Importance of culturing primary lymphocytes at physiological oxygen levels

Abstract

Although studies with primary lymphocytes are almost always conducted in CO 2 incubators maintained at atmospheric oxygen levels (atmosO 2 ; 20%), the physiological oxygen levels (physO 2 ; 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO 2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO2 than at physO2. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO2 than at atmosO2; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO2 that at atmosO2. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.glutathione ͉ low oxygen ͉ nitric oxide ͉ proliferation

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

“…Likewise, the nrdD mutant grew indistinguishably from SK36 under these same conditions. We have recently shown that growth of S. sanguinis sodA (superoxide dismutase) and ssaB mutants in rabbit serum under 12% O 2 , the level present in arterial blood (44), closely mimics the results of virulence studies performed in the rabbit model. 4 Neither the nrdI nor nrdHEKF mutants were recovered after 24 h of 37°C incubation under these conditions (data not shown).…”

“…Loss of NrdF expression was confirmed by Western blot (data not shown). As would be expected for a strain whose only remaining RNR is a class III (anaerobic) enzyme (15,43), the mutant could not be propagated in the presence of 6% O 2 , our usual microaerobic growth condition, which is also similar to dissolved O 2 concentrations in venous blood (44). Indeed, this mutant routinely failed to grow in BHI broth under an atmosphere of as little as 1% O 2 (data not shown).…”

Glut1-mediated glucose transport regulates HIV infection

“…Thus, although lymphocytes encounter fluctuations in O 2 levels in vivo, the physiological range of O 2 levels to which they are exposed is at least two to six times lower than the 20% O 2 levels maintained in standard incubators. Culturing at atmospheric O 2 levels has been shown to result in altered cell proliferation rates and other skewed T-cell responses (16,(19)(20)(21)(22)(23). Thus, because the goal of in vitro studies is generally to reveal information of in vivo significance, our studies here focus on findings with peripheral blood T cells cultured at physiological O 2 levels.…”

Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease

“…1). However, the implied caution has largely been ignored, keeping the entrenched culture methods in use despite the rapidly growing recognition that all cell types sense and respond to even small shifts in oxygen levels (1)(2)(3)(4)(5)(6).…”

“…In the overwhelming majority of such studies today, cells are cultured in incubators that are gassed with a mixture of CO 2 and air, which maintains the internal oxygen level essentially at atmospheric oxygen (atmosO 2 ) levels (atmosO 2 , 20-21% O 2 ,). The biomedical community (or at least some portion thereof) has, of course, been aware that these oxygen levels are 2-to 4-fold greater than the oxygen levels that lymphocytes and most other cells encounter in vivo (Fig.…”