Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos.

When dissociated cells of neural retinae of 9-day-old chick embryos were cultured in Eagle's minimum essential medium supplemented with dialyzed fetal calf serum, both the proliferation and differentiation of the neural retinal cells were inhibited. These cells remained quiescent and flattened. When ascorbic acid was added to such a medium, the cells started to grow and differentiated into lentoid bodies and pigmented cells after about 10 days.     

Novel method for the establishment of cardiomyocytes derived from rat embryonic stem cells in vitro

Cardiomyocytes were differentiated from embryonic stem cells (ES cells) derived from spontaneous dwarf rats (SDR) in vitro. The two-cell stage embryos were cultured in alpha-MEM supplemented with 10% fetal calf serum and embryotrophic factors (ETF). ETF were isolated from the conditioned medium of the SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. When two-cell stage rat embryos developed into tri-laminal germ disc embryos (flat type), colonies composed of small round cells were isolated by the colonial isolation method and used to establish an ES cell line. The ES cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum and 1 ng/mL of leukemia inhibitory factor. Embryoid bodies were made by the hanging-drop method using 1 x 10(7) ES cells/mL. The embryoid bodies differentiated and grew to form an embryonic monster in ETF-supplemented medium using Rose's circumfusion apparatus for about 1 month. The anlages of beating hearts in embryonic monsters were collected using a glass capillary. The anlages were cut into small pieces using razor blades and dissociated with trypsin-EDTA/PBS(-) solution. The resultant single cells were cultured in growth medium and used to establish a myocardial cell line. The cell line was subcultured for more than 25 passages and confirmed as showing the morphological and ultrastructural characteristics of cardiomyocytes.     

Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.     

The differentiation of L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of culture conditions

We report here that it is possible to induce differentiation in a subline of L5 myoblast line (L5/A10) by manipulating the culture media. When L5/A10 myoblast are cultured in F14 supplemented with 10% fetal calf serum the cells grow with a division time of 12 h and reach confluency at a cell density of approximately 2.4 X 10(5) cells per cm2, without undergoing differentiation, characterized, morphologically, by formation of multinucleated fibers, and biochemically, by the synthesis of muscle specific proteins such as creatine phosphokinase or myokinase. However, cells, grown in F14 + 10% fetal calf serum, will undergo regular differentiation after a limited number of division when transferred to F14 medium supplemented with limiting concentrations (1-2%) of fetal calf serum. Investigations of the biochemistry of myoblast differentiation in cell culture will be facilitated by the availability of a cell line that can undergo differentiation under controlled conditions.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39.

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.     

Swine serum increases hybridoma stability

Three hybridoma cell lines producing monoclonal antibodies to lambda phage were cultured for 9 months in Dulbecco's modified Eagl's medium supplemented with 10% fetal calf serum or with 5% swine serum. Under these conditions all the clones had the same proliferation rates and maximum population densities. The cultuvation in the medium with swine serum enhanced the stability of hybridomas: all clones retained the ability to produce antibodies throughout the experiment; one of the clones examined enhanced the activity by 10(3)-10(4) times. When the same three clones were cultured in the medium with the fetal calf serum, two of them lost their ability to produce antibodies 3-15 weeks after the beginning of the experiment.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture

The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20–21, 23–24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 μg/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20–21, 23–24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20–21 or 23–24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.     

Altered formation of DNA replication intermediates in cells growing in different culture conditions

In human melanoma cells one can detect two discrete DNA replication intermediates, 10-kb DNA intermediates and Okakzaki fragments. Both intermediates are seen when cells are rapidly growing in medium supplemented with fetal calf serum. When the medium is supplemented with newborn calf serum, one can detect Okakzaki fragments but not 10-kb DNA intermediates. In contrast we do not detect changes in the replicon sizes in the two media.     

Fibronectin is not a serum spreading factor for HeLa cells

The spreading of HeLa cells on plastic substratum is mediated by fibronectin-depleted foetal calf serum but not by fibronectin isolated by gelatin-Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37 degrees C in fibronectin-supplemented (10 micrograms ml-1) serum-free medium. Long-term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin-free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.     

In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [³H]serine and [¹⁴C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell.     

Growth of Myeloma MPC-11 Cells in Serum-free Growth Factor Supplemented Medium

A myeloma MPC-11 cell line, which originated in a mouse plasma cell tumor, was able to proliferate in the absence of serum in a synthetic medium supplemented with transferrin, ethanolamine and selenium. The cells showed almost the same growth rate in this medium as in medium with 10% fetal calf serum added. Immunoglobulins secreted into the serum-free medium by the cells were easily separated from medium components by polyacrylamide gel electrophoresis.     

18-hydroxylation in the Y-1 adrenal cell line: response to ACTH and to culture conditions.

The 18-hydroxylation of deoxycorticosterone in the Y-1 adrenal cell line was studied under various incubation and cell culture conditions and compared to 11 beta-hydroxylation. Repeated incubation of the substrate increased both 18- and 11 beta-hydroxylation in the Y-1 cells. Furthermore, both 18- and 11 beta-hydroxylation were increased with increased serum concentration and prolonged incubation time. While the increase in 11 beta-hydroxylation seemed to be independent of the type of serum, 18-hydroxylation was much more important in cells cultured in fetal or newborn calf serum supplemented medium than in those cultured in horse serum supplemented medium. As expected, ACTH treatment increased 11 beta-hydroxylation; however, it decreased 18-hydroxylation. The different regulation of these two hydroxylating pathways by ACTH, point to a heterogeneity of the cytochrome P-450(11) beta of the Y-1 cell line.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Stimulation of Ornithine Decarboxylase Activity in Neural Cell Culture: Potential Role of Insulin

Abstract: Age-dependent decreases in the levels of ornithine decarboxylase activity were observed in the optic lobes, cerebral hemispheres, and midbrain-diencephalon of 6–17-day-old chick embryos. In dissociated cell cultures from chick embryonic brains a similar pattern of declining ornithine decarboxylase activity with time in culture was observed. Ornithine decarboxylase activity in the dissociated brain cell cultures was stimulated by changing the culture medium. The peak stimulatory effect was shown to occur 12 h after changing the medium. Although serum-free medium stimulated ornithine decarboxylase activity slightly, the presence of serum in the medium was the primary stimulatory factor. Both fetal calf serum and heat-inactivated fetal calf serum produced dose-dependent stimulation of ornithine decarboxylase activity. Dialyzed fetal calf sera stimulated ornithine decarboxylase, but to a lower level than that produced by nondialyzed sera. Insulin (0.5–10 μg/ml) stimulated ornithine decarboxylase activity in a dose-dependent manner in serum free medium. In addition, 10² M-L-asparagine stimulated ornithine decarboxylase activity in serum-free medium.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

Nutrient deprivation increases vulnerability of endothelial cells to proinflammatory insults

Nutrient deprivation is a stimulus for oxidative stress and is an established method for induction of cell autophagy and apoptosis. The aims of this study were to identify conditions that evoke superoxide production in cultured human umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 medium under conditions of serum starvation (serum-free M199 medium), low serum (medium containing 2% fetal calf serum), and high serum (medium containing 20% fetal calf serum). HUVECs were also incubated under proinflammatory conditions, in medium supplemented with 50 ng/ml tumor necrosis factor-α (TNF-α) or neutrophils preactivated with 10 nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased fourfold in serum-starved HUVECs compared to cells incubated in 20% medium, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca²⁺ uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% medium compared to 2% medium alone, but unchanged with TNF-α in 20% medium. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low-serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation and suggest that proinflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.