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.

Inactive Publication Date: 2017-02-01
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FBS is a heterogeneous substance with complex components and the risk of introducing contamination and allergens, so it is not suitable for clinical application
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Method used

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  • Cell cryoprotectant and cryopreservation method
  • Cell cryoprotectant and cryopreservation method
  • Cell cryoprotectant and cryopreservation method

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to the field of cells, in particular to a cell cryoprotectant and a cryopreservation method. The cell cryoprotectant is prepared from DMSO, human albumin and a serum-free medium. The cryprotectant does not contain animal serum, the risks of introducing contamination and allergens are avoided, and the higher clinical safety is achieved compared with a conventional cell cryoprotectant. Meanwhile, the GMSCs cryoprotectant can well keep the activity of cryopreserved cells.

Description

technical field [0001] The invention relates to the field of cells, in particular to a cell cryopreservation solution and a cryopreservation method. Background technique [0002] Periodontal disease has caused irreversible damage to the supporting tissues around the teeth, and the current treatment methods widely used for periodontal regeneration have failed to fully realize the physiological and functional regeneration of periodontal tissue. The development of periodontal tissue engineering technology provides new ideas and space for the repair and regeneration of periodontal defect tissue. Since periodontal ligament stem cells were discovered, their good stem cell properties have been widely recognized, but their sources are limited, and periodontal ligament tissue needs to be extracted to obtain periodontal ligament tissue, which limits its clinical application potential. In the research of tissue engineering, it has always been the focus and core of the research to seek...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 陈海佳王一飞葛啸虎戚康艺王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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