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.     

Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.     

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     

Development and differentiation of mouse blastocysts in serum-free medium

Numerous studies have shown that the in vitro development and differentiation of mouse blastocysts require serum, but the number and nature of serum factors involved remains unclear. In this article, we describe a culture medium, EM-2, containing as a source of protein only commercially purified bovine serum albumin (BSA) and fetuin. This medium supports hatching, attachment and outgrowth of mouse blastocysts. Although attachment and outgrowth are delayed in EM-2 medium 12–15 and 5–8 h, respectively, these events occur at frequencies comparable to those observed in serum-containing media. Trophoblast cells are capable of differentiating in this medium: they synthesize Δ⁵,3β-hydroxysteroid dehydrogenase and their nuclei become polyploid. The inner cell mass also appears to differentiate to some extent in EM-2 medium as evidenced by the appearance of cells with characteristics of parietal endoderm. The fetuin factor is necessary at least for trophoblast outgrowth and the albumin factor is required for the survival and/or growth of the inner cell mass. It is, however, not evident from these studies whether the serum fractions used are actually involved in the induction of differentiation, or whether the early differentiative steps in the mouse blastocyst are preprogrammed and require for expression only a normal cellular metabolic rate.     

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.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.     

Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium

Isolated hepatocytes from adult rats were cultured for 3 days in a serum-free synthetic medium. Supplementation with fibrinogen digests, glucagon and insulin remarkably increased DNA synthesis in hepatocytes. DNA synthesis began to increase at 35 h and reached a maximum at 41 to 54 h after plating. At this time, cells were morphologically identifiable as hepatocytes. Glucagon could be replaced by dibutyryl cyclic AMP or isobutyl-methyl-xanthine. Addition of amiloride (a Na⁺ influx inhibitor) during the initial 22 h completely inhibited DNA synthesis. These results suggest that influx of Na⁺ during early prereplicative period and increase in cellular cyclic AMP levels during late prereplicative period are necessary for the induction of DNA synthesis in hepatocytes.     

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.     

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.     

Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium

Cell culture with serum-containing medium has potential problems associated with contamination of infectious agents. This study demonstrates for the first time the feasibility of regenerating cartilage tissues in vivo by implantation of chondrocytes cultured in vitro in a chemically-defined, serum-free medium. Chondrocytes cultured in the serum-free medium grew similarly to those in a serum-containing medium. Implantation of chondrocytes cultured in the serum-free medium and seeded on to polymer scaffolds resulted in the regeneration of cartilage tissues with histological aspects similar to those of cartilage tissues regenerated from chondrocytes cultured in serum-containing medium.     

Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagle's MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.     

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 and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Degranulation and cytokine expression in human cord blood-derived mast cells cultured in serum-free medium with recombinant human stem cell factor

Human cord blood-derived mast cells (HCMC) grown in medium with serum and recombinant human stem cell factor (rhSCF) with or without interleukin (IL)-6 are less mature than human skin mast cells (HSMC). We found that c-kit-positive HCMC cultured for 8-10 weeks with rhSCF in serum-free medium became sensitive to basic secretatogues and expressed the serine protease, chymase, which is preferentially expressed in HSMC. The HCMC release beta-hexosaminidase (beta-HEX) within 1 min of stimulation with compound 48/80 or substance P, and release was suppressed by pertussis toxin. Approximately 34% of the HCMC in the serum-free culture stained positively with chymase antibody. Chymase and c-kit levels, and responsiveness to basic secretagogues, increased substantially after an additional 2 weeks in a serum-free environment with rhIL-6 and rhSCF. Moreover, Fc(epsilon)RI-dependent activation of the HCMC resulted in induction of cytokines and cyclooxygenase-2. These results show that HCMC can differentiate into a phenotype morphologically and functionally similar to HSMC if exposed to SCF in serum-free medium.