Isolation and culture of dental pulp mesenchymal stem cells

• A brief introduction of Dental Pulp Stem Cells（DPSCs）

Dental pulp stem cells is a kind of mesenchymal stem cells, fibrous cells isolated in pulp tissue within the tooth, self-renewal and differentiation into bone, fat, cartilage, muscle and nerve cell types. There are many kinds of dental stem cells, found in a variety of dental stem cells, have considerable potential in the teeth have been found a variety of stem cells, including: stem cells from human exfoliated deciduous teeth, pulp stem cells，stem cells from apical papilla and periodontal stem cells and so on. Therefore, at present, the bank has teeth, to keep off the teeth, can isolate stem cells as a possible future application of deciduous teeth, also can be found in permanent teeth in dental pulp stem cells and has been shown to differentiate into odontoblasts, promote dentin regeneration effect applied to the treatment of dental caries.

• Isolation and culture of dental pulp mesenchymal stem cells
  

1. Isolation and culture of dentalpulp stem cells:

(1) The fresh, fresh pulp is placed in a 10 cm petri dish, with DPBS cleaning 3 times and operating procedures

Avoid pollution as much as possible.

(2) Then add 5ml complete culture medium (complete medium configuration: DMEM/F12 + 20% Plus Advanced Growth Supplement (StemBo Bioscience.) + 1% l-glutamine + 1% NEAA + 1% double reactance) infiltrating pulp tissue and shearing it with aseptic surgery

(3) To cut up it organization block is fixed in a petri dish, at 37 ℃ and 5% CO2 incubator incubation for 30 min to make organization block stick in a petri dish, wall.

(4) The 2 ml complete medium was added to the 6 cm petri dish.

(5) Change the fluid at 48 h, and then replace it every 3 to 4 days.

Training about 10 ~ 14 days there will be cells from the tissue block, the cells after reaching 80% ~ 90%, with pancreatic enzyme - EDTA or TrypLE in 37 ℃ digestion, represented by 1:3 proportion.

2. Dental pulp MSC transfer and culture:

(1) Absorb the medium in the dish, then add the appropriate amount of DPBS to wash it gently.

(2) Based on petri dishes right amount to join the pancreatic enzyme - EDTA, at 37 ℃ and 5% CO2 incubator role in 2 ~ 3 min, use

The straw was blown to the bottom of the petri dish, and the cells were observed to fall off completely.

(3) According to the use of trypsin - EDTA, the total culture medium was added to the cell, and it was mixed with the cell and then the 1200 x RPM centrifuge for 3 ~ 5min.

(4) The cell mass was suspended with 3ml ~ 10ml full media.

(5) Carried out in accordance with the 1:3 through the proportion of 1:4 batches or cell density according to the 30000 ~ 60000 / ml cells inoculated to T25 and T75 subculture in cultivation bottles or other containers, into the cultivation in 37 ℃ and 5% CO2.

(6) The culture medium was changed every 2 days, and 2 ~ 4 days were cultivated. When the fusion degree of the cells reached about 80 %, the cells were harvested from 1 ~ 3 of the operation steps.

(7) Cells cryopreserved: cell mass suspended in right amount (0.5 ~ 1 x 106 cell/ml) pulp MSC freeze-stored liquid (90% Plus Advanced Growth Supplement + 10% DMSO) 2 ~ 8 ℃ refrigerator placed 30 min, 24 h - 80 ℃ refrigerator, into liquid nitrogen tank cryopreserved.

* identification analysis: using flow analyzer to identify CD73 and CD90

Note: this method is only for reference. Users can make reasonable adjustments according to their own experience.

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