Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

The overall goal of this procedure is to isolate the epithelial cell population from a human amnion membrane. This is accomplished by first stripping the amnion membrane from the corion membrane and the placenta in the second step. The amnion membrane is washed of all traces of blood and then digested with an animal product free trypsin enzyme solution.

In the final step, the tissue slurry is filtered to collect the isolated epithelial cells. Ultimately, the purity of the amnion epithelial cell population can be assessed by flow cytometric analysis. The main advantage of this technique over existing methods is that it doesn't require exposure of cells to animal animal products, meaning that these cells are useful for clinical trials.

To isolate the amnion epithelial cells begin by placing the placenta onto a sterile surface within a class two biological safety cabinet. Then use sterile HBSS to wash as much blood as possible from the placenta surface. Next, situate the placenta so that the umbilical cord and placental membranes are facing up and manually strip the amnion membrane from the corion membrane of the placenta working around the edge of the membrane towards the umbilical cord.

Take care to remove any pieces of contaminating corion membrane from the amnion, and then use sterile scissors to cut the amnion membrane approximately two centimeters from the base of the umbilical cord and place the membrane in a 500 milliliter receptacle containing 250 milliliters of HBSS seal the container and shake it thoroughly to wash the tissue, and then use sterile forceps to transfer the membrane into a 15 centimeter Petri dish. Now, cut the membrane into five centimeter long vertical pieces and wash the pieces three to five more times as just demonstrated until the membrane becomes translucent with no contaminating blood. When all the blood has been removed, transfer the tissue pieces into 50 milliliters of Enzymatic Digest solution, taking ker to minimize the carryover of any HBSS and incubate the amnion tissue in a warm room at 37 degrees Celsius with gentle agitation to remove any of the remaining blood after 15 minutes, drag each piece along the inside rim of the container to drain off the old digestion solution and add the pieces to 100 milliliters of fresh enzymatic digest solution.

After an hour digestion under the same conditions, transfer the membranes into a new container of Enzymatic Digest solution. Taking care to drain the tissues as just demonstrated and gently agitate the membranes for a final 60 minutes following the second digestion. Strain the cell suspension through a 70 micrometer filter, and then spin down the cell slurry for 10 minutes At 1000 Gs and room temperature, gently resuspend the pellet in four milliliters of soybean based enzyme inhibitor to create a single cell suspension, and then add HBSS to the cells to a final volume of 10 to 20 milliliters.

Determine the number of viable cells by triam blue exclusion, and then to cryopreserve the isolated amnion epithelial cells. First, bend them down for five minutes at 700 cheeses and room temperature. Then re suspend the pellet at one times 10 of the six to one times 10 of the seventh cells per milliliter in animal product free cryo-preservation media and pipette a suitable volume of cells into o ringed cryo-preservation vials.

Finally place the vials into a controlled rate freezing apparatus where cooling occurs at approximately one degrees Celsius per minute until minus 80 degrees Celsius is achieved, and then transfer the vials into liquid nitrogen for long-term storage. Many animal product free media perform suitably. However, the optimal DMSO concentration is approximately five to 10%Typically, the expected viability post thaw is five to 10%less than the pre cryopreservation viability and can be significantly greater for the inexperienced user.

Cell culture can be performed with human amnion epithelial cells to enable the characterization or differentiation of the cells for further downstream analysis as they maintain their epithelial phenotype in serum free culture media. In addition to possessing immunoregulatory and anti-inflammatory properties, human amnion epithelial cells have been shown to be highly multi potent, in vitro and in vivo, differentiating into cell lineages representative of the three primary germ layers After its development. This technique paved the way for researchers to perform clinical trials for conditions such as lung disease and graft versus host disease.