![]() ![]() Interestingly, in spite of their varying colony forming efficiency values, all of the cells from the different perinatal sources express similar levels of pluripotency markers by quantitative RTPCR analysis except the cord-placenta junction derived mesenchymal stromal cells, which expressed the highest levels of expression for all the tested pluripotency markers. The median flourescence intensity ratios, however, indicate that Wharton's jelly and cord-placenta junction derived cells are very similar or higher than the cord lining in fetal placenta derived cells. Flow side a metric analysis of the single cell suspensions isolated from these perinatal tissues indicates that the percentages of positive cells for specific mesenchymal stem cell markers from all four tissue sources are similar to those expressed by standard bone marrow derived mesenchymal cells. The colony forming efficiency value of the cord-placenta junction cells, however, is similar to that observed for bone marrow mesenchymal stem cells suggesting that cord-placenta junction derived cells have higher proliferitive and self-renewal capabilities. The cells from the cord lining and Wharton's jelly cultures exhibit colony forming efficiency values of 59 and 80 colonies respectively. Collect the cells by centrifugation, resuspending the pellet in culture medium for subculture at a one times ten to the fourth cells per centimeter squared seeding density. Then neutralize the reaction with one to two milliliters of culture medium. When the cell growth reaches 70 percent confluency, dissociate the cells in one to two milliliters of trypsin solution per flask, rotating the flask for an even coating with the enzyme solution and incubate the cultures for three minutes at 37 degrees Celsius. Change the culture medium after three days and examine the xplants by phase contrast microscopy for cell outgrowth. Next, add nine milliliters of culture medium for a two to three day incubation at 37 degrees Celsius. Then carefully aspirate the supernatant and plate 15 to 20 partially digested tissue pieces per sample into individual 75 square centimeter tissue culture flasks. Allow the tissue pieces to settle for three minutes. Use a phase contrast microscope to observe the partial digestion by visualizing the release of cells from the tissue.Īt the end of the partial digestion period, neutralize the trypsin with an equal volume of culture medium and transfer the samples into individual 50 milliliter conical tubes. When all the tissues have been dissected, replace the PBS in each dish with three to five milliliters of trypsin and use scissors to cut each tissue sample into one to two millimeter pieces for a 30 minute incubation at 37 degrees Celsius and five percent carbon dioxide. Then, remove the blood vessels, transferring any remaining perivascular jelly under and around the blood vessels into the Wharton's jelly dish and place the remaining cord lining tissue in its own dish. Use a scalpel to scrape the Wharton's jelly away from the blood vessels and interepithelium of the subamnion. ![]() Next, cut the umbilical cord longitudinally to completely expose the blood vessels and the surrounding Wharton's jelly without disturbing the epithelium. Make a second incision below the junction to separate the cord-placenta junction from the placenta.Īnd split the separated tissues into individual petri dishes. Then use forceps to grasp the fetal end of the umbilical cord and use scissors to carefully make an incision at the top of the cord-placenta junction. When all the blood clots have been removed, carefully examine the sample to identify the different anatomical regions. Begin by placing the sample in a 150 millimeter petri dish on ice in a bio safety cabinet and use a needle and syringe to rinse the tissue several times with ice cold PBS. This method can provide insight into the characteristics of mesenchymal stromal cells from different segments and specific niches of the cord and placenta. Multi-potent cord mesenchymal stromal cells derived from this technique can be used to treat various diseases and disorders since they are more primitive than mesenchymal stem cells isolated from adult tissues. The main advantage of this technique is that it productively yields large numbers of high quality homogenous cell populations from distinct perinatal tissue sources. This method can help advance the field of stem cell biology, alternative medicine by obtaining special quality mesenchymal stem cells, non-invasively. The overall goal of this protocol is to isolate mesenchymal stromal Cells from four distinct perinatal tissue sources, the umbilical cord lining, Wharton's jelly, cord-placenta junction, and fetal placenta. ![]()
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