Methods for growth of cultured cells in serum-free medium

Mitochondria prepared from skeletal muscle are typically contaminated with sarcoplasmic reticulum fragments and salt-soluble proteins. These contaminants can be removed by density-gradient centrifugation in Percoll. The resulting mitochondria retain both their original state three respiratory rates and their respiratory control ratios. The method has also been adapted to prepare mitochondria from very small tissue samples.     

A simple freezing medium for serum-free cultured cells

Methylcellulose was found to protect serum-free cultured cells from the deleterious effects of freezing and thawing. We have formulated a simple medium suitable for freezing serum-free cultured cells that consists of 0.1% methylcellulose, 10% dimethylsulfoxide, and MEM or any other serum-free culture medium.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Enhanced multiplication and progesterone production by porcine granulosa cells cultured in a serum-free medium

Granulosa cells harvested from pro-estrous follicles of porcine ovary were grown in medium 199 supplemented with 0.4, 1 and 10% of growth-promoting calf serum proteins (GPP), and their multiplication and hormonal activity were compared with those of sister cultures carried in medium 199 supplemented with 10% calf serum. The medium containing the growth-promoting proteins was always superior to the whole-serum medium with regard to cell multiplication, activity of Δ5,3 hydroxysteroid dehydrogenase detected histochemically in the cells, and production of progesterone estimated by radioimmunoassay in the medium. It was inferior when it came to estrogen secretion in the beginning of the cultivation when calculated on a per cell basis.     

Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium.

Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

A serum-free medium that supports the growth of cultured skeletal muscle satellite cells

Summary A serum-free medium has been devised that supports the proliferation and differentiation of primary cultures of rat skeletal muscle satellite cells for up to 4 d. The medium consists of a mixture of Dulbecco's modified Eagle's medium and MCDB-104 plus insulin, dexamethasone, pituitary fibroblast growth factor, Deutsch fetuin, and linoleic acid. In addition to promoting the formation of myotubes from satellite cells, a decrease in fibroblast contamination of these cultures was observed when cultures grown in serum-free medium were compared to cultures grown in serum-containing medium. This work was supported by the Arizona Agriculture Experiment Station, Project No. R11, U.S. Public Health Service Grant R01 AG03393, Lilly Research Laboratoires, and Merck Institute for Therapeutic Research. This communication is Arizona Agriculture Experiment Station Journal Paper No. 3966.     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Cardiomyocytes cultured in serum-free medium: Growth and creatine kinase activity

Primary cultures of newborn rat heart cells were grown for up to 3 weeks in serum-free medium supplemented by insulin, hydrocortisone, transferrin and fetuin. The cells resumed spontaneous beating at 20 h post plating. Mean rates of beating on the second and third day were 79.5 and 94 beats per min, respectively. Cell proliferation occurred during the first 3 days of culture with maximal rates of DNA and protein synthesis on the second day. The highest values of creatine kinase activity were observed on days 2–5 and the three cytoplasmic isozymes, MM, MB and BB, were present in the cultures in proportions similar to those of the newborn heart, indicating stability of the differentiated state of the cells. The relative amount of each isozyme remained unchanged throughout the experiments, MM constituted 70–90% of enzyme activity, MB contributed up to 30% and BB did not exceed 15% of activity. The very low proportion of BB and the lack of increase in this isozyme with age of culture support our earlier morphological observations that non-myocytes do not overgrow the culture.     

Rat glioma cells (C6) cultured in serum-free defined medium

Rat glioma C₆ cells were adapted to and maintained in serum-free medium for 11 months. Morphological differentiation with extended dendrite processes was observed. This phenomenon is reversible if serum is re-supplemented and is protein or RNA synthesis dependent. The formed cytoplasmic processes rapidly retract when colchicine or vinblastine sulfate is added. db-cAMP is found able to stimulate the extension of cytoplasmic processes of cells cultured in medium containing serum, but no further stimulation was observed in cells adapted to serum-free medium. The serum-free adapted cells retain the ability to synthesize the acidic S-100 protein and the production rate is 25% higher than the cells cultured in serum-supplemented medium. The serum-free adapted cells have a longer population doubling time but the metaphase chromosomes show the same karyotype and modal number as that of C₆ cells continuously cultured in serum-supplemented medium.     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Adipocyte conversion of CHEF cells in serum-free medium

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serum-free medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, greater than 90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.     

Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum-free medium

A serum-free, hormone-supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum-supplemented media. To quantitatively compare cell behavior in SFHM with serum-supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+-linked sn-glycerol-3-phosphate dehydrogenase, malate dehydrogenase, NAD+-linked isocitrate dehydrogenase, NADH-cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum-supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: 1) many enzyme activities that may represent dedifferentiation are elevated by serum; 2) NAD-linked glycerol-3-phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; 3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; 4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 micrograms/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.     

Adrenergic receptor characteristics of cardiac myocytes cultured in serum-free medium: comparison with serum-supplemented medium

Neonatal rat ventricular myocytes cultured in serum-free medium coexpress both alpha 1 and beta 1 receptors as determined by radioligand binding studies. In cells exposed to serum for 48 hr surface area increased 3.69 fold, but the maximum number of binding sites ([125I]-iodocynanopindolol) only increased 1.5 fold from 12956 +/- 7579 to 19676 +/- 5181 sites/cell (n = 5, p less than .05) yielding a value of 2.48 sites/um2 for cells grown in serum-supplemented medium compared with 6.96 sites/um2 for cells grown in serum-free medium. Thus serum-induced hypertrophy is associated with a decrease in beta 1 receptor density relative to cell size; however, adenylate cyclase response is unaffected. This cell culture system constitutes an excellent model for studying interventions that may influence the regulation of cardiac myocyte hypertrophy by nonhemodynamic factors, particularly through the adrenergic receptor system.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.