Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Abstract

The possible role of extracellular calcium ([Ca+2]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca+2]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca+2]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca+2]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca+2]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca+2]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca+2]e was approximately 2 microM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca+2]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca+2]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca+2]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca+2]e can contribute to cytotoxicity, the presence of [Ca+2]e might not influence cryopreservation-induced cytotoxicity.

The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells

“…The action of DMSO as a cryoprotectant is based on the fluidizing of the cellular membrane [15]. DMSO penetrates into the cells, influencing the structure and conformation of the cellular membrane and proteins, resulting in decreased cellular activity [16,17]. The influence of an ethanol concentration higher than 5% on cell viability was also found.…”

Preliminary Report: Evaluation of Storage Conditions and Cryococktails during Peripheral Blood Mononuclear Cell Cryopreservation

“…These findings are consistent with previous reports describing improvements in postthaw viability in various cell systems. [35][36][37] With the identification of a significant level of CIDOCD playing a role in PBMC cryopreservation failure, we investigated the relative contributions of necrosis and apoptosis. Based on the information obtained from the viability studies, we elected to utilize VAA analysis as our primary assessment tool for these studies ( Fig.…”

Loss of Calpastatin Leads to Activation of Calpain in Human Lens Epithelial Cells

“…Therefore, we repeated the experiments, but we used the calcium ionophore, ionomycin, instead of external calcium. Ionomycin elevates cytoplasmic calcium by promoting Ca 2þ entry through the plasma membrane 17 and from the internal organelle stores. 18 Note that the basal level of calcium in DMEM/FBS medium was approximately 1.8 mM Ca 2þ .…”