Use of polyethyleneoxide and hydroxyethylstarch as blood plasma substitutes in the cryopreservation of testis interstitium cells in mice

Abstract

Purpose of the study: to investigate the impact of hydroxyethyl starch (HES) and polyethylene oxide (PEO) on the indicators of preservation of murine testis interstitial cells (IC) under cryopreservation. Materials and methods. To isolate IC the enzymes were used: 0.2 mg/ml collagenase and 0.1 mg/ml DNase. The obtained cell suspension was cryopreserved in the solutions that contained 0; 0,7; 1,4; 2,1; 2,8 M of dimethyl sulfoxide (DMSO) and/or 10%, 20% fetal cow serum, 10 mg/ml PEO or HES. The samples (1 ml) were cooled at a rate of 1 °C/min to -80 °C then stored in liquid nitrogen (-196 °C). They were warmed at 37 °C in the water bath. Cryopreservation solution was removed. The number of cells and their preservation were assessed before and after with the assistance of Goryaev’s camera. Viability of IC, Leydig cell preservation and preservation of metabolic activity were measured with trypan blue dye, histochemical staining for 3β-hydroxysteroid dehydrogenase activity. Results. It was shown that 1,4 M DMSO without supplements favored IC preservation. Addition to the cryopreservation solution 10% and 20% of fetal cow serum or 10 mg/ml HES increased total preservation of IC by more than 10% and Leydig cell cryopreservation by an average 15%. HES 10 mg/ml may decrease DMSO concentration to 0,7 M. This combination had the best indicators of total preservation of IC, preservation of viable cells and Leydig cells: 75,8 (53,3; 93,3), 55,6 (45,1; 69,4), 57,1 (40,2;70,3) %, respectively. PEO was ineffective. Conclusion. High-molecular weight synthetic polymers such as HES can substitute protective properties of blood serum under cryopreservation and allow decreasing effective concentration of permeable cryoprotective such as DMSO.

Keywords

• cryopreservation
• leydig cells
• testes
• polyethylene oxide
• hydroxyethyl starch