The Role of Carbon Dioxide as an Essential Nutrient for Six Permanent Strains of Fibroblasts

Abstract

The results of these studies demonstrate that carbon dioxide is required for the growth and maintenance of strains of fibroblasts derived from human tissues, strains FS4-705 and U12-705, from mouse tissue, strain L-705, and from rabbit tissues, strains RM3-56, RS1-56, and RT-56 in a chemically defined medium containing phosphite buffer in place of bicarbonate and supplemented with dialyzed serum and dialyzed embryo extract. Under these conditions, the cells fail to proliferate at a significant rate and begin to degenerate within 5 to 10 days when the flasks are not stoppered. Suflficient carbon dioxide is produced by the ceils to promote growth as indicated by the fact that maximal proliferation is obtained in the same phosphite media when stoppered flasks are employed. With the exception of RS1-56, all the remaining strains tested can be propagated serially in open flasks containing phosphite medium prepared with whole serum and embryo extract. The rate of growth under these conditions, however, is only one-half to onethird that obtained in stoppered flasks containing phosphite medium or the conventional bicarbonate medium.An earlier report from this laboratory described the propagation of several strains of mammalian cells in media containing fixed buffers such as tris(hydroxymethyl)aminomethane and glycylglycine in place of the conventional carbon dioxidebicarbonate system (1). Studies on the practical application of fixed buffers in tissue culture media were subsequently extended to include other compounds which are compatible with calcium and other cations and which are effective buffers in the p H range 7.0-8.0. In the course of these investigations, it was observed that several strains of fibroblasts proliferate at a reduced rate in certain media containing sodium phosphite (2) in place of bicarbonate when the flasks were dosed only by means of loosely fitting metal caps instead of stoppers. These observations suggested that carbon dioxide might be an essential nutrient for fibroblasts propagated in vitro. Some support for this * Aided by a grant (No. E-1547) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. speculation was indicated by other investigations which demonstrated that carbon dioxide is beneficial for the outgrowth of cells from explanted rat kidney (3, 4) and essential for explants of chick embryo tissue (5, 6). The results to be presented demonstrate that carbon dioxide is essential for the proliferation of six permanent strains of fibroblasts, derived from human, mouse, and rabbit tissues, when propagated in media containing dialyzed chick embryo extract and dialyzed horse serum. Materials and MethodsThe following strains of cells were propagated serially as described previously (7-9) : human fibroblasts, strains FS4-705 and U12-705 (8), rabbit fibroblasts, strains RM3-56, RS1-56, and RT6-56 (9) and mouse fibroblasts, strain L-705 (10). Medium 705 is composed of solution 703 (11) supplemented with 5 pe...

The nutrition of animal cells in vitro

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Expansion of mesenchymal stem cells under atmospheric carbon dioxide

“…This formulation was selected to minimize the potential cytotoxic effects associated with HEPES buffer . A low level (5 mM) of NaHCO 3 was added to the MOPS‐HEPES buffered medium, because bicarbonate is reported to be an essential cell nutrient for mammalian cells …”

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Observed Differences in Co2 Requirements Between Mammalian and Salmonid Fish Cell Lines

“…Although the salmonid cell cultures did demonstrate a growth requirement for C02 , their ability to grow in bicarbonate-free Tris-buffered medium with free gas exchange with the air sets them apart from most mammalian cells . Swim and Parker (12) reported that strains of fibroblasts derived from human, mouse, and rabbit tissues failed to proliferate at a significant rate when cultured in a medium without bicarbonate in unstoppered flasks .…”