Rainbow trout leukocyte culture: A simplified method

Summary The addition of 10% fetal bovine serum to Leibovitz’s L-15 culture medium resulted in marked growth of peripheral blood leukocytes from rainbow trout,Salmo gairdneri. Culture medium without serum or with 20% homologous serum did not induce substantial growth. In contrast to what has been reported by others, oxygenation of the culture medium was found not to be required for excellent cell growth.     

Evaluation of medium supplements for in vitro cultivation of Wuchereria bancrofti

Third-stage larvae (L3) of Wuchereria bancrofti molt to the fourth stage in an in vitro culture medium composed of NCTC 135 and Iscove's modified Dulbecco's medium (1:1; v/v) supplemented with 10% human serum and a mixture of anti-bacterial and anti-mycotic agents. In the present investigation this culture medium was used to examine the effects of different concentrations of human serum, medium supplements, and serum replacements on larval growth, development, and molting. Several medium supplements and serum replacements were evaluated including hemin, Nutridoma, and a mixture of soybean lipids, bovine serum albumin, and transferrin. The supplements tested could not support larval growth and development in the absence of serum and they did not have an enhancing effect on larval growth and development in combination with human serum. A medium supplement of 30% human serum resulted in molting of 80-94% of L3s and optimum growth to the mid to late fourth stage. This culture system provides an excellent alternative to experimentally infected animals as a source of larvae undergoing the third molt and fourth-stage larvae for screening potential anti-filarial compounds and for immunologic and biochemical studies.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Growth of mouse fibroblast-like cells in an autoclavable medium

A relatively simple, autoclavable medium was developed that would support the growth of the mouse fibroblast (MFL) cell line in suspension culture. This medium was prepared in three stages with decreasing quantities of serum. As the serum was reduced from 5% to none, the amount of Bacto-Peptone was increased from none to 0.5%. The reduction and finally elimination of serum did not affect proliferation adversely, but actually seemed to produce more rapid growth and higher levels of cell population.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture,clonal growth in low density culture,and stimulation to enter S phase in resting culture

Summary A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum.

In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy. However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium. This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate. The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats. Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos. Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations. At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation. When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis. Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures. There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs. 50%). Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations. By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.     

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells

17 beta-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degree of estrogenic action in endocrine-dependent breast cancer cell lines.     

Liposomes replace serum for cultivation of fermenting mycoplasmas

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Evaluation of storage conditions for refrigerated rabbit skin

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

适合棉铃虫细胞HzAm1生长的培养基筛选及低血清驯化

昆虫细胞-杆状病毒系统是昆虫杀虫剂生产和医用外源基因表达的有效工具。昆虫细胞的无血清或低血清培养是十分必要的。从三种商业化的培养基TC-100、GRACE和IPL-41中筛选出了最适合棉铃虫细胞HzAm1生长的基础培养基TC-100。以该培养基为基础,将血清用量从常用的10%降至1%,同时补加一定量的水解乳蛋白以及酵母提取物等,对棉铃虫细胞HzAm1进行驯化培养,效果良好。     

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Cell shape and growth regulation in skeletal muscle: Exogenous versus endogenous factors

Passive stretch (10–12%) of tissue-cultured avian skeletal myotubes in serum-containing medium stimulates myotube growth in a manner analogous to hormonal stimulation of adult muscle. The resulting increase in myotube length is accompanied by marked reduction in the number of surface microvilli seen by scanning electron microscopy. We investigated the possible involvement of exogenous growth factors in the transduction of stretch-induced alterations in cell shape into the concurring biochemical changes that are associated with cell growth. We show that the acute stimulation of myotube amino acid transport and protein synthesis by stretch are independent of serum growth factors in the culture medium by evidence obtained from serum dose-response experiments. The myotubes synthesize and secrete high molecular weight factors into their culture medium, which regulates myotube amino acid transport and protein synthesis. Stretch of the myotubes did not alter the appearance of these factors in the culture medium. The initial growth-related biochemical alterations induced by myotube stretch in vitro thus depend only on events intrinsic to the cells. However, subsequent stretch-induced growth of the myotubes occurs only in serum-containing medium. There are both serum-independent and serum-dependent steps in the transduction of the stretch stimulus into myotube growth.     

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However,in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.     

Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.