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Isolation and culture method of dental pulp mesenchymal stem cells

A technique for isolating and culturing mesenchymal stem cells, applied in the field of cell culture, can solve problems such as the inability to conduct large-scale production of dental pulp mesenchymal stem cells, and achieve the effects of improving the success rate of separation, improving quality, and simple and efficient operation.

Active Publication Date: 2021-03-19
重庆市博康生物工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the present invention provides a method for isolating and culturing dental pulp mesenchymal stem cells to solve the technical problem in the related art that the large-scale production of dental pulp mesenchymal stem cells cannot be carried out

Method used

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  • Isolation and culture method of dental pulp mesenchymal stem cells
  • Isolation and culture method of dental pulp mesenchymal stem cells
  • Isolation and culture method of dental pulp mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Isolation and Culture of Deciduous Dental Pulp Mesenchymal Stem Cells

[0055] 1. Experimental operation:

[0056] Step S0, taking deciduous teeth as a deciduous tooth sample, first cleaning the deciduous tooth sample with physiological saline, soaking the deciduous tooth sample in 75% alcohol solution for 60 seconds for disinfection, and then washing and disinfecting the deciduous tooth sample again with physiological saline to obtain the pretreated deciduous tooth sample deciduous tooth samples, and place the treated deciduous tooth samples in a protective solution, and store them at 4-8°C for later use; wherein, the protective solution includes streptomycin with a mass concentration of 100-500 mg / L and 60-300 mg of streptomycin at a mass concentration DMEM / F12 medium of / L streptomycin;

[0057] Step S1, take out the deciduous tooth sample and crack it, then take out the pulp tissue in the deciduous tooth sample, spread out the length of about 6-8mm, cut t...

Embodiment 2

[0064] Example 2 Detection of Dental Pulp Mesenchymal Stem Cells

[0065] 1. Experimental operation:

[0066] CD34, CD45, HLA-DR, CD73, CD105, and CD90 of the target dental pulp mesenchymal stem cells were fluorescently labeled, and then the fluorescently labeled target dental pulp mesenchymal stem cells were detected by flow cytometry, and get as Figure 4 and Figure 5 The resulting data are shown.

[0067] 2. Result analysis: by Figure 4 It can be seen that the CD34, CD45 and HLA-DR of dental pulp mesenchymal stem cells are all negative, and the positive expression rate is less than 2%. Depend on Figure 5 It can be seen that CD73, CD105, and CD90 of dental pulp mesenchymal stem cells are all positive, and the positive expression rate is higher than 95%. It can be seen that the dental pulp mesenchymal stem cells cultivated by the method for isolating and culturing dental pulp mesenchymal stem cells of the present invention meet the international standard for mesenchy...

Embodiment 3

[0068] Example 3 Growth Experiment of Dental Pulp Mesenchymal Stem Cells

[0069] 1. Experimental operation:

[0070] Take an appropriate amount of target dental pulp mesenchymal stem cells for cell growth experiments to determine the growth curve of the target dental pulp mesenchymal stem cells, and obtain the following Figure 6 The growth curve graph shown.

[0071] 2. Result analysis: by Figure 6 It can be seen that with the growth of culture time, the number of cells also increases rapidly, and the number of cells can reach up to 4*10 7 Therefore, it can be seen that the proliferation ability of dental pulp mesenchymal stem cells is strong, and they have strong cell viability.

[0072] In summary, the technical solution of the present invention cultivates dental pulp stem cells by forming a glass microsphere-dental pulp tissue complex, which improves the cell separation rate, and the probability of successful cell separation can reach more than 95%; it improves the se...

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Abstract

The invention provides an isolation and culture method of dental pulp mesenchymal stem cells. The method comprises the following steps of: S1, taking out a dental sample, clamping and cracking the dental sample, then taking out dental pulp tissues in the dental sample, chopping the dental pulp tissues, and immersing the chopped dental pulp tissues in a culture medium for later use; S2, putting sterilized glass beads into a container, and soaking the glass beads in a complete culture medium for later use; S3, shallowly burying the dental pulp tissues prepared in the step S1 into the glass beadsprepared in the step S2, and performing culturing for 1-3 hours to obtain a glass bead and dental pulp tissue complex; and S4, carrying out subculture on the glass bead and dental pulp tissue complexprepared in the step S3, and collecting the glass bead and dental pulp tissue complex by trypsin to obtain the target dental pulp mesenchymal stem cells. According to the isolation and culture methodof the dental pulp mesenchymal stem cells provided by the invention, the technical problem that large-scale production of the dental pulp mesenchymal stem cells cannot be carried out in related technologies is solved.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for isolating and culturing dental pulp mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells with strong self-renewal ability and multi-lineage differentiation ability. Mesenchymal stem cells come from a wide range of sources, and can be extracted from dental pulp, bone marrow, umbilical cord blood, umbilical cord, fat, hair follicles and other tissues; , nerve cells and other cell tissue differentiation. Mesenchymal stem cells have many advantages: they can support hematopoiesis and promote hematopoietic stem cell implantation, regulate immunity, promote tissue repair by paracrine, and are easy to isolate and culture; and compared with primitive embryonic stem cells, their safety performance is higher. Based on the above advantages, mesenchymal stem cells have been widely used clinically in graft-versus-host dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0664C12N2509/00C12N2533/12
Inventor 陈瑜潘华峰
Owner 重庆市博康生物工程技术有限公司
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