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Culture medium for inducing differentiation of dental pulp stem cells into osteoblasts, and preparation method and application of culture midium

A technology of dental pulp stem cells and osteoblasts, applied in the field of stem cells, can solve the problems of high cost, long induction time, and more energy invested by personnel, and achieve the effect of low cost and short induction time

Inactive Publication Date: 2020-02-21
GUANGZHOU SALIAI BIOLOGICAL GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fetal bovine serum is expensive, and foreign imported fetal bovine serum is difficult to purchase
Moreover, when the induction medium containing fetal bovine serum induces the differentiation of stem cells, the induction time is longer than that of serum-free induction differentiation medium
Therefore, the resulting cost is higher and the effort of personnel is more

Method used

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  • Culture medium for inducing differentiation of dental pulp stem cells into osteoblasts, and preparation method and application of culture midium
  • Culture medium for inducing differentiation of dental pulp stem cells into osteoblasts, and preparation method and application of culture midium

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preparation example Construction

[0034] The present invention also provides a method for preparing the above culture medium, comprising the following steps:

[0035] BSA, reduced glutathione, β-mercaptoethanol, SITE, β-glycerophosphate, L-ascorbic acid, dexamethasone and low-sugar DMEM medium added with amino acid concentrate were mixed to obtain a medium for dental pulp stem cells. Osteoblast differentiation medium.

[0036] Wherein, the mixing condition is that the temperature is 25±5°C.

[0037] The present invention also provides a method for inducing differentiation of dental pulp stem cells into osteoblasts, comprising the following steps:

[0038] The expanded and cultured dental pulp stem cells were inoculated in the above medium for inducing differentiation of dental pulp stem cells into osteoblasts for induction culture.

[0039] Wherein, the expanded and cultured dental pulp stem cells are prepared according to the following method:

[0040] After the pulp tissue is cut into pieces, it is digest...

Embodiment 1

[0053] Isolation and Expansion of Dental Pulp Stem Cells

[0054] 1) Materials:

[0055] Select the third molars of 12-18-year-old healthy donors who need to be extracted for orthodontics. Place the teeth in 4°C pre-cooled PBS, move them to the cell chamber, split the crown and root under sterile conditions, take out the pulp, and scissor The pulp tissue about 2 mm from the root tip was removed, and the pulp tissue was fully shredded under PBS infiltration.

[0056] 2) Digestion:

[0057] Add an appropriate amount of collagenase (3mg / mL) and Dispase (4mg / mL) 1; 1 to the mixed digestion solution, place it in a constant temperature shaker at 37°C for 30-40min, after digestion, centrifuge at 1000r / min for 5min, discard the supernatant . Add an appropriate amount of PBS to resuspend and centrifuge at 1000r / min for 5min, discard the supernatant.

[0058] 3) Vaccination:

[0059] Add culture medium (DMEM culture medium containing 10% fetal calf serum) to resuspend the cells, in...

Embodiment 2

[0065] (1) Preparation of serum-free osteogenic differentiation medium:

[0066] Serum-free osteogenic differentiation medium: containing 5g / L BSA, 1.5mg / L reduced glutathione, 0.1mmol / L β-mercaptoethanol, 1% (V / V) SITE, 1M β-glycerophosphate, 20 μM -Ascorbic acid, 1mM dexamethasone and the low-sugar DMEM medium that added the amino acid concentrate (the glucose concentration in the low-sugar DMEM medium is 1g / L. The amino acid concentrate is added with 1.5 mL of MEM amino acid solution and 0.5 mL of MEM non-essential amino acid solution).

[0067] The SITE is formed by mixing equal concentrations of Sibiricoside-A aqueous solution and THF aqueous solution at a volume ratio of 1:1, and the volume concentration of Sibiricoside-A solution and THF solution is 2%.

[0068] (2) Osteogenic differentiation:

[0069] Control group: take P in the logarithmic growth phase 4 Substitute dental pulp stem cells, the cells were seeded in a 6-well plate, the control group was set up with 3...

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Abstract

The invention provides a culture medium for inducing differentiation of dental pulp stem cells into osteoblasts. The culture medium comprises 4-5.5g / L of bovine serum albumin (BSA), 1.5mg / L of reducedglutathione, 0.08-0.12mmol / L of beta-mercaptoethanol, 0.08%-1.5%(V / V) of SITE, 0.8-1.2M / L of beta-glycerophosphoric acid, 15-25[mu]M / L of L-ascorbic acid, 0.5-1.2mM / L of dexamethasone and the balanceof low-sugar dulbecco's modified eagle medium (DMEM) culture medium added with an amino acid concentrated solution. The culture medium for inducing differentiation of dental pulp stem cells to osteoblasts is a serum-free culture medium, and the culture medium can be used to optimize a method for inducing differentiation of dental pulp stem cells to osteoblasts. The induction time is short and cost is low, and the invention provides a new selection for culture of seed cells in bone tissue engineering.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a culture medium for inducing differentiation of dental pulp stem cells into osteoblasts, a preparation method and application thereof. Background technique [0002] Dental pulp stem cells (Dental Pulp Stem Cells, DPSC) refer to undifferentiated cells or precursor cells that exist in the dental pulp tissue of the adult body with self-renewal and multi-directional differentiation potential, and have similar biology to other tissue adult stem cells. It has the characteristics of multi-directional differentiation potential, wide range of sources, safety and small immune rejection. Experiments have confirmed that DPSCs have multi-directional differentiation potential, and DPSCs express bone-derived proteins such as type I collagen, osteonectin, osteopontin, osteocalcin, and type I collagen. [0003] Human bone marrow mesenchymal stem cells (BMMSCs) have been proven to...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N2500/38C12N2500/42C12N2500/44C12N2501/39C12N2501/998C12N2501/999
Inventor 王小燕岳坤张海照张满
Owner GUANGZHOU SALIAI BIOLOGICAL GENETIC ENG CO LTD
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