Cell cryoprotectant and cryopreservation method
A cryopreservation method and cryopreservation solution technology, applied in the field of cells, can solve the problems of introducing pollution and allergens, complex components, adverse reactions, etc.
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Embodiment 1
[0062] Example 1: Primary isolation and culture of GMSCs
[0063] 1) Source of raw materials: the gingiva or impacted tooth extracted by clinical orthodontic eruption and the excision package
[0064] The gum tissue that surrounds the permanent teeth. Patients are required to have no gingival hyperplasia, inflammation and use of drugs that cause gingival hyperplasia. The excised gingival tissue was quickly immersed in 4°C pre-cooled PBS containing 3 times double antibody.
[0065] 2) Rinse: Rinse 3 times with PBS containing 3 times the double antibody, and then soak the gingiva in the serum-free medium of mesenchymal stem cells (containing penicillin 100u / mL and streptomycin 100u / mL) for 5 minutes;
[0066] 3) Digestion: cut the gum tissue into 1cm 3 Add an appropriate amount of collagenase-Dispase enzyme 1:1 mixed digestion solution (collagenase 3g / L, Dispase enzyme 4g / L), and place it in a constant temperature shaker at 37°C for 60min. After digestion, centrifuge at 1000...
Embodiment 2
[0080] Preparation of cryopreservation solution: prepare cryopreservation solution before cell collection, the formula is DMSO: mesenchymal stem cell serum-free culture medium: human albumin injection (20%) = 1:3:6. Refrigerate at 4°C for later use.
[0081] Cell collection: Select the 3rd generation GMSCs, when the cells are 80-90% full, use a pipette to discard the old culture medium, add 2-3mL 0.25% trypsin, digest for 1-3 minutes, observe under the microscope when the cells shrink and become round, Immediately add 5-10 mL of serum-free medium for mesenchymal stem cells to terminate the digestion, collect the cells, centrifuge at 1000 r / min for 5 min, and discard the supernatant.
[0082] Freezing: use the prepared freezing solution to press 1.5×10 6 Frozen cells at a density of 1 / mL. Dispense into 2ml cryopreservation tubes, 1mL per tube, put them into a program cooling box to restore room temperature, place them in a -80°C ultra-low temperature refrigerator, and transfer...
Embodiment 3
[0084] Preparation of cryopreservation solution: Prepare cryopreservation solution before cell collection, the formula is DMSO: mesenchymal stem cell serum-free culture medium: human albumin injection (20%) = 1:4:5. Refrigerate at 4°C for later use.
[0085] Cell collection: Select the 3rd generation GMSCs, when the cells are 80-90% full, use a pipette to discard the old culture medium, add 2-3mL 0.25% trypsin, digest for 1-3 minutes, observe under the microscope when the cells shrink and become round, Immediately add 5-10 mL of serum-free medium for mesenchymal stem cells to terminate the digestion, collect the cells, centrifuge at 1000 r / min for 5 min, and discard the supernatant.
[0086] Freezing: use the prepared freezing solution to press 1.5×10 6 Frozen cells at a density of 1 / mL. Dispense into 2mL cryopreservation tubes, 1mL per tube, put them into a program cooling box to restore room temperature, place them in a -80°C ultra-low temperature refrigerator, and transfer...
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