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 of human foreskin fibroblasts in a serum-free,defined medium without platelet-derived growth factor

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Wheresas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), trasferrin (10 μg/ml), thrombin (1 μg/ml), ascorbic acid (10 μg/ml), and hydrocortisone (5 × 10^?5M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblst growth factor, nor somatomedin activity increased proliferation. This serum-free medium designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.     

Differentiation of newborn rat adipocyte precursors in defined serum-free medium

Summary Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment. This work was supported by grants from the Juvenile Diabetes Foundation, File #185221 and from the National Institutes of Health 1 PO1 CA37589. Editor’s Statement This paper extends to primary cultures the serum-free methods previously applied to studies of adipocyte differentiation in established lines. The observation that serum can block differentiation in this system suggests the existence of previously unrecognized circulating plasma or platelet factors affecting adipocyte differentiation, and the model developed provides an assay for the identification of these factors.     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Characterization of sodium currents in mammalian sensory neurons cultured in serum-free defined medium with and without nerve growth factor

Summary The influence of nerve growth factor (NGF) on Na currents of rat dorsal root ganglia (DRG) was studied in neurons obtained from newborns and cultured for 2–30 hr inserum-free defined medium (SFM). Cell survival for the period studied was 78–87% both with and without NGF. Na currents were detected in all cells cultured for 6–9 hr. They were also detected after 2 hr in culture in 21.5% of the cells cultured without NGF (–NGF cells), and in 91.5% of the cells cultured with NGF (+NGF cells). Current density of the -NGF cells was 2.3 and 2 pA/m² after growth for 2 and 6–9 hr, respectively, compared to 3.0 and 3.9 pA/m² for the +NGF cells. The +NGF cells were separated into fast (F), Intermediate (I) and slow (S) cells, based on the Na current they expressed, while -NGF cells were all of theI type.F, I andS currents differed in their voltage-dependent inactivation (Vh ₅₀=–79, –28 and –20 mV), kinetics of inactivation (tau _h =0.55, 1.3 and 7.75 msec), and TTX sensitivity (K _i=60, 550 and 1100nm). All currents were depressed by [Ca] _o with aKd _Ca of 22, 17 and 8mm forF, I andS currents, respectively. Current density ofF andS currents was 5.5 and 5 pA/m² for theI current. The concentration-dependent curve ofI currentvs. TTX indicated thatI current has two sites: one withF-like and another withS-likeK _i for TTX. Hybridization ofF andS currents yieldI-like currents. Thus, the major effect of NGF on Na currents in SFM is the accleration of Na current acquisition and diversity, reflected in an increase of either theS orF type in a cell.     

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.     

Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline

cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murine cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.     

Culture of quiescent arterial smooth muscle cells in a defined serum-free medium

An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10^?6 M) and transferrin (5 m?g/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10^?4 M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.     

The growth of mouse melanoma cells in hormone-supplemented, serum-free medium.

A mouse melanoma line, M2R, derived from the B₁₆ transplantable tumor can be grown in a hormone-supplemented serum-free medium. Cells grown in medium supplemented with insulin, transferrin, testosterone (or progesterone), follicle-stimulating hormone, nerve growth factor (NGF) and leutinizing releasing hormone show growth rates equivalent to that seen in medium supplemented with 5% fetal calf serum (FCS). This hormone-supplemented medium will support the growth of cells indefinitely. In cells grown in hormone-supplemented medium, insulin is shown to increase incorporation of glucose into glycogen and fatty acids. Transferrin is shown to act in part as an iron transport protein, although another role in growth stimulation cannot be eliminated. It is suggested that progesterone stimulates growth via an androgenic breakdown product and thus acts in a manner similar to testosterone.     

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.     

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.     

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.     

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.     

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.     

The growth of spinal ganglion neurons in serum-free medium

The presence of serum in the culture medium affects the morphology of spinal ganglion neurons. With serum present, neuronal cell bodies are rounded and axons are predominantly straight. In serum-free medium axons curve all along their lengths, while both cell bodies and growth cones are spread on the substratum. Such “curved” axons straighten if serum is added to the culture dish.Serum-free medium may increase the adhesion of neurons to the substratum. This can account for the curved morphology of axons in serum-free medium, since such axons may represent a “history” of the movement of the growth cone during axon elongation.     

Studies on the cultivation of mammalian cell lines in a serum-free,chemically defined medium

Summary An improved chemically defined, serum-free medium for the cultivation of a variety of continuous cell lines has been developed. Eight lines of human origin, three lines of nonhuman primate origin, and five lines of rodent origin have been cultured serially for as long as a year in the medium. Growth rates of several serial lines resulted in as much as 20- to 30-fold increases per week. Hormones such as insulin, cortisol, and thyroxine significantly improved growth of cultures in the defined medium. Vitamin B₁₂ and biotin were required for growth. Lipids such as oleic acid, lecithin, and cholesterol also promoted growth of several cell lines. Virtually all continuous cell lines tested grew well upon initial transfer into the serum-free defined medium. Most cell lines could be serially subcultured rapidly with little evidence that selection of rare cell types was necessary for growth in the defined medium. However, a few cell lines such as the BHK-21 (hamster) cell and the AKR (mouse embryo) cell required prolonged periods (4 to 8 weeks) of culturing before rapid growth occurred. Primary cell cultures and other diploid cells such as human fibroblast (strain WI-38) could not be subcultured successfully in the present medium.     

Mycoplasma provides for the spreading of opossum kidney cells in a serum-free,defined medium

Summary During the course of investigating the growth and differentiation of opossum kidney cells in serum-free medium, it was observed that a mycoplasma contamination (M. hyorhinis) contributed to the spreading of cells. Contaminated cells, seeded on collagen-coated plates, spread out and grew to confluency in Dulbecco’s modified eagle’s medium/Ham’s F12 nutrient mixture containing insulin, transferrin, sodium selenite, and bovine serum albumin fraction V. In addition, differentiated characteristics, including parathyroid hormone-inhibitable, sodium-dependent phosphate transport, were expressed by these cells grown in this medium. After the infection was eradicated, the contamination-free cells would not spread out and proliferate in the same serum-free medium as they had done in the presence of mycoplasma. Normal cellular development, however, was attained after cells were plated in serum-free medium that included fetuin. Cells once again spread out, grew to confluency, and were able to express their differentiated characteristics.