Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: Cloning efficiency,growth in suspension,and selection of drug-resistant mutant phenotypes

Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10⁻⁷ M linoleic acid, 1×10⁻⁸ M H₂SeO₃) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN and AO.     

Natural killer activity in man under different experimental conditions]

The cytotoxic activity of natural killer (NK) cells against labelled with 3H-uridine target cells of standard human erythromyeloblast line K562 in 63 healthy donors was studied in 14-hr cytotoxic test. The cytotoxic reaction was realized in complete medium supplemented with different types of serum such as foetal calf serum (FCS), autologous or homologous sera in different schemes of incubation. It has been shown that NK cell activity was augmented by 50 per cent (p < 0.05) in the presence of homologous serum added to mononuclear cell suspension for 1 hr at 37 degrees C in comparison with the effect of FCS presence. Thus, the serum of healthy donors contains some factors which not only reflect an individual genotypic information of a donor, but also can be recognized by NK cells and significantly change the cytotoxic NK cell activity. The data obtained exclude possibility of using a homologous serum when positive controls for natural cytotoxic reactions are planned.     

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively; P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Effect of Arginine-deficient Media on the Herpes-Type Virus Associated with Cultured Burkitt Tumor Cells

After incubation at 37 C for 2 or more days, Basal Medium Eagle (BME) supplemented with 25% fetal calf serum (FCS) was not able to support adequate growth of EB3 and other lines of Burkitt tumor cells. The medium did, however, increase, by a factor of about 10, the number of cells synthesizing herpes-type virus with which the cultures were persistently infected. Not every lot of FCS produced these effects, nor were these effects seen when BME and FCS were incubated separately for 7 days before the medium was completed. At 37 C, appropriate lots of FCS interacted with several of the amino acids present in BME; this interaction resulted in an inhibition of cellular growth, whereas interaction with arginine yielded the virus-enhancing effect. Arginine-free BME, supplemented with 25% FCS and used without prior incubation, prevented cellular replication and promoted viral infection to a similar extent as did preincubated complete medium. Replenishment of arginine reduced, but did not regularly abolish, the virus-enhancing activity of preincubated media. RPMI-1629 medium was less affected by preincubation with FCS because it contained twice the amount of arginine that BME contained. The FCS factors which act upon arginine and other amino acids are not dialyzable and are partially resistant to heating at 56 or 60 C for 30 to 60 min. Calf and horse sera appear to be devoid of these activities. The nature of these interactions, as well as the mechanism by which arginine deficiency enhanced the viral infection, remains to be ascertained.     

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.     

Interaction of cultured mammalian cells with WR-2721 and its thiol, WR-1065: implications for mechanisms of radioprotection

An isothermal microcalorimeter was used to measure changes in heat flow when radioprotective drugs were added to cultured mammalian cells. The heat produced when WR-2721 was added continued for at least 90 min. WR-2721 was dephosphorylated by the cells to thiol (WR-1065) which oxidizes to disulphide. In the microcalorimeter, thiols give an immediate burst of heat due to this oxidation. A biological oxygen monitor revealed that WR-1065 and cysteamine rapidly consumed all the oxygen in culture medium. (10 mM WR-1065 deoxygenated medium in 2 min.) Rapid consumption of oxygen by radioprotective thiols indicates that they will not co-exist with oxygen for long in cells. This has two important implications with respect to mechanisms of radioprotection: (1) oxygen in tissues will be consumed rapidly and could result in local hypoxia; and, (2) at modest doses of protective agents the thiol will be consumed in oxic cells and hence very little will be available for reactions such as hydrogen donation. Our results indicate that anoxia is probably the principal mechanism of protection by aminothiols in mammals and aerated cells. This has major implications for clinical applications of radioprotectors and these are discussed.     

The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes

The influence of estrous cow serum (ECS) or fetal calf serum (FCS) and their interaction with gonadotropins on in vitro bovine maturation and fertilization was evaluated. The addition of ECS or FCS to the medium significantly increased the percentage of oocyte maturation over that of Ham's F-10 medium alone (P<0.05). The addition of follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) or of FSH plus hCG to the medium provided no advantage in maturation over serum alone. However, FSH plus HCG added to the maturation medium significantly improved the frequency of pronucleus formation in both groups compared with a medium to which no gonadotropins were added (P<0.05). Both ECS and FCS (as medium supplements) promoted oocyte maturation. Although the addition of a combination of FSH plus hCG to the medium did not increase the rate of oocyte maturation, it did contribute to the high incidence of subsequent pronucleus formation.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes.

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture

Small preantral follicles (40 to 90 microns in diameter) from domestic cats were cultured for 10 d using different media (M199 and Dulbecco's MEM) and protein (FCS and BSA) supplements. Culture efficacy was determined by Hoechst 33258 staining and estimation of Brom-desoxyuridine (BrdU)-incorporation into oocytes and granulosa cells. Culture in M199 + FCS and in DMEM + FCS resulted in 21.6% and 38.1%, respectively, of morphologically intact preantral follicles. Adding BSA increased the rate of normal follicles to 51.7% in M199 and to 58.6% in DMEM. Oocytes were found in 40% of the follicles, when DMEM and/or BSA supplementation was used, while M199 with FCS induced acute loss of oocytes in 85% of the follicles. About 10% of the oocytes contained degenerating chromatin. Measurement of BrdU-incorporation during culture allows for quick and effective assessment of follicle viability in vitro. Comparison of M199 and Dulbecco's MEM, both with FCS or BSA and DMEM with or without pyruvate/lactate, indicated that Dulbecco's MEM + BSA without pyruvate and lactate is the best medium for culture of cat follicles. However, further research of suitable medium supplements is needed.     

Lambing rate using vitrified blastocysts is improved by culture with BSA and hyaluronan

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.     

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 °C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.