Detecting changes of testicular interstitial cell membranes with a fluorescent probe after incubation and cryopreservation with cryoprotective agents

Abstract

Membrane alterations are among central factors predetermining cell survival during cryopreservation. In the present research, we tested some serum-/xeno-free cryoprotective compositions including dimethyl sulfoxide (Me₂SO) and polymers for their osmotic impact and toxicity towards testicular interstitial cells (ICs). IC survival was determined after their contact with Me₂SO, dextran (D40), hydroxyethyl starch (HES), polyethylene glycols (PEG1500 and PEG400), or after cryopreservation and cryoprotective agent (CPA) removal. A ratiometric fluorescent membrane probe 2-(2′-hydroxyphenyl)-5-phenyl-1,3-oxazole (probe O1O) was applied to assess changes in the plasma membrane of ICs. The cell survival study has shown that Me₂SO decreased IC survival in a time- and dosage-dependent manner. The CPA decreased the metabolic activity of ICs, thus implying its toxic effect on the living cell as a whole. Using probe O1O, we have demonstrated that the toxic effect also influenced the plasma membrane. IC membranes were not altered after incubation with 0.7 M Me₂SO. The presence of D40, HES, or PEGs in such Me₂SO containing media resulted in plasma membrane hydration and damage to the membranes of cells incubated with PEGs. Cryopreservation caused pronounced membrane dehydration of the survived ICs even after CPA removal in PEG-containing media and low indicators of IC survival. Interestingly, cryopreservation with the best cryoprotective media supplemented with 0.7 M Me₂SO and 100 mg/ml D40 resulted in minimal membrane alterations, thus implying its higher ability to protect membranes during cryopreservation.