Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

The overall goal of this procedure is to culture human mesenchymal stem cells on porous bone matrix. This is accomplished by first isolating mesenchymal stem cells from human amniotic membrane or AM hcs. Next, the porous bone matrix discs are prepared to favor the correct distribution of nutrients for cells to grow.

Then the AM hcs are cultured on the porous bone matrix discs. Finally, colony forming units are counted and morphology. Cell proliferation and cell adhesion are all analyzed.

Ultimately, cellular morphology, number of colony forming units, cell proliferation and cell adhesion are analyzed through optic microscopy staining with vital dyes and scanning electron microscopy respectively. The implications of this technique extend to our therapy of bone injuries because it's possible to deliver the mesenchymal stem cells into macroporous biomaterials that mimic the natural bone. To isolate mesenchymal stem cells from human amniotic membrane, hold the umbilical cord with one hand, and with the other hand detach the amniotic membrane, which looks like a translucent sheet.

If the choon is present in the sample, manually dissect it to separate it from the amnion tissue. Use five milliliters of PBS to wash the sample three times to remove residual blood and use simple forceps to remove blood clots with a scalpel. Make small cuts in the amniotic membrane to generate fragments of approximately 0.5 square centimeters.

To digest the membrane, place the fragments in a 50 milliliter conical tube and add five milliliters of 0.125%trypsin 0.5 millimolar EDTA solution. Incubate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes before spinning at 200 Gs and 25 degrees Celsius for five minutes. Then discard the supernatant.

Next, add 15 milliliters of collagenase type two solution and incubate for two hours at 37 degrees Celsius with occasional shaking according to the protocol of lava at al. Immediately after digestion, add 15 milliliters of PBS and centrifuge at 200 Gs and 25 degrees Celsius for 10 minutes. Discard the supernatant and use PBS to wash the cell pellet before spinning again for 15 minutes.

After discarding the supernatant, add 10 milliliters of H-G-D-M-E-M to the pellet and resuspend by gently shaking. To expand mesenchymal stem cells. Seed two milliliters of cell suspension in a culture flask and add two milliliters of H HG DMEM incubate.

And after five to seven days, remove non-adherence cells by changing the culture medium. Then every three days for an additional seven to 10 days, change the culture medium until the cells are 90%confluent. Add 0.125%tripsin EDTA and incubate for five minutes before aspirating the cell suspension and transferring it to a 15 milliliter conical tube.

After spinning the cells and discarding the supernatant, use HG DMEM to resuspend the cell pellet culture. The cells in 2 75 square centimeter flasks and maintain the culture at 90%confluence. Repeat the ization and rep plating until the ninth passage or subculture.

Then recover the cells for flow cytometry or other studies. To prepare porous bone matrix discs. Begin by adding three milliliters of HG DMEM without FBS to each NKB disc and incubate overnight in a humidified atmosphere according to FAA etal the following day.

Place each disc in a 50 milliliter conical tube and spin at 200 GS for five minutes to remove the excess culture medium, then transfer to a 24 well culture plate. Add 500 microliters of H-G-D-M-E-M and ensure that no air bubbles are present on the discs to favor the correct distribution of nutrients and cells. Incubate for 30 minutes at 25 degrees Celsius from three subcultures slowly and continuously.

Seed a cellular suspension on the surface of the pre wedded biomaterial disc and incubate for 30 minutes After the incubation, carefully add 1.5 milliliters of pre-war H-G-D-M-E-M and maintain the culture for three days to carry out a cell proliferation assay, add 150 microliters of vital colorant AB to the cell disc samples according to the manufacturer's guidelines. Immediately measure the absorbance at 570 nanometers with a reference wavelength of 600 nanometers. Incubate the cells for seven days measuring the absorbance every 24 hours.

Measure colony forming units by culturing 100 cells per 100 millimeter tissue culture disc in H-G-D-M-E-M and incubate for 14 days. Use PBS to wash the culture and use 0.5%crystal violet in methanol to stain the cells at room temperature for five to 10 minutes. Use PBS to wash the plates twice before using a microscope to count the cells as seen in these flow cytometry histograms.

The cell population isolated from amniotic membrane was strongly reactive to the surface mesenchymal markers CD 90, CD 73, and CD 1 0 5 and reacted negatively to the hematopoietic markers CD 34 and CD 45 indicating that the AM HSCs were in fact mesenchymal. As shown in these scanning micrographs, the surface of the biomaterial was covered with spherical cells on day one and the AMCs apparently attached to the bovine matrix surface on the seventh day. Via fill podal processes at higher magnification, the cells establish contact with each other.

This graph shows a small decrease in relative absorbance units of bovine matrix. After one day of incubation, the presence of the scaffold induces an increase in the cell proliferation, statistically significant in comparison to the culture performed in the absence of bovine matrix using the colony forming unit assay to analyze stem cells. This graph illustrates that the AM HSCs isolated as demonstrated in this video, and plated at varying densities preserved their ability to form discrete colonies at 14 days in culture.

After watching this video, you should have a good understanding of how to deliver the mesenchymal stem cells and to macroporous biomaterials.