Renewal of the culture medium induces a temporary decrease in the gap junctional communication accompanied by degradation and re-establishment of gap junctions in the V79-4 Chinese hamster cell line.
To replace the culture medium with a fresh one is a routine action of the mammalian cell culture technique. It is generally assumed that this act per se does not cause any significant physiological response of a cell population that would significantly interfere with the experimental procedures in culture. However, in this series of experiments we demonstrate that the exchange of the culture medium for a fresh one may induce a significant temporary decrease in the GJIC, assessed by the dye coupling method in the V79-4 Chinese hamster cell line. This effect is accompanied by a degradation of gap junctions and their re-establishment assessed by the semi-quantitative immunocytochemistry of connexin43. The minimum value of GJIC was reached 45 min after the exchange of the medium. Afterwards, GJIC grew up again, reaching the standard value 3 or 4 h later. This effect does not just result from the exchange of medium as a mechanical action, is not caused by the change of pH and is of quantitative character. The fresh medium loses its capability to reduce GJIC after 3 h of conditioning with the same cells. We found that the value of the early inhibition of GJIC observed during the first 2 h of treatment with the inhibitor of GJIC-EG (applied together with a fresh culture medium)--was indistinguishable from the effect of the exchange of medium itself. Only after that point of time is the EG-induced inhibition of GJIC definitely distinguishable. The results demonstrate that a simple exchange of the culture medium, which is generally implemented in various experiments in culture, may cause serious physiological, biochemical and even morphological responses of cells and thus affect the final results of experiments in culture, especially regarding the early effects of drugs. Consequently, to avoid an undesirable response of the cell population reported in this paper we recommend to apply or remove drugs using a medium conditioned with the same cells for at least 3 h.
Research Institute of Child Health, Department of Cell Biology, Brno, Czech Republic.
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Vítek
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Oborilová
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Mazura
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Benesová
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism; D006224:Cricetinae; D003470:Culture Media; D017077:Culture Media, Conditioned; D017629:Gap Junctions / Q000502:physiology*; D007150:Immunohistochemistry; D007546:Isoquinolines; D015398:Signal Transduction / Q000502:physiology*
D018031:Connexin 43; D003470:Culture Media; D017077:Culture Media, Conditioned; D007546:Isoquinolines; C017475:lucifer yellow