Cell cycle progression delay in conditioned medium does not play a role in the repair of X-ray damage in Chinese hamster V79 cells

We tested our hypothesis that the lower survival of X-irradiated cells in growth medium (GM) relative to that in conditioned medium (CM) is due to differences in nutrient concentration levels rather than to differential effects on cell progression and growth. Chinese hamster V79 cells in log and unfed plateau phase, grown in Eagle's minimal essential medium (MEM) with 15% serum (100% GM), were irradiated. Before plating, cells were incubated in situ in various concentrations of MEM with serum (GM, normal cell progression) or MEM without serum or in CM (no cell progression). Cell survival was the lowest in 100% MEM with or without serum and increased with the decrease in MEM and serum concentrations, reaching a plateau in 40% MEM or 40% growth medium (40% MEM with 6% serum), similar to that in conditioned medium. Growth kinetics was the same in 40 and 100% growth medium, but the D0 of cells in 40% growth medium was higher than that of cells in 100% GM. Similarly, the D0 of cells in 40% MEM was higher than that of cells in 100% MEM, although cell progression was absent in both media. The radiation sensitivity of cells was the same in 40% GM with progression and in 40% MEM and CM with no progression. Cells in low-nutrient media were flatter than those in 100% MEM or GM. There was a correlation between the nutrient concentration in the medium postirradiation and the D0. This correlation was independent of the presence or absence of serum and thus independent of cell cycle progression. The cell morphology which is dependent on the nutrient concentration appears to influence the ability of a fraction of cells to repair their radiation damage.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth

Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS.
In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Action mechanisms of physiological doses of androgen or pharmacological doses of estrogen in growth stimulation of Shionogi carcinoma 115 in mice

Shionogi carcinoma 115 (SC115) had been accepted for 20 yr as an androgen-dependent mouse mammary tumor. However, we recently found that the growth of SC115 tumors in vivo is also stimulated by pharmacological doses of estrogen through estrogen receptor. In the present study, action mechanisms of androgen or high doses of estrogen in the growth stimulation of SC115 were examined using a cloned cell line (SC-3) derived from the SC115 tumor. In serum-supplemented [2% steroid-free fetal calf serum-Eagle's minimum essential medium (MEM)] and serum-free [HAM F-12: MEM (1:1, v/v) containing 0.1% bovine serum albumin] media, testosterone (Test, 10(-9)-10(-6) M) significantly increased both cell number and DNA synthesis of SC-3 cells (by up to 10-fold), whereas oestradiol-17 beta (10(-12)-10(-6) M) had no such effects; the Test-induced growth was completely inhibited by the addition of a 100-fold molar excess of cyproterone acetate (CA). The serum-free medium cultured with SC-3 cells in the presence or absence of 10(-8) M Test was collected [conditioned medium (CM) or conditioned medium without Test (CM-)], and then Test in CM was removed by Gel filtration using Sephadex G-100 or inactivated by the addition of a 100-fold molar excess of CA. In the serum-free culture system, the addition of the CM without Test activity significantly enhanced both number of SC-3 cells and DNA synthesis in the cells, whereas CM(-) had no such effects. The present findings suggest that growth-stimulatory activities of androgen and high doses of estrogen on SC115 cells are mediated by growth factor(s), secreted from SC115 cells through androgen receptor and from some of nontransformed cells through estrogen receptor, respectively.     

Effects of serine and glycine on proliferation of an Ishikawa cell variant

The proliferation of cells on an Ishikawa human endometrial adenocarcinoma line variant (Ishikawa-Var I) is markedly influenced by the medium used to culture them, viz. MEM vs BM (basal medium; DMEM/Ham's F12, 1/1, with additional glutamine and HETES), under serum-free conditions. Components of BM which are not present in MEM were systematically tested in order to identify those that might account for these differences. Cells were cultured for various periods of time, up to 8 days, in serum-free MEM to which the components to be tested were added. Cell population densities were evaluated using a fluorometric DNA assay when the cells were grown in multiwell plates, or by cell counting when the cells were cultured in plastic dishes. It was found that addition to MEM of a mixture of the amino acids that this medium lacks, significantly increased cell density. By testing individual amino acids at the concentrations present in BM, it could be demonstrated that addition of serine alone was sufficient to obtain the densities achieved with BM. Glycine, a metabolic precursor of serine, had a similar but smaller effect. None of the other missing compounds of BM was effective. Effects of serine on DNA synthesis were also estimated by measuring incorporation of [3H]thymidine for 1 h after a 24 h culture period in MEM. The effect of serine was similar and additive to that of 1% charcoal-treated fetal bovine serum. A serine concentration dependence studied either with this method or measuring DNA/well after 8 days in culture showed detectable effects at 0.005 mM concentration and maximal responses at about 0.025 mM. These findings are of potential importance in studies on regulatory mechanisms of cell proliferation. A possibility to be explored, for instance, is that serine added to the medium increases intracellular phosphatidylserine concentrations leading to increases in the activity of protein kinase C, a stimulator of cell proliferation in some systems.     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO Chinese hamster ovary - hGH human growth hormone - dFCS dialyzed fetal calf serum - dhfr dihydroforate reductase - MTX methotrexate     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (delta crystallin) during so-called 'transdifferentiation' in these cultures. MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive delta crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; delta crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F-12 cultures. Medium 199 also blocks delta crystallin accumulation. The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of delta crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.     

The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium.

The growth of L-60TM cells (a suspension culture adapted L-cell) on media composed of MEM (minimum essential medium (Eagle)) and bactopeptone autoclaved together or separately under a variety of conditions has veen determined. It has been found that MEM autoclaved with 0.5% bactopeptone at 15 psi for 20 min, cooled and then neutralized with NaHCO3, consistently supported good cell growth of L-60TM and L-929 cells. Similar results were obtained when the MEM and bactopeptone were autoclaved separately. The cells grew initially as a monolayer, subsequently becoming a stationary suspension. Some experiments were carried out with agitated suspension culture of L-60TM cells in the autoclaved MEM-bactopeptone combination with and without added methylcellulose and results were obtained which indicate that large scale suspension culture is possible in this system. Other peptones were also found to support cell growth. The autoclaved MEM-bactopeptone combination also supported the growth of Chang liver and Vero cells. The Chang liver cells rapidly dissociated from the plastic surface but the Vero cells remained sufficiently securely attached so that it was possible to grow them near to confluency in roller bottles.     

Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.     

Identification of suitable culture condition for expansion and osteogenic differentiation of human bone marrow stem cells

This study was undertaken in order to identify the best culture strategy to expand and osteogenic differentiation of human bone marrow stem cells (hBMSCs) for subsequent bone tissue engineering. In this regard, the experiment was designed to evaluate whether it is feasible to bypass the expansion phase during hBMSCs differentiation towards osteogenic lineages by early induction, if not identification of suitable culture media for enhancement of hBMSCs expansion and osteogenic differentiation. It was found that introduction of osteogenic factors in alpha-minimum essential medium (??MEM) during expansion phase resulted in significant reduction of hBMSCs growth rate and osteogenic gene expressions. In an approach to identify suitable culture media, the growth and differentiation potential of hBMSCs were evaluated in ??MEM, F12:DMEM (1:1; FD), and FD with growth factors. It was found that ??MEM favors the expansion and osteogenic differentiation of hBMSCs compared to that in FD. However, supplementation of growth factors in FD, only during expansion phase, enhances the hBMSCs growth rate and significantly up-regulates the expression of CBFA-1 (the early markers of osteogenic differentiation) during expansion, and, other osteogenic genes at the end of induction compared to the cells in ??MEM and FD. These results suggested that the expansion and differentiation phase of the hBMSCs should be separately and carefully timed. For bone tissue engineering, supplementation of growth factors in FD only during the expansion phase was sufficient to promote hBMSCs expansion and differentiation, and preferably the most efficient culture condition.     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Abstract. Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (δ crystallin) during so-called 'transdifferentiation' in these cultures.
MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive δ crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; δ crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F–12 cultures. Medium 199 also blocks δ crystallin accumulation.
The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of δ crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.     

A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.     

Development of an animal‐component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h^?1 were achieved in IPT‐AFM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of insulin, thyroxine and oestradiol on growth of normal human diploid cells and human heteroploid HeLa cells

Insulin (5 micrograms/ml), thyroxine (1 microgram/ml) and beta-oestradiol (0.1 microgram/ml), if added separately to the medium, did not significantly influence the growth of normal human diploid LEP 19 cells cultivated in Eagle's MEM medium with 1% foetal calf serum. Combined, they did not influence the growth of LEP 19 cells in serum-free MEM medium either. Under the same cultivation conditions, insulin caused 37%, thyroxine 24% and a mixture of insulin, thyroxine and beta-oestradiol 325% (without serum) or 15% (with 1% foetal calf serum) stimulation of the growth of human heteroploid HeLa cells. Isolated beta-oestradiol significantly inhibited the growth of HeLa cells.     

Age dependent production of a competence factor by human fibroblasts

Several cell types such as Balb/c 3T3 have been shown to require platelet-derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin-C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12-wk embryo, newborn, and 3-yr-old were exposed to serum-free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3-yr-old cells with the addition of plasma alone, whereas conditioned medium from the 3-yr-old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3-yr-old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3-yr-old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age-dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.     

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).