Stable,position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures

Summary Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA’s effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21–22 and 23–24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA’s effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.     

Microtiter micromass cultures of limb-bud mesenchymal cells

Summary A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (μTμM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This μTμM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d μTμM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the μTμM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation. This work was supported by grants RR08006-13 (DFP) and HD05505 and HD18577 (MS) from the National Institutes of Health, Bethesda, MD. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa 52242 (maintained by NIH grant NO1-HD62915).     

The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas

Summary We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10⁻⁶ or 10⁻⁵ M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10⁻⁶ M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10⁻⁵ M) of retinoic acid and more than three times that of the control grafts.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Beneficial effect of nicotinamide on the proportion of insulin cells in developing chick pancreas

Previous studies have suggested that nicotinamide increases the number of insulin cells both in vivo and in vitro. However, the question remains as to whether there is in fact an increase and whether this increase is caused by the proliferation of progenitor cells, or by replication of existing insulin cells. In order to investigate this, the endodermal component of dorsal pancreatic buds of 5-day-old chick embryos was cultured on Matrigel in a serum-free medium (Ham's F12-ITS) to which nicotinamide, at a concentration of 5 and 10 mM, respectively, was added. Control explants were cultured in Ham's F12-ITS medium without nicotinamide. After 7 days in culture the buds were incubated with bromodeoxyuridine (BrdU) and then processed for immunocytochemistry. Localization of insulin, BrdU and glucagon was carried out on adjacent serial sections. The proportion of insulin cells was 6.76, 11.32 and 16.86% in control, 5 and 10 mM nicotinamide-treated explants, respectively. Hence adding nicotinamide to the culture medium induced a 1.7- and 2.5-fold increase in the proportion of insulin cells when compared to the controls. These proportions were significantly different from that of control explants (P < 0.05). However, a very small number of insulin cells were found to be proliferating, suggesting that the increase in the proportion of insulin cells had resulted from stimulation of progenitor cells and not proliferation of existing insulin cells.     

A serum-free medium for hybridoma cell culture

The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA bovine serum albumin - CS calf serum - DMEM Dulbecco's modified Eagle's medium - ELISA enzyme-linked immunosorbant assay - McAb monoclonal antibody - PEG polyethylene glycol - SFM serum-free medium     

A fluorometric assay for the measurement of endothelial cell density in vitro

Summary A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

Development of a new serum-free medium,USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes

Summary A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. Ths new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum. This work was supported by grant AM18983 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD.     

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.     

NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

Determination of L-ascorbic acid levels in culture medium: Concentrations in commercial media and maintenance of levels under conditions of organ culture

Summary The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified New circulator gassed with 95% O₂+5% CO₂ was 1.5 hr.; and when gassed with 20% O₂+5% CO₂+75% N₂, about 2 hr. In Petri dishes gassed with 20% O₂+5% CO₂+75% N₂, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at 0°C and about 9% per day when stored at 5°C. When medium with an initial content of 300 μg per ml was stored at room temperature, the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which allows the provision of a relatively constant level of L-ascorbic acid to an explant by taking advantage of the slow oxidation of L-ascorbic acid at 0°C.     

Vero细胞无血清培养技术的研究与应用

Vero细胞是世界卫生组织和我国生物制品规程认可的疫苗生产细胞系。随着对疫苗质量和安全性要求的不断提高,用无血清培养基取代含血清培养基培养Vero细胞已成为病毒疫苗生产的一个重要发展趋势。Vero细胞无血清培养的技术关键是研发或选择能支持细胞以贴附培养方式生长的无血清培养基。微载体培养是贴附依赖性细胞系规模化培养和病毒疫苗生产的有效技术途径。我们对Vero细胞无血清培养基的研发、Vero细胞无血清培养及病毒疫苗生产工艺做了讨论,对该领域存在的问题和发展策略进行了展望。