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Parodontium stem cell cryoprotectant and cryopreservation method thereof

A technique for periodontal ligament stem cells and cryopreservation method is applied in the field of periodontal ligament stem cell cryopreservation and its cryopreservation. High clinical safety, the effect of avoiding the introduction of xenogeneic substances

Inactive Publication Date: 2019-04-12
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used cell cryopreservation solution or the commercially available cell cryopreservation solution is usually composed of fetal bovine serum or serum substitutes and dimethyl sulfoxide and other substances, but fetal bovine serum is a heterogeneous substance with complex components. And there is a risk of introducing contamination and allergens, and the composition of serum substitutes is not clear and expensive, which is not suitable for clinical application, especially in cell therapy, the presence of heterologous proteins may cause unknown adverse reactions, Seriously affect the treatment outcome

Method used

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  • Parodontium stem cell cryoprotectant and cryopreservation method thereof
  • Parodontium stem cell cryoprotectant and cryopreservation method thereof
  • Parodontium stem cell cryoprotectant and cryopreservation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, mixture proportioning

[0064] (1) Trehalose working concentration: 500mg / L, 1000mg / L, 1500mg / L;

[0065] (2) Acetamide working concentration: 10%, 20%;

[0066] The experimental group is A: trehalose + acetamide + DMEM / F12 + DMSO + cells;

[0067] The control group is B: DMEM / F12+DMSO+ cells; or FBS+DMSO+ cells.

[0068] The specific composition is as follows:

[0069] Concentration ratio of each component in table 1 cryopreservation solution

[0070]

[0071]

Embodiment 2

[0072] Example 2. Using the parameters in Example 1 to freeze and preserve the cells, recover the cells and determine the relevant state of the cells

[0073] The final density of frozen cells was 10 6 cell / mL, the frozen storage volume is 1.0 mL / tube, and the frozen storage is carried out according to the following procedures: 2 hours at 4°C, 30 minutes at the liquid nitrogen port, and then put into liquid nitrogen.

[0074] Resuscitate the cells after 4 weeks of cryopreservation. Resuscitate 3 tubes in each group. Take out the cryopreservation tube from liquid nitrogen and put it directly into warm water at 37°C. Shake it from time to time to make it melt as soon as possible in a short time. Centrifuge after melting and discard. Clear, add 1mL of culture medium to each tube, count and calculate the average value. The activity distribution is as follows:

[0075] Table 2 Cell viability after cryopreservation and recovery of periodontal ligament stem cells

[0076]

[00...

Embodiment 3

[0082] Embodiment 3, identification of osteogenic ability

[0083] Take the P5 cells cultured in group A5, and use 5×10 4 Cells / well were inoculated in 6-well plates, divided into osteogenic induction group and control group, and cultured in complete medium. The osteogenic induction medium (containing 500ng / mL BMP-2, 10% FBS, 100nmol / L dexamethasone, 20mmol / Lβ-sodium glycerophosphate, 10mg / L vitamin C) was replaced after the adherent cells in the osteogenic induction group grew and fused. L-DMEM), the control group continued to culture with complete medium and conventional induction medium (L-DMEM of 10% FBS, 100nmol / L dexamethasone, 20mmol / L β-sodium glycerophosphate, 10mg / L vitamin C). The culture medium was changed every 3 days, and Alizarin red staining was performed at 4 weeks for identification. Osteogenic effect see figure 2 .

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Abstract

The invention relates to the technical field of stem cell cryopreservation, in particular to a parodontium stem cell cryoprotectant and a cryopreservation method thereof. The parodontium stem cell cryoprotectant is prepared from, 500-2000 mg / L of trehalose, 5-20vt% of acetamide, 5-15vt% of DMSO and the balance a basal culture medium. Compared with a conventional cell cryoprotectant, the parodontium stem cell cryoprotectant and the cryopreservation method thereof have the advantages that the cryoprotectant has a less damage effect on cells during cell cryopreservation, the cell viability is higher, the cryopreservation effect of the cryoprotectant is obviously superior to that of the conventional cell cryoprotectant; on one hand, the cost of the cell cryopreservation can be reduced to a certain degree, on the other hand, the introduction of heterologous matter can be avoided, and the clinical safety is higher.

Description

technical field [0001] The invention relates to the technical field of stem cell cryopreservation, in particular to a periodontal ligament stem cell cryopreservation solution and a cryopreservation method thereof. Background technique [0002] Periodontitis is mainly a chronic inflammation of periodontal supporting tissues caused by local factors. The age of onset is more common after the age of 35. If gingivitis is not treated in time, the inflammation can spread from the gums to the deep layer to the periodontal ligament, alveolar bone and cementum and develop into periodontitis. Because there are no obvious symptoms in the early stage, it is easy to be ignored. When the symptoms appear, they are already serious, and even the teeth cannot be preserved. Periodontal ligament stem cells are adult stem cells derived from periodontal ligament. They have the ability to self-replicate, can produce different types of mature cells with specific phenotypes and functions, can mainta...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 葛啸虎陈海佳黄幸王小燕李学家
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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