Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P > 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.
A Simple and Highly Effective Method for Slow-Freezing Human Pluripotent Stem Cells Using Dimethyl Sulfoxide, Hydroxyethyl Starch and Ethylene Glycol
