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

Abstract

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. 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.

Protocol

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

1. Human tissue collection and preparation.
   1. 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 ....

Results

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 t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Fisher Scientific	SH3001304
Single-edged razor blades	Fisher Scientific	12-640
15 cm culture dishes	BD Biosciences	353025
Fetal Bovine Serum (FBS)	Life Technologies	16000044_3616914956
DMEM with L-Glutamine	Corning	10-013-CV
MEM Vitamins	Corning	25-020-Cl
MEM Nonessential amino acids	Corning	25-025-CI
Penicillin-Streptomycin 	Corning	30-002-CI
Shaker	Thermo-Fisher	MaxQ 4450
Centrifuge	Eppendorf	5702
Incubator	Thermo-Fisher	Forma Series II Water Jacketed CO2 Incubator Model 3100
T-75 Flasks	BD Biosciences	353136
T-175 Flasks	BD Biosciences	353112
Pipet tips	Rainin	P2, P10, P20, P200 and P1000
Microscope	Zeiss	Axiovert 200m
Anti-Integrin αVβ3 Antibody, clone LM609	EMD Millipore	MAB1976
Beta 1	Oncosynergy	OS2966
Alpha 5 [CD49e]	ID Pharmingen	555615
Beta 4 [CD104]	EMD Millipore	MAB 2058
Pipet tips	Corning	Filtered tips P2, P10, P20, P200 and P1000
Glass flask