Periodontal ligament stem cell cryoprotectant and cryopreservation method thereof

Abstract

The invention belongs to the field of cell cryopreservation and particularly relates to a periodontal ligament stem cell cryoprotectant and a cryopreservation method thereof. The cryoprotectant disclosed by the invention is prepared from glycerinum, mesenchymal steam cell serum-free medium and human serum albumin. The cryoprotectant disclosed by the invention can effectively solve the technical defects that an existing cryoprotectant has low use effect and a survival rate of post-resuscitation periodontal ligament stem cells is low. The periodontal ligament stem cell cryoprotectant disclosed by the invention can keep activity of cryopreserved cells; furthermore, the survival rate of the post-resuscitation periodontal ligament stem cells is obviously improved, and expression of surface markers of the stem cells and differentiation potential of the stem cells are not affected.

Description

technical field

[0001] The invention belongs to the field of cell cryopreservation, and in particular relates to a cryopreservation solution and a cryopreservation method for periodontal ligament stem cells. Background technique

[0002] Periodontal disease is not only the main cause of tooth loss in adults, but also a major oral disease that endangers the health of human oral cavity and even the whole body. Periodontal disease is an infectious disease stimulated by chronic inflammation of periodontal tissue. It is an inflammation caused by microorganisms in dental plaque in a corresponding microenvironment. It damages periodontal tissue, especially the destruction of alveolar bone and periodontal tissue. Loss of tissue, eventually leading to loosening of teeth and resorption of alveolar bone. The goal of periodontal treatment is to treat periodontal inflammation, improve the microenvironment to regenerate new connective tissue attachment and support tissue, and complete th...

Examples

Embodiment 1

[0033] Primary isolation, culture, identification and passage of PDLSCs were carried out.

[0034] Primary isolation and culture of PDLSCs includes the following steps:

[0035] 1. Material collection: Take the third molars of 12-18-year-old healthy donors that need to be extracted for orthodontics, and quickly immerse them in PBS that has been pre-cooled at 4°C and contains 3 times the volume of double antibodies.

[0036] 2. Rinsing: Rinse the tooth body with PBS containing 3 times the double antibody from the root to the crown, thoroughly remove blood stains, repeat 3 times.

[0037] 3. Scraping of periodontal tissue: Scrape the periodontal ligament tissue of the middle and lower 1 / 3 segment of the root with a scalpel blade in one direction, transfer it into a centrifuge tube, and cut the tissue block into about 1mm with ophthalmic scissors 3 size.

[0038] 4. Digestion: add an appropriate amount of collagenase-dispase enzyme 1:1 mixed digestion solution (collagenase 3g / L...

Embodiment 2

[0049] The preparation of the cryopreservation liquid of embodiment 2: the glycerol with the volume percentage content of the cryopreservation liquid of embodiment 2 being 10%, the mesenchymal stem cell serum-free culture medium with the volume percentage content of the freezing liquid of embodiment 2 of 30% and the volume percentage of the frozen storage solution of Example 2 is 60% human albumin (human serum albumin mass concentration is 20%), after mixing, the frozen storage solution of Example 2 is prepared, and it is refrigerated at 4°C for subsequent use .

Embodiment 3

[0051] Preparation of the cryopreservation solution in Example 3: Serum-free culture of the mesenchymal stem cells in which the volume percentage of the cryopreservation solution in Example 3 is 10% glycerol and 40% by volume solution and the volume percentage of the frozen storage solution of Example 3 is 50% human albumin (the mass concentration of human serum albumin is 20%), and after mixing, the frozen storage solution of Example 3 is prepared, and the frozen storage solution of Example 3 is prepared at 4°C in a refrigerator Refrigerate and set aside.

technical field

[0001] The invention belongs to the field of cell cryopreservation, and in particular relates to a cryopreservation solution and a cryopreservation method for periodontal ligament stem cells. Background technique

[0002] Periodontal disease is not only the main cause of tooth loss in adults, but also a major oral disease that endangers the health of human oral cavity and even the whole body. Periodontal disease is an infectious disease stimulated by chronic inflammation of periodontal tissue. It is an inflammation caused by microorganisms in dental plaque in a corresponding microenvironment. It damages periodontal tissue, especially the destruction of alveolar bone and periodontal tissue. Loss of tissue, eventually leading to loosening of teeth and resorption of alveolar bone. The goal of periodontal treatment is to treat periodontal inflammation, improve the microenvironment to regenerate new connective tissue attachment and support tissue, and complete th...

Examples

Embodiment 1

[0033] Primary isolation, culture, identification and passage of PDLSCs were carried out.

[0034] Primary isolation and culture of PDLSCs includes the following steps:

[0035] 1. Material collection: Take the third molars of 12-18-year-old healthy donors that need to be extracted for orthodontics, and quickly immerse them in PBS that has been pre-cooled at 4°C and contains 3 times the volume of double antibodies.

[0036] 2. Rinsing: Rinse the tooth body with PBS containing 3 times the double antibody from the root to the crown, thoroughly remove blood stains, repeat 3 times.

[0037] 3. Scraping of periodontal tissue: Scrape the periodontal ligament tissue of the middle and lower 1 / 3 segment of the root with a scalpel blade in one direction, transfer it into a centrifuge tube, and cut the tissue block into about 1mm with ophthalmic scissors 3 size.

[0038] 4. Digestion: add an appropriate amount of collagenase-dispase enzyme 1:1 mixed digestion solution (collagenase 3g / L...

Embodiment 2

[0049] The preparation of the cryopreservation liquid of embodiment 2: the glycerol with the volume percentage content of the cryopreservation liquid of embodiment 2 being 10%, the mesenchymal stem cell serum-free culture medium with the volume percentage content of the freezing liquid of embodiment 2 of 30% and the volume percentage of the frozen storage solution of Example 2 is 60% human albumin (human serum albumin mass concentration is 20%), after mixing, the frozen storage solution of Example 2 is prepared, and it is refrigerated at 4°C for subsequent use .

Embodiment 3

[0051] Preparation of the cryopreservation solution in Example 3: Serum-free culture of the mesenchymal stem cells in which the volume percentage of the cryopreservation solution in Example 3 is 10% glycerol and 40% by volume solution and the volume percentage of the frozen storage solution of Example 3 is 50% human albumin (the mass concentration of human serum albumin is 20%), and after mixing, the frozen storage solution of Example 3 is prepared, and the frozen storage solution of Example 3 is prepared at 4°C in a refrigerator Refrigerate and set aside.

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