Effects of cryoprotective agents on osmotic tolerance limit of testicular interstitial cells

Abstract

Background: Cryopreservation is a multistep process, which includes stages affecting biological material mechanically, osmotically and toxically. The use of cryopreservation of biological materials is cost-effective and affording long-term storage at cryogenic temperatures. It also guarantees the stability of the genetic component of cells and reduced contamination of the biological material. Objectives: The objective of the research is to evaluate the effects of cryoprotective agents (CPAs) (dimethyl sulfoxide (DMSO), dextran (D40), hydroxyethyl starch, polyethylene glycols (PEG1500 and PEG400), and fetal bovine serum) and their combinations on the interconnection between the osmotic tolerance of testicular interstitial cells (ICs) and cryoprotection. Materials and Methods: The osmotic tolerance limit (OTL) of ICs and the toxic effect of the CPA were investigated in the phosphate buffer saline based media of different osmolarities: isosmotic (300 mOsm), hypo-osmotic (225 mOsm), hyperosmotic (600 mOsm). Similar osmotic conditions can develop during cryopreservation of cells in the temperature interval from +4 to -30 °C. Results: The indicators of cell survival after incubation in the media differed depending on osmolarities of incubation media. They were compared with the indicators obtained after cooling ICs to -30 °C followed by warming and CPA removal. We have shown that the non-toxic additive D40 increased the OTL of ICs in hypo-osmotic medium and decreased negative effects of DMSO on the cells. These effects were accompanied by high indicators of ICs survival obtained after cooling ICs to -30 °C with 100 mg/ml D40 and 0.7 M DMSO. Conclusions: These results unveil the mechanisms of cryoprotection of 0.7DMSO+D40 and partially explain the superiority of 0.7DMSO+D40 media shown in our previous works compared with other investigated media. Understanding the mechanisms of cryodamage and cryoprotection of 0.7DMSO+D40 paves a way toward the development of new serum-/xeno-free cryoprotective compositions and improvement of cryopreservation protocols for cell suspensions that include many types of cells. Further studies are required to reveal the effects of DMSO on membranes and intracellular metabolic processes.

Keywords

• osmotic tolerance
• toxicity
• dimethyl sulfoxide
• dextran
• hydroxyethyl starch
• polyethylene glycol
• mechanisms of cryoprotection