Mesothelial Cell Isolation: A Technique to Isolate Primary Mesothelial Cells From Human Omentum Tissue Specimen

Human primary mesothelial cells, or HPMCs, line many visceral organs like the omentum or the layer of fatty tissue present in the peritoneal cavity.

To isolate the mesothelial cells, begin by immersing a human omentum specimen in a suitable buffer to remove the excess red blood cells or RBCs. Next, place the omentum in a glass dish and mince it into pieces. This dissociates the tissue, releasing HPMCs along with the contaminating RBCs.

Transfer the pieces into a conical tube and allow them to remain still for the desired time. The minced tissue rich in low-density fat rises to the top, while the dense cellular components settle at the bottom. Next, aspirate the liquid at the bottom, containing HPMCs and RBCs, into a fresh tube.

Centrifuge to collect the cells. Remove the buffer-containing supernatant. Resuspend the pellet in a mesothelial growth medium and incubate for the desired duration. The media facilitates the selective growth of HPMCs over RBCs.

Observe the cells using a light microscope to confirm the growth of cuboidal-shaped human mesothelial cells.

Upon acquisition of a surgical human omentum specimen, immediately immerse the sample in room temperature PBS. Within 2 hours of the immersion, transfer the omentum to a 50-milliliter conical tube containing 20 milliliters of fresh PBS. Transfer the remaining PBS wash containing the primary human mesothelial cells or HPMC and red blood cells into a new 50-milliliter conical tube and spin down the cells.

Then, pour the tube contents into a 10-centimeter sterile culture dish and use two scalpels to mince the tissue into 5 cubic millimeter pieces. To isolate the primary human mesothelial cells or HPMC, transfer the omentum pieces into a 50-milliliter conical tube and wait 1 minute for the solid pieces to float to the top. The HPMC will settle at the bottom of the tube with the red blood cells.

Use a pipette to transfer the PBS wash containing mesothelial cells and red blood cells into the tube containing the pelleted cells and spin down the cells. Then, add 20 milliliters of fresh PBS to the omentum and collect the PBS-containing HPMC and RBC three more times as just demonstrated to the original pelleted cells.

After the final spin, plate the cells in a 75 square centimeter flask in 15 milliliters of full growth medium. To isolate additional HPMC from the omentum sample, shake the remaining solid tissue in 20 milliliters of PBS at 200 RPM and 37 degrees Celsius. After 10 minutes, let the sample rest for 1 minute to allow the solid tissue to rise to the top of the tube.

Then, collect the HPMC and RBC from the bottom of the tube as just demonstrated. Now, spin down the cells from the secondary wash and plate them in a separate 75 square centimeter flask in 15 milliliters of full growth medium. To isolate any remaining HPMC, shake the tissue for another 10 minutes this time in a mix of PBS and 10 milliliters of trypsin-EDTA, and plate these HPMC and RBC in a 75 square centimeter flask.

Then, incubate the cultures at 37 degrees Celsius and 5% carbon dioxide in a humidified environment. Feeding the plated cells with 15 milliliters of fresh full growth medium on days 3 and 5 without removing the spent medium. The HPMC can be identified by their cuboidal shape and their expression of cytokeratin-8 and vimentin.