Density‐dependent effects of oxygen on the growth of mammalian fibroblasts in culture

Abstract

Various concentrations of oxygen were used to determine the optimum culture medium Po, for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T-15 flasks were seeded with =Z 2 X lo4 cells (E 1.3 x lo3 celldcrn'), the highest plating efficiencies and cell yields were obtained with a culture medium Po, of 40-60 mm Hg.At higher inoculum sizes (lo5 cells per T-15) used routinely for mass cultures, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% 0, (growth medium Po, 125-135 mm Hg) and those gassed with 1% O2 (growth medium Po, zz 40-60 mm Hg). The enhanced clonal growth observed at the latter Po, results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% 0,. A slight extension of lifespan was observed in WI-38 cells serially subcultured with a gas phase of 1% 0,.Numerous reports of the deleterious effects of excessive or insufficient concentrations of oxygen on cell growth appear in the literature (McLimans e t al., '68, '72; Balin et al., '76a). As an essential respiratory gas, oxygen dissolved in the culture medium must be sufficient to sustain a normal respiration rate but must not be at a partial pressure such that the cell is damaged (Swartz, '73). Generally, organ cultures require a gas phase containing up to 95% oxygen, and most cell cultures, whether of one or several layers, are routinely grown with a gas phase containing a n atmospheric oxygen concentration, 16 to 20% depending on altitude. An even lower concentration of l-3% oxygen is required for both enhanced clonal growth (Richter et al., '72; Taylor et al., '74) and chromosome stability (Parshad and Sanford, '71; Parshad e t al., '77). To explore further this relationship between cell numbers and oxygen requirements, several concentrations of gaseous oxygen and inoculum sizes were used to assess the Po, of the culture medium that results in optimal proliferation of attached cells. In contrast to the report of Balin et al. ('76a) MATERIALS AND METHODS Cells, culture media, and test gasesHuman Phase I1 WI-38 cells (CCL-75) were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland. A culture of human WI-26 cells was a gift from Monroe Vincent, Microbiological Associates (MA), Bethesda, Maryland. Stock cultures of WI-26 and WI-38 were grown in minimum essential medium (Eagle, '59) (MEM) supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and the "nonessential amino acids" a t a final concentration of 0.1 mM; all MEM and additives were obtained from MA. Lowpassage adult human lines BiRil (CRL 1104) and WilMac (CRL 124) were obtained from ATCC and were grown in Dulbecco-Vogt's modified MEM (DV) obtained from Schwaid Mann, Orangeburg, New York. Cells of line NCTC 9032 are fibroblast-like and derived from an explant culture of normal adult human s...

Effects of sulfhydryl groups and oxygen tension on the cell proliferating activity of bovine serum albumin in culture.

“…Several studies have reported the relationship between the cell growth of human diploid fibroblasts and oxygen tension in the culture environment (1,2,14). We have found that albumin is a component of serum that is affected by oxygen tension when we cultured human fibroblasts in serum-free, BSA-containing medium.…”

Oxygen-dependent growth declining and effect of vitamin E for human diploid fibroblasts in serum-free, BSA-containing culture.

“…It is, however, difficult to determine optimum oxygen tension of the culture, because the concentration of oxygen dissolved in medium differs from that in gas phase and because oxygen tension at growth surface of substrate-attached cells is affected by cellular density and depth of medium layer (Werrlein and Glinos 1974). But the optimum oxygen tension is estimated below 60 mm Hg (about 7-8% 02) from other reports (Balm et al 1976;Taylor et al 1978) and oxygen tension in peripheral tissue in vivo (Caligara and Rooth 1961;Evans and Naylor 196667). Therefore, we compared cell growth and lifespan of the cells in culture under both aerobic and hypoxic (7%) environments in this study.…”

“…Several studies have reported relationship between the cell growth or in vitro lifespan of human diploid fibroblasts and oxygen tension in the culture environment (Balmn et al 1976;Packer and Fuehr 1977;Taylor et al 1978). Human diploid fibroblasts in culture present a unique model for examining the effect of environmental oxygen, because established cells are difficult to rule out the possibility of their adaptation to aerobic environment.…”

“…Many studies have been so far reported concerning the effect of environmental oxygen on cultured mammalian cells in serum supplemented medium (Taylor et al 1974(Taylor et al , 1978Balmn et al 1976;Packer and Fuehr 1977). However, it has been known that serum is a powerful antioxidant (Vidlakova et al 1972).…”

Optimization of a MRC-5 Cell Culture Process for the Production of a Smallpox Vaccine

“…One hypothesis is that a nutrient(s) in the medium is provided at a higher amount and is supporting the cells nutritionally better than at a lower amount (i.e., lower volume of media). Another speculation is that MRC-5 cells proliferate more strongly under a lower oxygen tension (Taylor et al 1978;Balin et al 1984) and this might occur as a result of the increased volume increasing the diffusion length for gas exchange thus decreasing oxygen levels at the monolayer in the T-flask (Randers-Eichhorn et al 1996). Further work using a dynamic gassed system with dissolved oxygen control is planned to prove this hypothesis.…”