Serum-free modified trichosel medium for the isolation ofTrichomonas vaginalis

A serum-free medium was developed for the isolation and cultivation ofTrichomonas veginalis. The basal medium was Trichosel fluid medium to which was added 20 g per liter of yeast extract, 2.5 ml per liter of whole milk, and 10 g per ml of cholesterol; the entire medium was then heat sterilized. This serum-free medium supported the growth and proliferation of small numbers of trichomonads and was successful for maintenance, serial passage, and clinical isolation of the organism from vaginal specimens.     

THE FAILURE OF PHENOL TREATED ESCHERICHIA COLI TO GROW ON MEMBRANE FILTERS

SUMMARY: Counts of Escherichia coli were done on nutrient agar (control), on membrane filters on nutrient agar and on membrane filters on filter paper pads. With untreated bacteria counts were similar under all conditions, though membrane filters on nutrient agar tended to give slightly low counts. Phenol treated bacteria gave much lower counts when membrane filters were used: the mean counts for 3 strains of the test organism with filters on nutrient agar varied from 35–65% of the control, while counts with filters on filter paper pads were somewhat lower, varying from 30–47% of the control. The low counts on membrane filters on filter paper pads were not due to adsorption of phenol by the filters or to a low concentration of nutrients in the growth medium.     

Cellular adhesion to collagen

BALB/3T3 cells were released from tissue culture plates with EGTA, and their rates of attachment to collagen gels polymerized on Millipore filters; were measured. Cell attachment in serum-free medium was 20–50% of that which occurred in medium containing 10% fetal calf serum (FCS). Cell attachment to gels pretreated with FCS and assayed in serum-free medium was identical with that of gels in FCS-containing medium. Thus, it seems there are two separate mechanisms of attachment to collagen; one involving direct attachment and a second mediated by a serum component(s) which binds to collagen.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

The embryolethality and teratogenicity of acrolein in cultured rat embryos

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.     

黄牛表皮细胞分离与体外培养最适条件的探索

为了建立黄牛表皮细胞分离与体外培养的最适条件,比较了组织块法与单细胞悬液法、不同蛋白酶(胰蛋白酶和分离酶)的消化以及有无血清培养基对细胞生长的影响,以克隆形成率来检测细胞生长和存活情况。结果证明：采用分离酶分离表皮细胞进行无血清培养是黄牛表皮细胞体外培养的最适条件。     

Isolation and Culture of Corn Stunt Spiroplasma in Serum-Free Medium

A serum-free medium (medium A₆), supports the primary isolation and cultivation in vitro of corn stunt spiroplasma (CSS). The growth characteristics of CSS in A₆ medium are similar to those in the conventional serum dependent, ketoglutarate sorbitol medium (SMK).     

胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.     

Development of a serum-free medium for growth of NS-1 mouse myeloma cells and its application to the isolation of NS-1 hybridomas

In this study, we have devised a hormone-supplemented, lipid-enriched serum-free medium, designated KSLM, that supports the growth of NS-1 mouse myeloma cells and have studied its applicability to the efficient isolation of antibody-producing hybridomas formed from the fusion of NS-1 myeloma cells and spleen cells from immunized mice. Our results show that KSLM medium, when used in conjunction with our hybridization protocol, allowed for the isolation, in a reproducible manner, of antibody-secreting hybridomas. Moreover, the yield of antibody-producing hybridomas was similar in KSLM medium and serum-supplemented medium. Here, we also report on the adaptation of NS-1 myeloma cells to growth in lipid-deficient KSLM medium. The use of the adapted myeloma cells (NS-1-503), instead of NS-1 myeloma cells, in fusion experiments not only permitted the isolation of antibody-secreting hybridomas in lipid-free KSLM medium but also resulted in a higher yield of antibody-producing hybridomas in both complete KSLM medium and serum-supplemented medium.     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable.Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast.Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×10⁸/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day.The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.     

Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.     

An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

Summary We have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum. This work was supported by the Japanese Ministry of Education, Science and Culture and in part by grants from the National Institutes of Health. Editor's Statement This article reports a creative technical application of the author's previous work on lipid metabolism in lymphoid cells allowing an efficient, alternative selection procedure for isolation of hybridomas.     

Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents

Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.     

Development of embryonic chick insulin cells in culture: Beneficial effects of serum-free medium, raised nutrients, and biomatrix

Summary A previous finding that insulin cells do not survive or differentiate in explants of embryonic avian pancreas cultured in collagen gel with a serum-containing medium has provided a model system for identification of conditions favorable for development of these cells. To this end, we here modify the substrate and the medium. The epithelial component of dorsal pancreatic buds of 5-d chick embryos was cultured for 7 d on Matrigel in serum-containing and in serum-free medium, the latter incorporating insulin, transferrin, and selenium, Endocrine cell types were distinguished by immunocytochemistry; insulin cell counts were expressed as a proportion of insulin plus glucagon cells. With serum-containing medium, Matrigel stimulated a significant increase in this proportion as compared with collagen gel—3.1% as against 0.2%; the serum-free medium further increased this proportion to 17.3%. Raising the level of essential amino acids approximately fivefold increased the latter figure somewhat (to 18.9%), but it was more than doubled (to 37.4%) by raising the glucose concentration from 10 mM to 20 mM. Raising the levels of amino acids and glucose simultaneously yielded a lesser increase (to 31.8%). Some cultures grown in collagen gel and serum-containing medium for 7 d were transferred to Matrigel and serum-free medium for a further 7 d. Insulin cell development recovered, indicating that progenitor cells had survived and were stimulated to develop by the improved conditions. This study indicates that components of the biomatrix and the medium (in particular, a raised glucose concentration) are important for the survival and differentiation of embryonic insulin cells.     

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm² by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm². With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.     

Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes.

Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.     

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions

Summary We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.     

Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture

We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.     

Retinoic acid can change normal differentiation of rat egg-cylinders cultured in vitro

The modified organ culture of rat egg cylinders provides favorable conditions for 2 weeks for the differentiation of main tissue types. To study the effect of retinoids on early rodent differentiation, retinoic acid (RA) was added in various concentrations to serum-supplemented or serum-free medium. Explant survival decreased when RA was added to serum-free medium. Although the cartilage was well differentiated even in cultures deprived of serum, RA inhibited chondrogenesis in all cultures without or with serum. The frequency of columnar epithelium was higher and its folds more often present when RA was added to the medium. Keratinization of squamous epithelium depended on the RA concentration added to the medium, and was almost absent when the concentration was high. Other tissues often present in serum-supplemented medium (such as neuroblasts and myotubes) were not affected by RA, a result that differs from those obtained in other experimental systems.