This protocol can help answer key questions about heterogeneity and functional properties of human amniotic epithelial cells isolated from amniotic membrane. While previous protocols isolated cells from co-amniotic membrane without discerning between regions, our protocol allows the culture of cell population isolated from three distinct anatomical regions. To separate the amniotic membrane by region, in a biosafety cabinet under sterile conditions, identify the umbilical amnion enveloping the umbilical cord, the placental amnion covering the decidua basalis, and the reflected region, which is considered the rest of the amnion that is not attached to the placenta.
To dissect the umbilical amnion region, use dissecting forceps to hold the portion of the amnion membrane that covers the junction of the placenta and the umbilical cord, and use a scalpel to dissect the region that surrounds the cord, while stretching to separate the region from the chorion. Then, deposit the separated tissue in a labeled beaker with 100 milliliters of saline solution. To dissect the placental amnion region, use a sterile cotton gauze to remove the blood clots from the surface of the chorion/amnion that covers the placenta.
And use dissecting forceps to grasp the membrane on the border between the placenta and the reflected region. Use a scalpel to cut along the circumference of the placenta and separate the placental amnion from the chorion, being careful not to cut any vessels from the placenta. Place the separated tissue in another labeled beaker with 300 milliliters of saline solution.
And separate the rest of the amniotic membrane that is not attached to the placenta from the chorion. Then, place the reflected region in another labeled beaker with 300 milliliters of saline solution. To wash the membranes, use tweezers to place them on a sterile surface to be able to discard the saline solution from each container of tissue.
Place the membranes back in the container and add 100 milliliters of fresh saline solution to the umbilical region and 300 milliliters of fresh saline solution to the placental and reflected regions. Use dissecting forceps to stir the membranes to remove any blood residue and place the membranes on the sterile surface to be able to discard the saline solutions. Then, wash the membranes at least two more times, as just demonstrated, until the tissues are translucent.
For enzymatic digestion of the different amniotic membrane regions, cut the reflected and placental regions into two or three fragments. And place the fragments and the umbilical region into individual 50-milliliter centrifuge tubes. Add 20 milliliters of 0.5%trypsin-EDTA to each tube of reflected and placental region tissue fragments and five milliliters of 0.5%trypsin-EDTA to the tube containing the umbilical region tissue.
Shake the tubes gently for 30 seconds. Then place the membranes on a sterile surface, discarding the trypsin-EDTA solution and replace it with 30 milliliters of fresh 0.5%trypsin-EDTA for the reflected and placental region tissue fragments and 15 milliliters of fresh 0.5%Trypsin-EDTA for the umbilical region tissue. Then place the tubes in a rotator inside an incubator for a 40-minute rotation at 20 rotations per minute at 37 degrees Celsius.
At the end of the incubation, transfer the entire volume of cell solution into new conical tubes. And add two times the volume of 37-degree-Celsius human AE cells medium to each tube on ice. Digest any leftover tissue fragments in the original tubes with fresh 0.5%trypsin-EDTA, as just demonstrated.
At the end of the second digestion, use a pair of dissecting forceps to hold one end of the amnion portion and a second pair of dissecting forceps to squeeze along the remaining tissue to remove any rows of epithelial cells that did not completely peel off during the previous incubation periods. Then, remove the membranes to be able to transfer the second digestion solution to a second set of centrifuge tubes. And inactivate the digestion enzymes with two times the volume of fresh 37-degree-Celsius human AE cell medium on ice.
For human AE cell isolation, sediment the cells by centrifugation. And resuspend the pellet in each tube with 10 milliliters of fresh 37-degree-Celsius human AE cell medium. Pull each pair of digestions into a single tube and filter the suspensions through 100-micrometer strainers to remove any extracellular matrix debris.
After counting, seed the human AE cells from each of the three regions into individual 100-millimeter plates at a three times 10 to the fourth cells per centimeters squared concentration in 37-degree-Celsius human AE cell medium, supplemented with 10 nanograms per milliliter of human epidermal growth factor. Then, incubate the cultures at 37 degrees Celsius under normoxic conditions in a humidified incubator until the downstream analysis of interest. After 48 hours of culture, human AE cells with an epithelial phenotype adhere to the surface of the plate, while the supernatant contains cell debris and floating cells that can be removed once the medium is changed.
It is advisable to discard the cultures and process another membrane upon identifying the presence of bacterial contamination, excessive erythrocytes due to insufficient washing of the membranes, deficiently or nonadherent cells, or cells with a fibroblast morphology. Human AE cell morphology depends on the origin of the cells, as cells from the reflected zone demonstrate a cuboidal morphology and grow in a cobbled monolayer, and cells from the placental and umbilical regions are flatter and squamous. Immunofluorescence against E-cadherin reveals that the primary cultures and subcultures maintain their epithelial phenotype, suggesting that there is no contamination of another cell type.
In addition, the cells are viable, as evidenced by their expression of the Ki-67 proliferation marker. Although the subpopulations from the amnion differ in their morphology and function, the expression and presence of the core of the pluripotency factors does not change in human AE cells derived from the placental and reflected regions. Since human amniotic epithelial cells are a possible source of pluripotent stem cells, we can challenge them through a line of specific definition protocols to elucidate whether these cells from each region have different potentials.
Previous to a started protocol, review the medical history of the patient to be sure that the tissue exhibits no microbiological characteristics of infection.
