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In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells

A technology of mesenchymal stem cells and culture methods, applied in the field of in vitro isolation and culture of tooth-derived mesenchymal stem cells, can solve problems such as insufficient knowledge of stem cells, and achieve the effect of maintaining cell uniformity, maintaining cell stemness, and good cell uniformity

Inactive Publication Date: 2015-04-29
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still quite a few children who lost the first opportunity to store umbilical cord blood stem cells or umbilical cord stem cells for their children due to their parents’ lack of knowledge about stem cells in the past. The development of deciduous dental pulp stem cell storage business can make up for this regret , buy an insurance for your child's health

Method used

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  • In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells
  • In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells
  • In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells

Examples

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Embodiment 1

[0034] Example 1 Preparation of tooth-derived mesenchymal stem cell culture medium.

[0035] The mononuclear cell culture fluid provided by the present invention includes basic culture fluid and newborn fetal bovine serum, specifically: α-MEM basic culture+10% newborn fetal bovine serum+L-ascorbic acid-2-phosphate sodium+L-glutamine + Penicillin / Streptomycin.

Embodiment 2

[0036] Example 2 Isolation and culture of tooth-derived mesenchymal stem cells.

[0037] Specific steps for the isolation and culture of dental-derived mesenchymal stem cells:

[0038] 1]. Tooth preservation solution: sterile saline, stored at 4 degrees for no more than 48 hours.

[0039] 2]. Specimen transportation and pretreatment: Scrape off the gums and surrounding tissues on the surface of the teeth, and then use iodine and 75% alcohol to clean the surface of the teeth in order to prevent the contamination of oral bacteria. Then wash with normal saline 5 times to remove iodine and alcohol.

[0040] 3]. Isolation and culture of cells: Wrap the tooth with sterilized gauze, place the pulp cavity towards the middle, place it in a vise, and gently crush the tooth to expose the pulp. The culture medium was added to the centrifuge tube, and the dental pulp was collected into the centrifuge tube with sterile tweezers, and then cut into pieces with scissors. Add 3 mg / mL type Ⅰ ...

Embodiment 3

[0045]Example 3 CFU-F assay of tooth-derived mesenchymal stem cells.

[0046] Primary CFU-F analysis: with 2 x 10 5 / cm 2 Dental pulp cells were planted in 3 wells of a six-well plate, culture medium was added, cultured in a standard incubator, and the medium was changed for 48 hours; the medium was changed every 3-4 days thereafter. When the adherent cells form more than 50 cell clones (about 7-11 days), count CFU-F under a microscope, or stain with toluidine blue to count the number of CFU-F. The average value of 3 wells is the number of CFU-F. Remarks: Due to the low yield of dental pulp cells, primary CFU-F analysis was not performed.

[0047] CFU-F analysis of passaged cells (dental pulp DPSCs): 100 cells were planted in 3 wells of a six-well plate, culture medium was added, cultured in a standard incubator, and the medium was changed every 3-4 days. After culturing for 10 days, count CFU-F under a microscope, or stain with toluidine blue to count the number of CFU-F...

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Abstract

The invention discloses an in-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells. The in-vitro separation and cultivation method comprises the following steps: (A), gingiva on a tooth surface and surrounding tissue are scraped off, and the tooth is stored in tooth preserving fluid; (B), a culture solution is added to a centrifuge tube, dental pulp is collected to the centrifuge tube and cut into pieces by scissors; (C), collagenase type I and dispase are added to digest dental pulp tissue; (D), single-cell suspension is prepared; (E), the obtained single cell is subjected to primary culture, and the planting density of primary cells is (1-5)*10<4> / cm<2>; and (F), the cells are subjected to subculturing. The tooth is crushed with a physical method to obtain the dental pulp tissue, the collagenase type I and the dispase are used for digestion, the single-cell suspension is obtained, primary planting and culturing are performed for 10-15 days, and then passage is performed; and the problems that the primary cells are mixed with other components of the tooth and aging occurs probably in a cell mass culture process are solved effectively.

Description

technical field [0001] The invention belongs to stem cell culture technology, in particular to a method for in vitro separation and culture of tooth-derived mesenchymal stem cells. Background technique [0002] In 2000, Gronthos first proposed the concept of dental pulp stem cells (DPSCs), and named a group of stem cells extracted from the dental cavity, which have the ability to form clones and a high proliferation rate in vitro, as DPSCs. In 2003, Miura isolated stem cells with multi-differentiation ability from the crown and root pulp of exfoliated deciduous teeth, and named them stem cells from human exfoliated deciduous teeth (SHED). When cultured in vitro, SHED has high proliferative ability and colony formation ability, and can differentiate into nerve cells, osteoblasts and odontoblasts, and can form bone and dentin after the cells cultured in vitro are transplanted back into mice. [0003] Studies on dental pulp stem cells have shown that dental pulp stem cells c...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 李陶曾桂芳何毅欣曾晓萍胡祥刘沐芸
Owner SHENZHEN BEIKE BIOTECH
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