Vitreous Cryopreservation Of Human Umbilical Vein Endothelial Cells By Alginate Hydrogel Microencapsulation

Cryopreservation is an effective method of long-term preservation of cells, tissues and organs, so it is widely used in the field of tissue engineering, regenerative medicine and assisted reproductive medicine. Cryopreservation is mainly achieved through the slow cooling method and the vitrified method. The slow cooling technology is relatively mature until now, and many biological samples have been realized preservation at low temperatures. However, the slow cooling method has some disadvantages that cells suffer various types of damage caused by intracellular ice formation and solution effects during cooling and recrystallization during warming. The vitrified method is achieved by using a higher concentration of cryoprotective agent and rapid cooling. This method is simple, convenient and time-saving, but a high concentration of cryoprotective agent will cause damage due to osmotic pressure and toxicity of solutions. Although we use multi-step addition and removal of cryoprotective agent, the damage cannot be avoided. Sometimes vitrification can be achieved during freezing, but if the concentration of cryoprotective agent is not enough high, it will appear devitrification during warming, which is lethal to cells and greatly reduces cell viability.Human umbilical vein endothelial cells (HUVECs) were used as a cell model in this paper, and osmotic tolerance limits of HUVECs and permeability coefficients of the cell membrane were measured. A low-cost, easy-to-use microfluidics-based microperfusion chamber was used to investigate the osmotic responses of cells in the presence of hypertonic saline solution and 1.5 M dimethyl sulphoxide (Me2SO) at 25, 14,5 and-2℃. Permeability coefficients of the cell membrane were gained by fitting 2-p equations, and the cell membrane permeability coefficients at a reference temperature of 273.15 K. were obtained by using Arrhenius relationship. Results showed that cells were within a safe range when osmolality was between 155 mOsm/kg and 921 mOsm/kg or when the normalized cell volume was between 1.39 and 0.53. The hydraulic conductivity and Me2SO permeability coefficient at a reference temperature of 273.15 K were 0.32× 10-14 m/Pa/s and 0.30×10-8 m/s, and the activation energies were 40.15 kJ/mol and 82.47 kJ/mol, respectively. Finally, the cell membrane permeability coefficients and osmotic tolerance limits of cell volume were used to simulate the addition and removal of cryoprotective agent.In order to solve these problems in the slow cooling method and traditional vitrified method, vitreous cryopreservation was used by alginate hydrogel microencapsulation. The results showed it greatly improved cell viability compared with the conventional vitrified method. Although it appeared devitrification during warming, it did not affect cell viability, so this method is effective.