Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Area of Science:

• Cell Biology
• Cryobiology
• Biochemistry

Background:

• Extracellular calcium ([Ca+2]e) is implicated in cellular toxicity.
• Cryopreservation involves cellular stress and potential damage.
• Understanding factors influencing cryopreservation cytotoxicity is crucial for cell viability.

Purpose of the Study:

• To investigate the role of extracellular calcium ([Ca+2]e) in cryopreservation-induced cytotoxicity.
• To evaluate different fluorescent probes for monitoring cytotoxicity during cryopreservation.
• To determine the primary indicators of cell survival after cryopreservation.

Main Methods:

• Madin-Darby canine kidney (MDCK) cells were used.
• Fluorescent multiple endpoint assays and indicator dyes (Fluo3, neutral red) were employed.
• Cells were subjected to cryopreservation with dimethylsulfoxide (DMSO) under varying extracellular calcium conditions.

Main Results:

• Increased intracellular calcium ([Ca+2]i) induced by ionomycin was cytotoxic, and neutral red effectively monitored this.
• During cryopreservation, plasma membrane integrity, not lysosomal function, correlated with cell survival.
• [Ca+2]e levels did not significantly affect plasma membrane integrity, neutral red retention, or cell survival post-cryopreservation.

Conclusions:

• Plasma membrane integrity indicators are superior to neutral red for assessing cryopreservation cytotoxicity.
• Dimethylsulfoxide (DMSO) may exhibit lysosomal toxicity during cell cryopreservation.
• Extracellular calcium ([Ca+2]e) does not appear to be a major factor in cryopreservation-induced cytotoxicity for MDCK cells.