ВИКОРИСТАННЯ ТРОМБОЦИТАРНОГО ЛІЗАТУ ЯК КОМПОНЕНТА КРІОЗАХИСНИХ СРЕДОВИЩ ДЛЯ КРІОКОНСЕРВУВАННЯ МЕЗЕНХІМАЛЬНИХ СТРОМАЛЬНИХ КЛІТИН

Abstract

Mesenchymal stromal cells (MSCs) due to their unique properties are widely used in regenerative medicine. Standard cryopreservation methods that are based on the use of penetrating cryoprotectant dimethyl sulfoxide (DMSO) and fetal bovine serum (FS) can ensure high cell survival, but limit the possibility of therapeutic application because of the risk of adverse reactions. The toxicity of high DMSO concentrations and FS immunogenicity require significant optimization of cryopreservation approaches. In the current study, freezing of human MSCs in cryoprotective media (CPM) with different compositions was performed. Twenty-four hours prior to freezing, cells were pretreated by addition of 100 mM sucrose into the culture medium. CPM with 200 mM sucrose were supplemented with 10% FS or 10% platelet lysate (PL) in the presence or absence of 1% DMSO. The cells frozen without any cryoprotectants were used as a negative control. The MSCs cryopreserved in media containing 10% DMSO and 10% FS were chosen as a positive control group. The MSCs were frozen in cryogenic vials with a cooling rate of 1 deg/min to -80°C with the following immersion into liquid nitrogen. The cell survival was determined by trypan blue staining; metabolic activity was assessed using the Alamar Blue test. It was revealed that viability of MSCs after freezing in CPM containing 200 mM sucrose, 10% FS or 10% PL without DMSO addition were 59±3.3% and 58±2.5%, respectively. The metabolic activity of cells in the PL group exceeded the results of the FS group by 12%. When 1% DMSO was added into the CPM containing 200 mM sucrose and 10% PL, the cell survival rate and metabolic activity were by 7% and 13% higher than in the presence of 10% FS. The obtained results indicate that replacement of FS with PL in the CPM without penetrating cryoprotectant DMSO allows to maintain MSCs viability and increase their metabolic activity after freeze-thawing.