Human dental pulp stem cells

Product attributes:

Organizational source: Dental pulp tissue

Product specifications: 5 × 105cells/T25 cell culture bottle

Cell Introduction:

Human dental pulp stem cellsSeparate from synovial tissue; The pulp tissue is located inside the pulp cavity of the tooth. The shape of the pulp cavity is roughly similar to the shape of the tooth. The pulp cavity in the crown is larger, called the pulp chamber, while the pulp cavity in the root is smaller, called the root canal. There are small holes in the apex, called the apical foramen. The pulp tissue mainly includes nerves, blood vessels, lymphatic and connective tissues, as well as odontoblasts arranged around the pulp, which play a role in dentin formation. Due to the different effects of pathogenic stimuli and differences in the body's resistance, dental pulp undergoes different pathological changes, which manifest as a series of different symptoms and signs in clinical practice. After a prolonged period of pulp congestion, it transforms into acute pulp inflammation. Mesenchymal stem cells (MSCs) originate from embryonic mesodermal tissue and have strong self replication and multipotent differentiation potential. They have the ability to differentiate into various terminal cells such as adipocytes, osteoblasts, chondrocytes, and muscle cells. The use of MSCs to repair cartilage injuries has great application prospects. Currently, MSCs can be isolated and prepared from tissues such as bone marrow, adipose tissue, synovium, bone, muscle, as well as amniotic fluid, umbilical cord, and umbilical cord blood.

Method Introduction:

The cells isolated in the company laboratory were prepared using collagenase digestion and low-density dilution cloning, with a total cell count of approximately 5 × 10? Cells/bottle.

Quality inspection:

The purity of the samples isolated from the company's laboratory by CD90 immunofluorescence identification can reach over 90%, and they do not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, fungi, etc.

Training Information:

Culture medium: containing FBS, growth additives, Penicillin, Streptomycin, etc

Fluid change frequency: Change the fluid every 2-3 days

Growth characteristics: Wall adhesion

Cell morphology: fibroblast like

Passage characteristics: Can be passed down for about 3-5 generations

Digestive fluid: 0.25%

Cultivation conditions: Gas phase: air, 95%; CO2，5%

How to freeze and preserve cells?

Freezing preservation method 1: Place the freezing tube at 4 ℃ for 30-60 minutes → (-20 ℃ for 30 minutes *) → -80 ℃ for 16-18 hours (or overnight) → store in a liquid nitrogen tank vapor phase for long-term storage.

Freezing preservation method 2: Place the freezing tube in a programmable cooling machine that has been programmed to cool down 1-3 ℃ to below -80 ℃ per minute, and then store it in a liquid nitrogen tank vapor phase for long-term storage. -At 20 ℃, it should not exceed 1 hour to prevent excessive ice crystals from causing massive cell death. This step can also be skipped and placed directly in a -80 ℃ freezer, but the survival rate is slightly reduced.

Cultivation operation:

1) Resuscitate cells: Quickly shake and thaw a cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add 4mL of culture medium and mix well. Centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1-2mL of culture medium, and blow evenly. Then add all cell suspensions to the culture bottle and culture overnight (or add the cell suspensions to a 10cm dish, add about 8ml of culture medium, and culture overnight). The next day, change the solution and check the cell density.

2) Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.

1. Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

2. Add 1ml of digestion solution (0.25% Trypsin 0.53mM EDTA) to a culture bottle and place it in a 37 ℃ incubator for digestion for 1-2 minutes. Then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, tap the culture bottle a few times, and add a small amount of culture medium to terminate digestion.

3. Add 6-8ml/bottle of culture medium, gently mix and aspirate, centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1-2mL of culture medium and blow evenly.
4. Divide the cell suspension into a new dish or bottle containing 8ml of culture medium at a ratio of 1:2.

3) Cell cryopreservation: When the cells are in good growth condition, cell cryopreservation can be performed. The following T25 bottles are classified;

When cells are frozen, discard the culture medium, wash with PBS once, and add 1ml. After the cells become round and fall off, add 1ml of serum containing culture medium to terminate digestion, which can be counted using a hemocytometer.

Centrifuge at 1000rpm for 4 minutes to remove the supernatant. Add 1ml of serum to resuspend cells, add serum and DMSO according to the number of cells, gently mix well, DMSO final concentration is 10%, cell density is not less than 1x106/ml, freeze 1ml of cell suspension in each cryovial, pay attention to labeling the cryovial.

3. Place the cryovial in a program cooling box, put it in a -80 degree freezer, and after 2 hours, transfer it to liquid nitrogen storage. Record the location of the freezer for future retrieval.

Experimental report:

The product is only used for scientific research:

1、 Separation and cultivation:

1. Under sterile conditions, extract atrial tissue from 1-3 day old SD rats, then wash the tissue block twice with PBS, and finally make it approximately 1mm3 in size;

2. Add 4mL of enzyme digestion solution (0.1% and 0.1% type I collagenase) to the tissue block, suspend for 10 seconds, digest at 37 ℃ for 10 minutes, then use a dropper to prepare a single-cell suspension, naturally precipitate and collect the supernatant, terminate digestion with 10 μ S medium, and place at 4 ℃;

3. Add 3-4 mL of enzyme digestion solution to the remaining tissue, suspend for 10 seconds, and digest at 37 ℃ for 10 minutes. Collect the supernatant according to the above method and stop digestion. Then, place it at 4 ℃ and repeat this step 2-3 times until the tissue is digested;

4. Filter the cell digestion solution through a 200 mesh stainless steel sieve, centrifuge at 1200r/min for 10 minutes, discard the supernatant, and suspend the precipitated cells in DMEM/F12 medium containing 10% FBS. Inoculate the cells into a 25cm2 culture bottle and incubate in a 37 ℃, 5% CO2 incubator;

5. After 1 hour of differential adhesion, aspirate the culture medium and inoculate it into a 6-well plate as needed for further cultivation.
2、 Immunofluorescence identification:
1. When the atrial myocytes grow to 80% fusion, discard the culture medium and wash the cells twice with warm PBS for 10 minutes each time. Then fix the cells with 4% paraformaldehyde at room temperature for 15 minutes;

2. Wash the cells twice with PBS for 10 minutes each time, and then permeate the membrane with 0.1% Triton X-100 at 4 ℃ for 15 minutes;

3. Wash the cells twice with PBS for 10 minutes each time, and then block the cells with 4% BSA at room temperature for 30 minutes;

4. Dilute the alpha actin primary antibody in a ratio of 1:100, and then incubate the cells overnight at 4 ℃ in a refrigerator;

5. Wash the cells with PBS three times, each time for 10 minutes. Dilute the secondary antibody against alpha actin in a ratio of 1:150 and place it at 37 ℃ for 1 hour;

6. Wash with PBS three times for 10 minutes each time, then observe the image under an inverted fluorescence microscope and take photos.
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