Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Effect of Fetal Calf Serum Fractions and Proteins on Division and Transformation of Trypanosoma (Schizotrypanum) cruzi in Vitro*

SYNOPSIS. Division and epimastigote-to-trypomastigote transformation of Trypanosoma cruzi were observed in O'Daly's SM medium supplemented, in place of whole fetal calf serum, with fractions of this serum, its partially purified proteins, or with mixtures of these fractions and proteins. In addition to their division-promoting effects, most but not all serum fractions stimulated [³H]thymidine uptake by the flagellates. As revealed by TEAE-cellulose column chromatography and immunoelectrophoresis, the serum fractions were altered during the logarithmic growth phase of the trypanosomes.     

Effect of steroid hormones on vaginal epithelial cells: Anin vitro model for steroid hormone action

Conditions have been standardized to maintain rat vaginal epithelial cellsin vitro with more than 95% viability. Cultured epithelial cells were used to study the effects of normal fetal calf scrum, estradiol and progesterone on the incorporation of [³H]-uridine in RNA and incorporation of [¹⁴C]-aminoacids in proteins. While fetal calf serum and estradiol stimulate the incorporation of both uridine and afno acids, progesterone did not show any effect. Estradiol treated vaginal cells show typical fcroridges (indicative of keratinization of cells) in contrast to estradiol deprived cells, which show microvilli on cell surface when examined in scanning electron microscope.     

A New Liquid Medium for Trypanosoma (Schizotrypanum) cruzi*

A new liquid medium, with fetal calf serum as the sole undefined component, was devised for the cultivation of Trypanosoma cruzi. The need for the serum is ascribed to its mitogenic proteins, which stimulate division of, and the uptake of [³H]thymidine by the parasites. In the new medium, T. cruzi has a cycle culminating in the appearance of up to 90% metacyclic forms in the stationary phase. This cycle is repeated on each serial transfer.     

An Improved Chemical Substitute for Fetal Calf Serum for the Micronucleus Test

The routine use of the micronucleus test in the mutagenicity evaluation of xe-nobiotics is limited by high cost and limited availability of fetal calf serum. On the other hand, there are disadvantages, such as hypotonic damage and clumping of cells, associated with the use of mineral medium substitutes for fetal calf serum. Alternatively, we recommend a chemical medium containing Hanks' buffered salt solution, 1% (w/v) bovine serum albumin, and 0.15% (w/v) ethylenediaminetetraacetic acid, final pH 7.4, to preserve morphology, density and homogeneity of bone marrow cells. Mast cell granules are efficiently removed from rat bone marrow preparations by washing twice with this medium. The morphological preservation of cells is further enhanced by fixation with 70 % (v/v) ethanol for 5 min. The proposed medium provides a cost-effective and convenient substitute for fetal calf serum with substantially improved quality of bone marrow preparations for the micronucleus test.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

Tri-iodothyronine (T3) stimulates the growth of Morris Hepatoma cells grown in culture

Cell cultures prepared from transplanted Morris Hepatoma #44 and host liver were grown in media containing 10% fetal calf serum depleted of iodothyronines. Addition of 200 nM triiodothyronine significantly stimulated the rate of cell replication and thymidine kinase activity of hepatoma cells. The responses of adult liver cells were similar but less marked. This study documents for the first time that the growth of hepatoma cells in vitro is thyroid dependent.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Release of low density lipoprotein receptors from human fibroblasts or HeLa cells by tryptic digestion.

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with $\text{6-[}{}^3\text{H]-glucosamine·HCl}$ and [${}^3\text{H}$]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.     

The regulation of cell growth: II. Some characteristics of a fetal calf serum factor (FF2) stimulating ornithine decarboxylase in mouse liver

A heat stable, non-dialysable fetal calf serum factor (FF₂), capable of stimulating ornithine decarboxylase in mouse liver, kidney and spleen, has been detected in fetal calf serum and commercial preparations of 81% pure fetuin.The factor has a molecular weight of approx. 17 500, contains sulfhydryl groups necessary for its activity, and is protease resistant.Stimulation of hepatic ornithine decarboxylase by the fetal calf serum factor is dose and time dependent and is blocked by both cycloheximide and by actinomycin D, if the latter is administered within 1 h of the factor. Theophylline enhances the effect of the fetal calf serum factor on ornithine decarboxylase in the liver and the factor stimulate ornithine decarboxylase in hypophysectomized and adrenalectomized rats.     

Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts.

NCTC 2071 cells, transformed mouse fibroblasts, did not respond to choleragen when grown in chemically defined medium. When grown in medium containing 10 percent fetal calf serum, however, the cells accumulated cyclic AMP upon exposure to the toxin. Gangliosides isolated from the fetal calf serum were as effective as whole serum in inducing choleragen responsiveness in the cells. The putative choleragen receptor, the monosialo-ganglioside GM1, could not be detected by chemical analysis in cells exposed to serum. 3H-Labeled GM1 was detected in these cells, however, following sequential exposure to galactose oxidase and sodium borotritide. Thus, uptake of minute amounts of GM1 from serum by these cells sensitized them to choleragen.     

Conditioned medium enhances neuritic outgrowth from rat spinal cord explants

Medium conditioned by primary cultures of fetal or neonatal rat skeletal muscle, fibroblasts, or lung cells dramatically increases the neuritic outgrowth from spinal cord explants. After 7 days in vitro, the outgrowth of neurites from 15- to 16-day fetal rat spinal cord slices grown in conditioned medium (CM) covers a 3- to 4-fold greater area than that from slices grown in fresh, nonconditioned (control) medium. Moreover, the pattern of neuritic outgrowth is markedly different in CM-treated slices. In control slices, the neurites form a tangled, dense network of neurites which usually extend only a small distance from the slice edge, while in CM-treated slices, the neurites form a more open network, with the majority of neurites extending radially for long distances (up to several millimeters) from the slice edge. The effect of CM on neuritic outgrowth is not due to a detoxification or modification of the serum in the medium, because increased neuritic outgrowth was observed in slices grown in medium conditioned in the presence or absence of 10% fetal calf serum. The outgrowth-enhancing factor(s) in CM has a high molecular weight, since all outgrowth-enhancing activity is retained by membrane filters with a nominal molecular weight cutoff of 10⁵ daltons. This factor(s) is stable at 58°C for 30 min, and does not appear to be βNGF or fibronectin.     

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells

Background aimsAs angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells.MethodsThe usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs).ResultsExpression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities.ConclusionsThe use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs.     

Serum‐free medium with osteogenic supplements induces adipogenesis in rat bone marrow stromal cells

Adult BMSCs (bone marrow stromal cells) contain MSCs (mesenchymal stem cells). MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and myoblasts and are thus considered useful in tissue engineering for therapeutic and clinical purposes. FCS (fetal calf serum) is usually included in the differentiation medium, but the clinical application of FCS may pose a problem for some patients. To improve the efficiency and safety of BMSC cultivation, the effect of SF (serum‐free) conditions on the osteogenic differentiation of rat BMSCs was examined. In the presence of 10% FCS and osteogenic supplements, the cells formed mineralized von Kossa‐positive deposits. Under SF conditions with osteogenic supplementation, however, the cells possessed cytosolic lipid vacuoles that could be stained with Oil Red O. The mRNA expression of PPARγ (peroxisome proliferator‐activated receptor γ), an adipogenic marker, increased under the SF condition with osteogenic supplements. These data indicate that rat BMSCs differentiate into adipocytes under SF conditions even with osteogenic supplementation and that FCS is needed to induce proper osteogenic differentiation in rat BMSCs.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other     

High-density lipoproteins decrease both binding of a polynuclear aromatic hydrocarbon carcinogen to DNA and carcinogen-initiated cell transformation

Lipophilic carcinogens partition into the three major classes of lipoproteins potentially present in serum used as a medium supplement for cell culture. Different serum lots sequester differing quantities of the procarcinogen benzo[a]pyrene, dependent on the serum lipoprotein concentrations. In the presence of high-density lipoproteins a mutagenic benzo[a]pyrene metabolite was bound to cellular DNA at decreased levels when compared to cells exposed to the mutagen in the absence of high-density lipoproteins. Fetal calf serum with low levels of lipoproteins, specifically, high-density lipoproteins, is associated with efficient methylcholanthrene-initiated transformation of C3H/10T1/2 cells, while calf serum with a significant concentration of high-density lipoproteins requires up to a 500% increase in methylcholanthrene concentration to achieve similar levels of transformation in this mouse embryo cell line. When concentrated serum lipoproteins or purified HDL were added to fetal calf serum containing MCA at μg/ ml, the C3H/10T1/2 transformation frequency was decreased compared to the transformation frequency achieved in the presence of 1 μg/ml of MCA in fetal calf serum without supplementation. The results suggest that high-density lipoprotein partitioning of lipophilic polynuclear aromatic hydrocarbon mutagens from the cell culture medium may effectively reduce the concentration of carcinogen available for interaction with cellular DNA in vitro, which, in turn, may be associated with decreased carcinogen-induced transformation of cells.     

Uptake of fetal proteins by Trypanosoma cruzi immunofluorescence and ultrastructural studies

Isolated proteins from fetal calf serum needed for trypanosomal growth were labelled with I¹²⁵, colloidal gold and fluorochrome tagged specific antibodies. The proteins were localized in the membranes and cytoplasm of parasites cutured in vitro in a defined liquid medium.     

Evaluation of storage conditions for refrigerated rabbit skin

An evaluation of various refrigerated (4 °C) storage solutions and conditions was conducted using rabbit skin. Two in vitro methods to assay skin viability are presented: one which directly measures basal cell viability and one which assesses the skin's ability to grow in culture following storage. The superiority of storage in nutrient medium supplemented with fetal bovine serum over conventional storage in saline is clearly demonstrated. Storage in nutrient medium with 10% fetal calf serum resulted in basal cell viabilities which were over 30% higher than viabilities of skin stored by conventional methods in saline. Skin stored in saline failed to grow in culture, while 100% of the cultures of skin stored in medium plus fetal calf serum grew. Although addition of fetal calf serum to the saline improved the basal cell viability, growth in culture occurred only when the skin was stored in a capped tube. Skin stored in medium without serum gave viability results which were not significantly different from the unstored control, but growth rates in culture did differ significantly from the control values. Our study shows that the viability of rabbit skin and its ability to grow in vitro are depressed when the tissue is maintained at 4 °C in saline or in petri dishes, and optimal when refrigerated in nutrient medium supplemented with FBS in a sealed tube.