Name: mesothelial cell isolation: a technique to isolate primary mesothelial cells from human omentum tissue specimen
Uploaded: 2023-04-30T13:26:19.000+00:00
Duration: 1 min 37 s
Description: Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

mesothelial cell isolation: a technique to isolate primary mesothelial cells from human omentum tissue specimen

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

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.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Isolation and Culture of Primary Untransformed Stromal Cells
<ol>
	<li>Human tissue collection and preparation.
	<ol>
		<li>Obtain specimens of human omentum, 2 cm3, removed during an abdominal surgery, and immediately immerse tissue in RT phosphate-buffered saline (PBS) (Figure 1A). A piece of omentum 3 cm x 2 cm typically yields 1 million primary human mesothelial cells (HPMC) and 200,000 primary human fibroblasts or normal omental fibroblasts (NOF).</li>
		<li>Spin down the PBS the tissue was immersed in at 0.5 x g for 3 min as soon as possible after collection (&#60; 2 hr in PBS) and transfer the tissue to fresh 20 ml PBS in a 50 ml conical tube. Cut up tissue into 5mm3 pieces by mincing with scalpels in a 15 cm diameter, sterile culture dish (Figure 1B).</li>
	</ol>
	</li>
	<li>Primary human mesothelial cell isolation
	<ol>
		<li>To isolate HPMC, transfer the minced tissue into 50 ml conical tubes and wait 1 min for the solid pieces to float to the top (Figure 1C &#38; D). HPMC and red blood cells (RBC) will be present in the liquid on the bottom. Use a pipette to remove the liquid from the bottom of the tube and into a new conical tube. Spin the liquid down at 0.5 x g for 3 min and aspirate the supernatant from the HPMC/RBC pellet.
		<ol>
			<li>Repeat the process three times: add 20 ml PBS to the minced tissue, allow tissue to rise, remove liquid from the bottom with a pipette and into the HPMC/RBC tube, spin down, and remove the supernatant. After all spins are completed and the supernatant is aspirated, the pellet will contain HPMC and RBC.</li>
		</ol>
		</li>
		<li>Plate all cells from the pellet into a 75 cm2 flask in 15 ml of full growth media (DMEM with 10% fetal bovine serum [FBS],1% MEM vitamins [93 mg/L], 1% MEM nonessential amino acids [81.4 mg/L], 1% penicillin streptomycin [Pen-Strep, 100 U/ml penicillin and 100 &#181;g/ml streptomycin]).</li>
		<li>Perform a secondary PBS wash to isolate additional HPMCs.
		<ol>
			<li>For the secondary PBS wash, shake the remaining solid tissue at 200 rpm, 37 &#176;C for 10 min in 20 ml of PBS, then wait 1 min for the tissue to float to the top. Remove the liquid from the bottom of the tube into a new tube, centrifuge at 0.5 x g for 3 min, and aspirate the supernatant.</li>
			<li>Plate all the HPMC/RBC from the secondary PBS wash in a separate 75&#160; cm2 flask in 15 ml of full growth media.</li>
		</ol>
		</li>
		<li>To isolate any remaining HPMC, shake the minced tissue at 200 rpm, 37 &#176;C degrees for 10 min in 20 ml of a 1:1 0.25% trypsin 25 mM EDTA:PBS solution by volume. Allow the tissue to rise, collect the liquid at the bottom of the tube into a new 50 ml tube, and spin down at 0.5 x g for 3 min.
		<ol>
			<li>Aspirate the supernatant and plate all the HPMC/RBC in a separate 75 cm2 flask in 15 ml of full growth media.</li>
		</ol>
		</li>
		<li>Culture the plated cells at 37&#176;C and 5% CO2 in a humidified environment. Feed plated cells by adding 15 ml of full growth media on days 3 and 5 without removing the spent media. HPMC can be cultured for 5-7 days before splitting.
		<ol>
			<li>Use low-passage HPMC (up to passage 2) in all experiments to minimize dedifferentiation and modification of original phenotype. Use a wide-field microscope preferably with a 20x objective with plastic differential interference contrast capabilities or 4-20x objective with phase contrast capabilities (phase contrast filters on microscope) for taking all images of cells, and a 10x objective ()in bright field to analyze immunohistochemical 3,3&#39;-Diaminobenzidine (DAB) staining.</li>
		</ol>
		</li>
		<li>Confirm that the HPMC are cuboidal and express cytokeratin 8 and vimentin by performing immunohistochemistry (Figure 2A, C, E).</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>Single-edged razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000044_3616914956</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axiovert 200m</td><td></td></tr><tr><td>Anti-Integrin &amp;alpha;V&amp;beta;3 Antibody, clone LM609</td><td>EMD Millipore</td><td>MAB1976</td><td></td></tr><tr><td>Beta 1</td><td>Oncosynergy</td><td>OS2966</td><td></td></tr><tr><td>Alpha 5 [CD49e]</td><td>ID Pharmingen</td><td>555615</td><td></td></tr><tr><td>Beta 4 [CD104]</td><td>EMD Millipore</td><td>MAB 2058</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Glass flask</td><td></td><td></td><td></td></tr></tbody></table>

Source: Peters, P. N. et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
This video describes the protocol to isolate primary human mesothelial cells from the human omentum specimen. The mesothelial cells can be used to establish an in vitro ovarian cancer model to investigate ovarian cancer cell adhesion, invasion, and proliferation.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20376/20376_Figure1.jpg" /> 
Figure 1. Isolation of primary human omental cells. (A) A piece of omentum is collected at surgery in PBS and transported to the laboratory. The tissue is pelleted at 0.5 x g x 3 min and transferred to fresh PBS. (B) The piece of omentum is transferred to a 10 cm petri dish, washed with PBS, and scalpels are used to cut t...

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

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.

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

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