The effects of various nutritional supplements on the growth,migration and differentiation ofxenopus laevis neural crest cells in vitro

Summary This study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested for cell migration and adhesion. This work was supported by establishment and major equipment grants from the Alberta Heritage Foundation for Medical Research to N. C. M. Nadine C. Milos is a Heritage Medical Research Scholar of the Alberta Heritage Foundation for Medical Research.     

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.     

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues). This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research. Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal and tumor prostate epithelial cells from the same system.     

The Cultivation of Symbiote-free Marine Ciliates in Axenic Medium

SYNOPSIS. Media have been developed for axenic cultivation of 10 strains belonging to 7 species of small marine ciliates. A medium containing cerophyl extract, proteose peptone, trypticase, yeast nucleic acid, biotin, calcium pantothenate folic acid, nicotinamide, pyridoxal HCl, riboflavin, thiamine HCl, and DL-thioctic acid in sea water supports the growth of Uronema nigricans strains Pc and 34/2, Parauronema virginiatum strains 2/1 and 19/1, Miamiensis avidus strains Ma and 19/3, Miamiensis sp. strain 1/1, a strain of "G" ciliate, and strains 33/8 and 34/7 of 2 unidentified species. By substituting a mixture of asolecithin, cephalin, and Tween 80 for cerophyl in the medium, luxurious growth of all except the strains of the 2 unidentified species can be obtained. A defined medium consisting of 18 amino acids, 5 purine derivatives, 8 vitamins, asolecithin, cephalin, and Tween 80 in synthetic sea water also has been developed for 6 of the strains: M. avidus Ma and 19/3, Miamiensis sp. 1/1, P. virginiatum 2/1 and 19/1, and U. nigricans Pc. In general the ciliates grow best at pH 7.2 in the dark at 27 C in media containing sea water of density = 1.015. Under these conditions maximum populations are reached in 4–5 days and range from several hundred thousand to 3 or 4 × 10⁶ depending upon the strain. Electronmicroscopic observations for the presence of endosymbiotes gave negative results.     

A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

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

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

Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport

Summary Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 μm). Short-circuit current (I_sc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed I_sc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 μm. Responses in I_sc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.     

Heterogeneic modulation of malignant behavior in human glioma cells in defined and serum-containing media

Summary Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG, D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However, glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth and invasion was heterogenously preserved in the defined media used. Depending on the assay to be used in the study of glioma cell behavior, the degree of medium supplementation has to be considered.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Chemically defined serum-free media for the cultivation of primary cells and their susceptibility to viruses

Summary Chemically defined media SFRE-199-1 for the growth and SFRE-199-2 for the maintenance of primary baboon kidney (Bak) cell cultures were formulated by supplementing medium M199 with insulin, sodium pyruvate, zinc sulfate, and increasing arginine-HCl, cysteine, cystine,l-glutamine,l-glutamic acid, glycine, histidine, tyrosine, and glucose to maximally active nontoxic concentrations. For prolonged maintenance of the cells, physiological pH control, and blocking of excessive lactic acid accumulation in the spent medium of the cell cultures, it was necessary to supplement the medium containing Earle's balanced salts withd-(+) galactose. The cells grew and were maintained equally well on glass or polystyrene surfaces. Selenium, when added to growth medium or substituted for insulin and zinc sulfate, did not stimulate cell growth. Electron microscopy showed that numerous dense particles, approximately 250 to 400 ? in diameter, with the appearance of glycogen, were found throughout the cytoplasm in the cells grown in SFRE-199-1 and maintained in SFRE-199-2. Echovirus types 1 to 3, poliovirus types 1 to 3, coxsackievirus types B2, B4, B5,Herpesvirus hominis type 1, simian herpesvirusH. simiae and SA8, and simian adenovirus SV34 when titrated in primary Bak cells and grown and maintained in SFRE-199-1 and 2, respectively, developed titers comparable to those obtained in conventionally grown and maintained cells. This study was supported in part by National Institute of Health Grant RR00361 and World Health Organization Grant V4/181/38. This laboratory serves as the NIH/WHO Collaborating Center for Reference and Research in Simian Viruses.