Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum

Summary An optimized basal nutrient medium, MCBD 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 μg/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg²⁺ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS. This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD.     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Clonal growth of Chinese hamster cell lines in protein-free media

Summary A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable “essential” role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly “defined” medium are discussed. This research was supported by Grant No. CA15305 from the National Cancer Institute.     

Clonal growth of human keratinocytes with small amounts of dialyzed serum

Summary A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca²⁺ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca²⁺ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with 10 μg/ml HC and 1.0 mg/ml FBSP. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation,identification, and growth characteristics

Summary Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum. The cell yield was approximately one million cells per square centimeter omentum. The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18. Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport. Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium. Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed. Seeding NHMC at densities less than 3000/cm² did not result in monolayer formation. The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages. However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Density-dependent inhibition of 2-deoxy-d-glucose uptake into glioma and neuroblastoma cells in culture

The transport of 2-deoxy-d-glucose (2-DG) into cultured human glioma (138 MG) and mouse neuroblastoma (C1300) cells has been studied in relation to the growth curves of the cells. An initial increase in the uptake of 2-DG into exponentially growing 138 MG cells, could be attributed to the number of days the cells were kept in culture rather than to their increased density. As the 138 MG cells reached confluency after 3 days the 2-DG uptake became density-dependent inhibited. In still denser cultures the cell growth was inhibited. This was accompanied by morphological ‘normalization’ of the cells and increased uptake of 2-DG. Uptake of 2-DG into C1300 cells was density-dependent inhibited throughout the cell growth cycle. As the cell density increased from 15 × 10³ to 130× 10³ cells/cm² the rate of uptake/cell decreased to one-fourth. At the latter cell density the cells entered stationary phase, without any major changes in morphology. The results suggest that spontaneously occurring tumour cells, such as glioma and neuroblastoma cells, can regulate the sugar transport in relation to cell density. This could be due to newly-acquired differentiated properties of the cells or to true contact-inhibitory phenomena.     

Clonal growth of chinese hamster cell lines in protein-free media

A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable "essential" role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly "defined" medium are discussed.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Growth of functional primary cultures of kidney epithelial cells in defined medium

Primary cultures of baby mouse kidney epithelial cells can grow without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium K-1) designed for an established kidney epithelial cell line, MDCK. The five supplements in Medium K-1 are insulin, transferrin, PGE1, T3, and hydrocortisone. Medium K-1 also supports the growth of kidney epithelial cell cultures from a number of animals, including man, without fibroblast overgrowth. Outgrowth of kidney epithelial cells from kidney explants was also observed with Medium K-1. Thus, the medium appears to be selective for epithelial cell growth. The physiological properties of primary cultures of baby mouse kidney epithelial cells were studied in detail. Baby mouse kidney epithelial cells grew at equal rates (0.5 doublings/day) in Medium K-1 and serum-supplemented medium. Medium K-1 also supported the formation of baby mouse kidney epithelial colonies at low cell densities. The dependence of baby mouse kidney epithelial colony formation upon the five factors in Medium K-1 was examined. These studies indicated that the formation of baby mouse kidney epithelial colonies in defined medium depended upon all the five supplements in Medium K-1, in a manner similar, although not identical, to MDCK colonies. Primary cultures of baby mouse kidney epithelial cells grown in Medium K-1 retained kidney cell-associated properties, including the ability to form multicellular domes, a phenomenon associated with transepithelial salt transport. Amiloride-sensitive Na+ uptake and the mucosal surface enzyme leucine aminopeptidase were also observed in baby mouse kidney cultures. Similar functions were observed in MDCK monolayers.     

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. I. Effect of liposome B components on colony growth

Summary An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64–24 with far less protein supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 μg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 μg/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 μg/ml liposome B (a mixture of soy lecithin, sphingomyelin, cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine or sphingomyelin. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by B.A.VdH. in partial fulfillment of the requirements for the Ph.D. degree. This research was supported by grant CA-15305 to R.G.H. and grant CA-30545 to T.K.-S., both from the National Institutes of Health, Bethesda, MD.     

Growth arrest by octanoate is required for porcine preadipocyte differentiation

A preadipocyte clonal line has been established from porcine subcutaneous tissue. This line, designated PSPA, showed a fibroblastic phenotype and kept on growing under a preadipose condition even after reaching confluence. When confluent cultures were stimulated with insulin, dexamethasone, biotin, pantothenate, and octanoate, growth was arrested, and the cells exhibited a marked increase in lipogenesis. However, adipose conversion was not induced upon exposure of PSPA cells to a standard hormonal mixture of mouse 3T3-L1 cells, and they continued dividing as did the preadipocytes in growth medium. By serially omitting each individual adipogenic agent from the PSPA differentiation medium, it was determined that octanoate was one of the most essential but the only factor able to induce growth arrest. Octanoate supplementation to 3T3-L1 medium increased the triglyceride accumulation of PSPA cells accompanied by growth arrest. Both RT-PCR and Western blot analysis supported the idea of octanoate as a potential agent with the antiproliferative activity requisite for porcine preadipocytes to enter terminal differentiation.     

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10⁶ cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Taurine and hypotaurine transport in neuroblastoma cells: effects of cations

The cation requirements of [³H]taurine and [³⁵S]hypotaurine uptake by cultured neuroblastoma C1300 cells were compared in Krebs-Ringer-Hepes-glucose medium. The uptakes were strictly sodium-dependent at both low and high taurine and hypotaurine concentrations. The omission of Ca²⁺ or Mg²⁺ ions affected uptakes only marginally. The optimal K⁺ concentration was equal to the physiological concentration, whereas abnormally high K⁺ levels inhibited similarly taurine and hypotaurine uptake. The sodium dependence curves of both uptakes were sigmoidal in character at low and high taurine and hypotaurine concentrations. Hill plots suggest that two Na⁺ ions are coupled with the transfer of one taurine or hypotaurine molecule into neuroblastoma cells. With respect to cation requirements taurine and hypotaurine transports are similar in cultured neuroblastoma cells and display features considered typical of the uptake of a neurotransmitter amino acid.