In Vitro Cell. Dev. Biol. 29A:208-214, March 1993 © 1993 Tissue Culture Association 0883-8364/93 101.50+0.00 EXTRACELLULAR CALCIUM DOES NOT CONTRIBUTE TO CRYOPRESERVATION- INDUCED CYTOTOXICITY LAURA S. RHOADS, ANNE M. DANKS, JOHN IM, ANNE WARNER, ROBERT L. ISAACSON, JOHN BAUST, AND ROBERT G. VAN BUSKIRK 1 Department of Biological Sciences (L S. R., J. L, A. IF., J. B., R. G. E. B.), and Psychology Department (A. M. D., R. L. L), State University of New York, Binghamton, New York 13902 (Received 12 August 1992; accepted 18 September 1992) SUMMARY 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)]-tetraaceticacid (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-rain exposure to ionomycin in the presence of 2 mM [Ca+2]e was approximately 2 #M 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 cryopreser- vation. 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 [CaS2]e can contribute to cytotoxicity, the presence of [Ca+2]e might not influence cryopreservation-indueed cytotoxicity. Key words: CytoFluor 2300; calcium cytotoxicity; cryopreservation; Calcein; CFDA; Fluo3; neutral red; multiple endpoint. INTRODUCTION The ability to properly cryopreserve cells and tissues demands a) that the mechanism(s) underlying cryopreservation-inducedcytotox- icity be elucidated and b) that a battery of assays be developed that can determine if a putative cryoprotective mixture can prevent cyto- toxicity that occurs during cryopreservation. We tested a fluores- cent muhiple-endpoint assay (16,27) as a process for understand- ing cryopreservation-induced cytotoxicity. In particular, we used this multiple probe approach to determine if a loss of calcium ho- meostasis, such as that which might occur during cryopreservation, could contribute to cytotoxicity induced by freezing single ceils. An increase in intracellular calcium (]Ca+2]i) is a common feature of both programmed cell death (apoptosis) and necrotic cell death. Apoptosis is the normal cell death that occurs during embryogene- sis, differentiation, and aging (1,7,9). Cells undergoing apoptosis 1 To whom correspondence should be addressed. 208 demonstrate a variety of morphologic changes ranging from the disruption of the nuclei to plasma membrane blebbing (30). In all cases of apoptosis studied, the cascade of events leading to pro- grammed cell death are thought to be initiated by a rise in [Ca+2]i (22,29,30). Necrotic cell death, on the other hand, is also charac- terized by a rise in [Ca+2]i and has been especially well studied in neurons where such rises have been implicated in neuronal cell loss due to ischemic injury (for a review see 18). The potential cytotoxic role of calcium in necrotic neuronal death is especially convincing. For instance, activation of the glutamate channels in hippocampal neurons by N-methyl-D-aspartate increases [Ca+2]i and subse- quently leads to necrosis as evidenced by morphologic changes (23). These cytotoxic effects were blocked by the removal of extra- cellular calcium ([Ca+2]e). Thus there is evidence that a loss of calcium homeostasis might be a common pathway of cell cytotoxic- ity (25). This latter hypothesis was tested using cryopreserved Madin- Darby canine kidney (MDCK) cells and a fluorescent multiple end-