Table 1 Freezing protocols
Protocols | Freezing protocol 1 | Freezing protocol 2 | Freezing protocol 3 | Freezing protocol 4 |
|---|---|---|---|---|
Cryo-vials type | 0.25 mL straws | 0.25 mL straws | 0.25 mL straws | 0.25 mL straws |
Diluents | Tris coconut water | Tris coconut water | Tris coconut water | Tris coconut water |
Cryoprotectant type | Glycerol and DMSO | Glycerol and DMSO | Glycerol and DMSO | Glycerol and DMSO |
Cryoprotectant concentrations | 8, 10, or 15% | 8, 10, or 15% | 8, 10, or 15% | 8, 10, or 15% |
Dilution rate | 1:2 | 1:2 | 1:2 | 1:2 |
Equilibration time | 4 h at 5 °C | 4 h at 5 °C | 4 h at 5 °C | 4 h at 5 °C |
Freezing rate | − 3 °C/min until − 50 °C, − 50 °C/min to − 130 °C, finally− 196 °C | 4 °C/min until − 50 °C, cool from − 50 to − 150 °C at a rate of 50 °C/min, finally − 196 °C | l °C/min, to − 20 °C − 20 to − 80 °C at a rate of 20 °C/min, finally – 196 °C | Vapor freezing at – 50 °C for 12 min, then immersed directly into liquid nitrogen at − 196 °C |