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Human dental pulp cell separation culture method

A technique for separating and culturing dental pulp cells is applied in the field of human cell culture, which can solve the problems that dental pulp stem cells are not easy to adhere to, affect the effect of cultivation, and consume a lot of time, so as to improve the effect of adherence and reduce time and economic costs. , the effect of improving the adherence rate

Pending Publication Date: 2020-09-15
北京昱龙摩尔国际生物医学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some difficulties in culturing dental pulp stem cells at present. When culturing dental pulp tissue, fibroblasts are often obtained instead of dental pulp stem cells, and dental pulp stem cells are not easy to adhere to the wall during the culture process, which seriously affects In the existing methods, cell adhesion is often promoted by pre-coating the culture dish or adding cohesin to the medium, but the former will consume a lot of time, while the latter is expensive, resulting in the cost of the existing method. higher

Method used

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  • Human dental pulp cell separation culture method
  • Human dental pulp cell separation culture method
  • Human dental pulp cell separation culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0041] A method for isolating and culturing human dental pulp cells, comprising the steps of:

[0042] S1: Collect completely extracted orthodontic teeth of healthy people (12-25 years old), clean the surface with 75% ethanol, drill grooves about 1 mm deep on both symmetrical sides along the long axis of the teeth with a split drill, and seal the teeth with a large steel wire. Cut it in half, take out the pulp tissue with dental tweezers; wash it with PBS buffer solution with 1% double antibody and cut it into pieces smaller than 1mm3, add 0.3% type Ⅰ collagenase and 0.4% neutral protease, mixed evenly, digested at 37°C for 1 hour, gently pipetting the discrete single-cell clumps to obtain dental pulp cells, and kept them for later use;

[0043] S2: Wash the dental pulp cells with PBS buffer, centrifuge at 1000r / min for 5min to collect the cells, and repeat the washing and centrifugation operations; resuspend the pellet with the primary medium added with glucomannan and sodium...

experiment example 1

[0046] identification test

[0047] (1) Immunofluorescence detection experiment (this experiment is carried out after more than 12 passages)

[0048] Dental pulp cells were cultured by the above method, and the passaged cells obtained were detected by immunofluorescence, the results showed vimentin, type I collagenase, type III collagenase, alkaline phosphatase, BMP and dentin-specific non-collagen Phosphoproteins were all positive, indicating that the cultured cells were indeed dental pulp cells.

[0049] (2) Cell morphology observation experiment

[0050] During the culture process, the above-mentioned cells were placed under an inverted phase-contrast microscope for continuous observation every day. The growth status and basic shape of the cells can be seen: the cells can grow on the iron wall on the second day of culture, and some colonies grow, and the colony growth cells are round. Shaped or oval, the cells are small and closely arranged, the cells at the edge of the c...

experiment example 2

[0054] Comparison experiment of wall attachment

[0055] The formulations shown in Table 1 were used to configure primary culture media with different proportions, and the same source of dental pulp tissue was cultured in the method provided in the examples, and the growth of cells in each group was compared; in addition, gelatin and transparent The Petri dish was coated with uric acid, and the control experiment was carried out in the same way. The growth of cells in each group is shown in Table 2 (where the adherence status was observed every 15 minutes, and the fusion status was observed every 12 hours).

[0056] Table 1 primary culture medium formula (unlisted ingredients in the table are all the same as the examples)

[0057]

[0058] Table 2 Cell growth

[0059]

[0060]

[0061] As can be seen from Table 2, the time for the adherence time and fusion degree of 1-5 groups to reach 80-90% are significantly lower than those of 6-11 groups, and are equivalent to 12...

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Abstract

The invention provides a human dental pulp cell separation culture method. Glucomannan and sodium alginate are added into a primary culture medium, so that cell adherence growth and replication in a primary culture process can be effectively promoted, and the adherence effect equivalent to that of a traditional coated culture dish or added laminin can be achieved; the pre-coating operation can beomitted, and compared with laminin, the glucomannan and the sodium alginate are more economical, so that the time cost and the economic cost are effectively reduced while the adherence effect is improved, and a large number of high-activity dental pulp stem cells are obtained; meanwhile, the culture medium provided by the invention does not need serum, can effectively promote the growth and replication of the dental pulp cells and improve the differentiation capacity of the cells through the cooperation of various components, and can overcome the defects of the serum on the premise of ensuringthe performance, thereby effectively improving the culture effect of the dental pulp cells.

Description

technical field [0001] The invention belongs to the technical field of human cell culture, and in particular relates to a method for separating and culturing human dental pulp cells. Background technique [0002] Dental pulp is a kind of loose connective tissue located in the pulp cavity, which has the ability to repair and regenerate. It contains nerves, blood vessels, lymph and connective tissue, as well as odontoblasts arranged around the pulp and odontogenic cells inside the pulp. Dental pulp stem cells. Dental pulp stem cells are adult stem cells that maintain an undifferentiated state. It has been proven to have multiple differentiation capabilities and can be used for tissue repair and regeneration of dentin, tooth body, and crown. important basis. However, there are still some difficulties in culturing dental pulp stem cells at present. When culturing dental pulp tissue, fibroblasts are often obtained instead of dental pulp stem cells, and dental pulp stem cells ar...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0664C12N2501/90C12N2500/05C12N2501/998C12N2501/999C12N2500/30C12N2509/00
Inventor 张晓南吴芳春谷涌泉侍晓云张斌吴刘兵
Owner 北京昱龙摩尔国际生物医学研究院
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