixture of M 199:MCDB 153 supplemented with 1.25% serum) supported radial outgrowths of cells from oral explants, as well as the subsequent transfer and growth of the cells in mass culture and at clonal density. Cells were typically fibroblastic in that they expressed vimentin uniformly, but did not express immunocytochemical markers of epithelial or endothelial cells. Cells derived in either LSM or HSM showed significantly higher colony-forming efficiency and clonal growth rate when transferred in LSM, as compared with HSM. Because cell migration occurred to a lesser extent in LSM, microscopic scoring of colony formation was also markedly facilitated. In both LSM and HSM, cellular low-molecular-weight thiols constituted about 30% of the total amount of sulfhydryls. Glutathione was present in about six- to seven-fold-higher amounts than cysteine--glutathione primarily in its reduced form and cysteine primarily in its oxidized form. A corrosion product of dental amalgam, i.e., Hg2+, decreased cell survival measured as colony-forming efficiency in a dose-dependent manner following either an acute (one h) exposure or continuous exposure (seven days). These studies demonstrated that human oral fibroblasts could be cultured at about one-tenth of the serum content that is commonly used.(ABSTRACT TRUNCATED AT 250 WORDS)

P8 Keratinocyte influence on melanocyte growth and differentiation in a TPA-free, low-serum medium

P8 Keratinocyte influence on melanocyte growth and differentiation in a TPA-free, low-serum medium

Induction of K-channel expression in a neuroblastoma cell line

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.

Baby hamster kidney (BHK-21/C13) cells can express striated muscle type proteins

Abstract When baby hamster kidney (BHK-21/C13) cell lines are subjected to low-serum medium, cell morphology changes from polygonal to elongated and occasionally fusion of cells is also observed. BHK-21 cells initially growing in Eagle's modified minimum essential medium (EMEM) containing 10% newborn bovine serum were induced to differentiate by changing the culture medium after the cells had grown to confluency. After this point the cells were grown in a low-serum medium (EMEM with 2% normal horse serum), for at least 4 days. The expression of different muscle-specific proteins (desmin, titin and skeletal muscle myosin) and of tropomyosins was studied in both polygonal and elongated BHK-21 cells using the indirect-immunofluorescence assay, two-dimensional (2D)-gel electrophoresis and immunoblot-ting. Filamentous staining was found with the desmin antisera in the polygonal cells and at all stages of BHK-cell elongation. While no reaction was seen with the titin and myosin antibodies in the polygonal cells, a punctate staining reaction for titin was detected 2 days after medium-change, although the cells had not yet elongated. After 4 days titin was found in a striated pattern. Filamentous staining was seen with the skeletal-muscle-specific myosin antibody at this stage. Confirmatory results were obtained from immunoblotting assays and 2D-gel electrophoresis of cytoskeletal preparations from undif-ferentiated and differentiated BHK cells. These latter experiments showed the initation of tropomyosin expression only in the differentiated cells. The positive staining with antibodies to skeletal muscle myosin and titin indicates a striated-muscle nature of the (elongated) BHK-21/C13cells.

Effect of free fatty acids and phospholipids on growth of and product formation by recombinant baby hamster kidney (rBHK) and Chinese hamster ovary (rCHO) cells in culture

Recombinant BHK and CHO cells producing human antithrombin III (rh ATIII) were used to investigate the utilization of phospholipids and free fatty acids from low-serum (0.1% FBS) culture medium. Both cell lines show distinctly different patterns of fatty acid utilization. For rBHK ATIII cells it is shown that under low serum conditions several different combinations of free fatty acids (bound to bovine albumin) elicit an identical growth stimulatory effect although individual consumption and production rates of fatty acids are different. Increased fatty acid concentrations lead to increased uptake rates without any further effect on growth rate being observed. Recombinant antithrombin III formation is found to be a function of combinations and concentrations of fatty acids present in the culture medium.

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line, TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.

Oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells from neonatal and adult rat optic nerve differ in their responsiveness to platelet-derived growth factor

We have investigated, in vitro, the mitogenic responsiveness to platelet-derived growth factor (PDGF) of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerve and their differentiation into oligodendrocytes. Progenitor cells from adult optic nerves differentiate into oligodendrocytes in a limiting concentration of foetal calf serum more slowly than in cultures of neonatal cells. Nevertheless, differentiation of oligodendrocytes from progenitors is nearly complete by 6 days in vitro, with 50% expressing galactocerebroside by 4–5 days. In these experiments, adult optic nerve cells were grown in medium containing PDGF, a potent mitogen for neonatal O-2A progenitor cells, and yet the decline in numbers of O-2A progenitor cells matches the rise in oligodendrocyte numbers. We suggest that this is because adult O-2A progenitor cells differ from their neonatal counterparts and do not show the same proliferative response in the presence of exogenous PDGF. We tested this hypothesis by a quantitative autoradiographic analysis of tritiated thymidine-labelled nuclei, comparing percentages of labelled adult and neonatal O-2A lineage glial cells in low-serum medium, in the presence or absence of PDGF, with their response to a monolayer of neonatal rat cortical type 1 astrocytes or astrocyte-conditioned medium. Whereas, adult O-2A progenitors responded to astrocyte monolayers and to conditioned medium from astrocyte cultures, there was no dose-dependent response to PDGF-BB over a wide range of concentrations. Antibodies to human PDGF neutralise the growth-promoting activity of astrocyte-conditioned medium for neonatal O-2A cells but do not neutralise astrocyte-conditioned medium stimulation of adult O-2A progenitor cells. This indicates that the principal astrocyte-derived growth factor(s) for adult O-2A progenitor cells is unlikely to be PDGF.

Differential Effects of Fibroblast Growth Factor on Insulin Receptor and Muscle Specific Protein Gene Expression in BC3H-1 Myocytes

BC3H-1 mouse muscle cells in culture were employed to study the mechanisms which regulate insulin receptor gene expression during differentiation. When BC3H-1 myoblasts were plated in low serum media (1% fetal bovine serum), cell division ceased. Within 1 week cells had the morphological features of myocytes and expressed muscle specific proteins such as creatine phosphokinase and the nicotinic acetylcholine receptor. It is known that following incubation in low serum media, the steady state mRNA levels for the key muscle transcription factor, myogenin are increased. Exposure of BC3H-1 cells to a 20-base myogenin antisense oligomer blocked morphological differentiation, and resulted in nearly complete inhibition of the expression of the acetylcholine receptor but not the insulin receptor(IR). In order to study further the relationship between differentiation and IR gene expression, fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation, was employed. FGF treatment inhibited the transcription of both myogenin and the acetylcholine receptor. However FGF did not inhibit the transcription of the IR. These studies indicate therefore that IR transcription increases during muscle cell differentiation, and suggest that during differentiation the control of IR gene expression differs from the control of muscle specific proteins.

Identification of Hypoxanthine and Inosine in Brain Dialyzable Fraction as Stimulators for Growth of Porcine Aortic Endothelial Cells in Response to Fibroblast Growth Factor in Either Dialyzed Serum Media or Low Serum Media

The rate of proliferation of porcine aortic endothelial cells (PAEC) in response to fibroblast growth factor (FGF) was largely retarded when incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with either 1% fetal bovine serum (FBS) or 10% dialyzed FBS in place of 10% FBS. Proliferation of endothelial cells in low serum media in response to FGF was enhanced to the level of media containing FGF plus 10% FBS by the addition of the dialyzable fraction from bovine brain homogenates. From the bovine brain dialyzable fraction, two active components were purified and identified as hypoxanthine and inosine. Either hypoxanthine or inosine, at a dose of 5 μM in DMEM with 1% FBS, maximally increased the incorporation of [3H]thymidine into DNA of PAEC in low serum media in the presence of FGF. However, no additive effect was observed when hypoxanthine and inosine were added simultaneously. The present data indicate that the proliferative action of FGF on PAEC can be potentiated by hypoxanthine and inosine.

自主開発低血清培地によるＬＡＫ細胞の高密度培養 濃縮回転培養法およびｈｏｌｌｏｗ‐ｆｉｂｅｒ ｓｙｓｔｅｍへの応用

Lymphokine activated killer (LAK) cells were cultured in low serum culture medium that was newly developed. The medium was composed of three basal media, i. e. Dulbecco's modified eagle (DME), F-12 (Ham) and RPMI-1640, and it was enriched with other supplements. The medium was applied with 1% gelatin and 3% AB serum for CRTC (concentrate rotary tissue culture) system, or with 1% AB serum for hollow-fiber system in LAK cells culture. It was demonstrated that the medium can be used to proliferate LAK cells to about thirty-fold the initial cell number with CRTC system, or to twenty one-fold with hollow-fiber system. Therefore, this medium can be applied for these culture systems to obtain a large number of LAK cells in low serum concentration.

Long-term growth of diploid human fibroblasts in low serum media

Hayflick and Moorhead demonstrated that diploid human fibroblasts have a limited life span when grown in media containing 10% bovine calf sera. Recent experiments have suggested that antigrowth factors in serum may be a potential contributor to the limited proliferative capacity of normal diploid cells. To reduce the concentration of inhibitory serum factors 10-fold, MRC-5 diploid fibroblasts were cultured in media with only 1% serum. Long-term culture in 1% serum requires the addition of purified growth factors to sustain proliferation. Although there are dramatic changes in cell morphology, we find that the long-term division potential of MRC-5 cells cultured in media containing 1% serum and growth factors differs little from that found with cells cultured in 10% serum. In contrast, MRC-5 cells cultured in 10% serum and added growth factors have a somewhat extended life span. These results suggest that negative growth factors are not responsible for the limited proliferative capacity of in vitro cultured human fibroblasts. Moreover, the evidence that human fibroblasts can undergo major changes in cell morphology and still retain a normal life span raises questions about the validity of using morphological changes as indicators of cellular senescence.

Stimulation of limb cartilage differentiation by cyclic AMP is dependent on cell density

Cyclic AMP (cAMP) has been implicated in the regulation of limb cartilage differentiation. This study represents an attempt to clarify potential mechanisms by which cAMP might regulate chondrogenesis. We have found that the ability of cAMP to stimulate limb cartilage differentiation in vitro is dependent on cell density. Dibutyryl cAMP (dbcAMP) elicits a striking increase in the accumulation of Alcian blue, pH 1.0-positive cartilage matrix, and a corresponding three- to fourfold increase in the accumulation of 35S-labeled glycosaminoglycans (GAG) by limb mesenchymal cells cultured in low serum medium at densities greater than confluence (i.e. micromass cultures established with 1–2 × 10 5 cells in 10 μl of medium). Moreover, dbcAMP causes a striking (two- to fourfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific sulfated proteoglycan in these high density, supraconfluent cultures. In contrast, cAMP does not promote the chondrogenesis of limb mesenchymal cells cultured at subconfluent densities (i.e. cultures initiated with 2.5–5 × 10 4 cells in 10 μl of medium). In these low density cultures, dbcAMP does not promote the formation of cartilage matrix, sulfated GAG accumulation or the accumulation of cartilage-specific mRNAs. These observations suggest that cAMP may exert its regulatory effect in part by facilitating cell-cell communication during the critical condensation phase of chondrogenesis.

Serum can act as a shear protecting agent in agitated hybridoma cell cultures.

Hybridoma cells, S3H5/gamma 2bA2, were grown in spinner flasks containing RPMI 1640 media at different levels of serum. Cells in low serum media (1% fetal bovine serum) were more sensitive to shear induced by mechanical agitation than cells in high serum media (10%), indicating that serum can protect cells from shear induced by mechanical agitation. The data suggest that serum alters the physiological properties of the cells to make them more shear-resistant, rather than by changing the physico-chemical properties of medium.

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.

Constitutive expression of c-myc oncogene confers hormone independence and enhanced growth-factor responsiveness on cells transformed by human papilloma virus type 16.

The effect of constitutive expression of the c-myc oncogene on the biological properties of cells transformed or immortalized by human papilloma virus type 16 (HPV16) was studied. Whereas transfection of HPV16 alone into primary baby mouse kidney (BMK) cells failed to generate any immortalized cell lines unless the tumor promoter phorbol 12-myristate 13-acetate was present, cotransfection of HPV16 with a plasmid that constitutively expresses murine c-myc (pSVc-myc-1) generated numerous rapidly growing colonies of cells. Cell lines transformed by HPV16 and pSVc-myc-1 did not require phorbol ester or steroid hormones for growth and were tumorigenic in syngeneic immunocompetent mice. Transfection of pSVc-myc-1 into established cell lines transformed by HPV16 and the v-fos oncogene increased the growth rate and saturation density of these lines severalfold, allowed growth in low-serum medium, and abolished the requirement of these cell lines for glucocorticoids or progestogens. Transfection of the EJ-ras oncogene into these lines did not significantly affect any of these properties.