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Primary separation and culture method for periodontal ligament stem cells

A technology of periodontal ligament stem cells and periodontal ligament cells, which is applied in the field of stem cell culture, can solve the problems of time-consuming, expensive, and long digestion time, and achieve the effects of improved enzymatic hydrolysis, low cost of pancreatic enzymes, and reduced costs

Active Publication Date: 2015-11-18
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the primary method of tissue culture takes a long time
Among the enzymatic hydrolysis methods, type I collagenase enzymatic hydrolysis and dispase enzymatic hydrolysis are expensive, and the digestion time is longer

Method used

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  • Primary separation and culture method for periodontal ligament stem cells
  • Primary separation and culture method for periodontal ligament stem cells
  • Primary separation and culture method for periodontal ligament stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Primary isolation of periodontal ligament of deciduous teeth

[0035] 1) The method of enzymatic hydrolysis with type I collagenase and dispase is compared with the present invention.

[0036] Take 6 deciduous teeth and perform the following operations respectively: place them in PBS containing double antibodies, transfer them to the laboratory, and put the teeth in 75% ethanol for 1 min for disinfection. Then add 10mL PBS containing double antibody and wash twice. Finally, the periodontal ligament tissue in 1 / 3 area of ​​the tooth root was scraped with a scalpel blade, placed in PBS, centrifuged at 400 g for 5 min, and the supernatant was removed to obtain the cleaned periodontal ligament tissue.

[0037] Divide 6 teeth into two groups, three teeth in each group, add mixed enzyme 1 of 10 times the volume of periodontal ligament tissue after cleaning to group 1, mixed enzyme 1 is type V collagenase with mass volume concentration of 0.1% and mass volume concentration 0...

Embodiment 2

[0057] Primary isolation of periodontal ligament stem cells from third molars (wisdom teeth)

[0058] The teeth were placed in PBS containing double antibodies, transferred to the laboratory, and the teeth were disinfected in 75% ethanol for 1 min. Then add 10mL PBS containing double antibody and wash twice. Finally, the periodontal ligament tissue in 1 / 3 of the root area was scraped with a scalpel blade, placed in PBS, centrifuged at 800g for 3 minutes, and the supernatant was removed. Add 10 times the volume of periodontal ligament tissue mixed enzyme, the mixed enzyme is a mixture of V-type collagenase with a mass volume concentration of 0.25% and trypsin with a mass volume concentration of 0.05%; After digestion disappeared, centrifuge at 800g for 10min, remove the supernatant, add 20mL PBS, pipette evenly, pass through a 70μm mesh sieve, sample and count, centrifuge at 800g for 5min, add complete medium, adjust the cell density to 1×10 4 , seeded in a 6-well plate, and ...

Embodiment 3

[0063] Primary isolation of premolar periodontal ligament stem cells

[0064] The orthodontically extracted premolars were placed in PBS containing double antibodies, transferred to the laboratory, and the teeth were sterilized in 75% ethanol for 5 minutes. Then add 10mL PBS containing double antibody and wash twice. Finally, the periodontal ligament tissue in 1 / 3 of the root area was scraped with a scalpel blade, placed in PBS, centrifuged at 200 g for 10 min, and the supernatant was removed. Add 10 times the volume of periodontal ligament tissue mixed enzyme, the mixed enzyme is a mixture of V-type collagenase with a mass volume concentration of 0.05% and trypsin with a mass volume concentration of 0.25%; 37°C, 100rpm for 22 minutes after the tissue is completely digested and disappears , centrifuge at 200g for 10min, remove the supernatant, add 20mL PBS, pipette evenly, pass through a 70μm mesh sieve, sample and count, centrifuge at 200g for 5min, add complete medium, adju...

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Abstract

The invention discloses a primary separation and culture method for periodontal ligament stem cells, which comprises the following steps: separating periodontal ligaments, carrying out enzymolysis on periodontal ligament tissues by a mixed enzyme of collagenase V of which the mass-volume concentration is 0.05-0.25% and pancreatin of which the mass-volume concentration is 0.05-0.25%, carrying out primary culture on periodontal ligament stem cells, and separating the periodontal ligament stem cells by adopting a limiting dilution method. The primary separation and culture method disclosed by the invention has the following advantages: (1) the cost is reduced because the cost of the pancreatin is lower than that of dispase; (2) the digestion time is shortened, the periodontal ligaments are dense connective tissues, the affinity of the collagenase V to the connective tissues is superior to that of collagenase I, and the collagenase V and the pancreatin are combined, so that the enzymolysis effect is obviously improved.

Description

technical field [0001] The invention relates to a method for primary isolation and culture of stem cells, in particular to a method for primary isolation and culture of periodontal ligament stem cells. It belongs to the field of stem cell culture. Background technique [0002] Stem cells are a type of cells with self-replication, self-renewal, and differentiation potential. They can be divided into embryonic stem cells and adult stem cells, which can be isolated from various adult tissues such as fat, bone marrow, umbilical cord, and placenta. The periodontal ligament is the connective tissue connecting the cementum and the alveolar bone, and has functions such as support, sensation, and nutrition. Recent studies have shown that undifferentiated mesenchymal stem cells also exist in periodontal ligament, and mesenchymal stem cells participate in the regeneration of periodontal tissue. [0003] Periodontal disease is a common and frequently-occurring disease in human oral di...

Claims

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Application Information

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IPC IPC(8): C12N5/074
Inventor 陈海佳王一飞葛啸虎麦锦连
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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