The overall goal of this procedure is to isolate, characterize, and differentiate human gentile pulse stem cells from permanent teeth by using two methods. This is accomplished by collecting healthy impacted wisdom teeth, cutting them around cement to enamel junction at the beginning. Dental poly stem cells D PSCs are isolated by two different methods.
In the first method, polyp tissues are enzymatically digested by incubating in collagenase type one plus disc space solution. Here we call them ed. Considering the isolation method in the second method, palp tissues are only placed into the flask without any digestion.
In this way, dpss begin to migrate from tissue into the flask. These cells named OG refer to the outgrowth isolation method. The second step of the procedure is the identification of stem cells using fall cytometry.
The third step of the procedure is the induction of odontoblast differentiation, and the final step of the procedure is the comparative evaluation of odontoblast differentiation between two groups by in red staining and QBCR. I'm RA Kde from Department of Stem Cells and Developmental Biology at Rian Institute. Today I'm going to show you the isolation characterization and comparative differentiation of human mesenchymal stem cells by using two methods.
Hello, I am Dr.Rezaian from Rio Institute. We are working on a project related to human dental pulp stem cells. These cells are kind of mechy stem cells, which are easy to obtain with minimum pain and morbidity.
Mechy stem cells specify with plastic aran ability, formation of colonies, and multiple differentiation capacity. Hello, I'm Dr.Ami from the Department of Stem Cells. At institute, we use this procedure in our lab to isolate stem cells for the purpose of research studies and future applications.
In regenerative medicine, the very first step for using this source of stem cells for future stem cell based therapy is to choose the best protocol for isolating stem cells from human part tissue. In order to achieve this goal, it is crucial to investigate certain aspects of cellular behavior under different stem cell isolation conditions. First, I'm going to isolate human dental pulp stem cells by using either enzymatic dissociation of pulp or outgrowth of stem cells from tissue implants.
Then characterize and differentiate them into ogen blast. So let's get started. First way, space and collagenase Type one dissolve both in PBS.
Then filter them using oh 0.2 micron syringe filter. Pull both into conical tube. Then add pener and PBS to achieve final concentration.
Transfer healthy wisdom teeth into the lab in the cold basic medium under the steel condition. Clean surface of the tooth by 70%ethanol before studying. Make sure to clean up the breeze from the surface of the tooth.
Cut tooth around the cement enamel junction by using sterilized dental disc to reveal pulp chamber. It should be considered that cutting process must be performed slowly to reduce overheating of dental tissue. Then gently separate pulp tissue from the crown means pulp tissue into one to two millimeter pieces with the Scarpa blade.
Transfer small pieces of pulp tissues into one milliliter enzyme solution for one hour at 37 degrees Celsius vortex every 30 minute to help breaking up pulp tissues. Afterwards, remove large aggregates by passing them through a 70 micron cell strain. Then add PBS containing PENER centrifuges in 1, 200 RPM for five minutes.
Remove the supernat carefully. Then we suspend the plate in the proliferation medium pm transfer it into the culture flask and add medium. Then incubate incubated.
Change the medium every three days until the cell confluence is achieved for the outwork method. Repeat the cutting process. After cutting tooth means pop tissues into one to two millimeter fragment.
Then place them into the culture. Flash with proliferation, medium and incubated. It should be considered that total volume of the proliferation medium must be support the attachment of all pieces for further cell outgrow.
Change the medium after observing outgrowth and then every three days until the cell confluence is achieved. For immunophenotyping, incubate both types of D PSCs with PE or FITC conjugated antibodies for 30 minutes at four degrees Celsius in dark. Then add PBS and centi views at 1, 200 RPM for five minutes.
Remove the supernatant and resus, suspend the plate in PBS and finally evaluate surface markers by using flow cytometer subculture, both types of DPCs for three passages. Then tripsin ice and transfer them to six red culture dishes at 60%Co fluency replace PM by odontogenic medium. Three wells are remain as a negative control by adding pm.
Change the medium every three days at day 21st wash cells with PBS. Then fix them by one milliliter per well. 10%formal gel height for 15 minutes at home temperature.
After 15 minutes, remove the fixative carefully and wind cells three times with distilled water. Then replace water by one milliliter per red alza in red stain solution. After 20 minutes, we remove excess dye and wash cells four times with deionized water.
Then add one millimeter per well water to prevent the cells from drying. After the staining, you can see three up rails turn into red compared to the controls under the microscope, you can see dis stain color absorption around the cell by large magnification for quantification of a red staining. After removing water, add one milliliter per well, 10%acidic acid, then incubated for 30 minutes with shaking.
Then gently scrape the cells from the plate by using a cell scraper. Then transfer them into the separate tubes. VOR takes vigorously for 30 seconds.
Then heat them to 85 degrees Celsius for 10 minutes. To avoid evaporation seal tubes with ParaView transfer tube to ice for five minutes, they incent used them at 20, 000 G for 15 minutes. Meanwhile, make Alzheimer red standard serial dilution according to the region protocol.
After the centrifugation, remove the snat and transfer them into the new tubes. Neutralize the pH with 10%ammonium hydrocyte. Then add standards and also samples into 96.
Well plate the then measure the absorbance at 405 nanometer and analyze data according to the standard serial dilution. Here you can see DPCs, which are isolated by enzymatic dissociation on day 10th, 15th and 18th, and also outgrown DPCs on day fifth, 10th, 13th and 18th. Both types of dpss in sase three are almost in the same size and morphology Immunophenotyping results show the presence of mesenchymal stem cell marker such as CD 44, CD 73, and CD 19, and the absence of hematopoietic and endothelial markers such as CD 34, CD 45, and CD 11 B.Interestingly, the expressions of CD 1 0 5 and CD 146 are more in outgrown d PSCs in comparison with D-P-S-C-E-D, the quantification of enzyme red staining on day 21st of odontoblast differentiation show more calcium deposition in D-P-S-C-E-D in comparison with without con dental top of stem cells.
QPCR results also indicate significant higher expressions of MEP and A LP as the mineralization markers in D-P-S-C-D compared to the outgoing dental of stem cells. Meanwhile, both genes are regulated during the differentiation and also the expression of DSPP as the odontogenic marker increased during the differentiation. However, there is no significant variation in the expression of DSVP in differentiated cells between ED and OG groups.
We've just showed you how to isolate human dental parts stem cells by using either enzymatic dissociation of pulp or outgrowth of stem cells from tissue explan. So that's it. I wish you good luck to try to use this procedure in your experiment.
Thank you.
