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. This method can help advance the field of stem cell biology, alternative medicine by obtaining special quality mesenchymal stem cells, non-invasively. The main advantage of this technique is that it productively yields large numbers of high quality homogenous cell populations from distinct perinatal tissue sources.

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. This method can provide insight into the characteristics of mesenchymal stromal cells from different segments and specific niches of the cord and placenta. 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.

When all the blood clots have been removed, carefully examine the sample to identify the different anatomical regions. 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. Make a second incision below the junction to separate the cord-placenta junction from the placenta.

And split the separated tissues into individual petri dishes. Next, cut the umbilical cord longitudinally to completely expose the blood vessels and the surrounding Wharton's jelly without disturbing the epithelium. Use a scalpel to scrape the Wharton's jelly away from the blood vessels and interepithelium of the subamnion.

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. 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. Use a phase contrast microscope to observe the partial digestion by visualizing the release of cells from the tissue.

At 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. Allow the tissue pieces to settle for three minutes. Then carefully aspirate the supernatant and plate 15 to 20 partially digested tissue pieces per sample into individual 75 square centimeter tissue culture flasks.

Next, add nine milliliters of culture medium for a two to three day incubation at 37 degrees Celsius. Change the culture medium after three days and examine the xplants by phase contrast microscopy for cell outgrowth. 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.

Then neutralize the reaction with one to two milliliters of culture medium. 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. The cells from the cord lining and Wharton's jelly cultures exhibit colony forming efficiency values of 59 and 80 colonies respectively.

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. 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 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.

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. Further, mesenchymal stromal cells isolated from all of the perinatal sources readily differentiate into adipogenic, chondrogenic, and osteogenic cell types as well as demonstrated trilineage differentiation. Although the potential of differentiation varies according to the mesenchymal stromal cell source.

Once mastered, this technique can be completed in two to three hours if it's performed efficiently. While attempting this procedure, it is important to practice lateral technique and avoid cross contamination between the tissue sources. This technique paved the way for researchers to explore the nature of stem cell niches in perinatal tissues.