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A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation, identification, and growth
A Pronk1, P Leguit, A A Hoynck van Papendrecht
1Laboratory of Surgery, University Hospital Utrecht, The Netherlands.
Abstract:
Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum. The cell yield was approximately one million cells per square centimeter omentum. The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18. Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport. Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium. Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed. Seeding NHMC at densities less than 3000/cm2 did not result in monolayer formation. The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages. However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells.
Insights
Researchers cultured normal human mesothelial cells (NHMC) from omentum. Optimal growth required specific serum concentrations and epidermal growth factor, but cells changed morphologically after passage 4.
Area of Science:
- Cell Biology
- Tissue Engineering
- Mesothelial Cell Research
Background:
- Normal human mesothelial cells (NHMC) are crucial for serosal membrane function.
- Understanding NHMC culture conditions is vital for research and potential therapeutic applications.
- Previous studies have explored mesothelial cell characteristics, but optimal serial passaging remains an area of investigation.
Purpose of the Study:
- To establish and characterize normal human mesothelial cell cultures.
- To determine optimal growth conditions and growth factors for NHMC.
- To assess the long-term stability and morphological changes of cultured NHMC.
Main Methods:
- Isolation of NHMC from human omentum.
- Identification using cytokeratin 6 and 18 monoclonal antibodies.
- Transmission electron microscopy for ultrastructural analysis.
- Culturing NHMC with varying serum concentrations and growth factors (EGF, hydrocortisone, insulin).
- Serial passaging up to 7 times to evaluate long-term culture behavior.
Main Results:
- NHMC yield was approximately 1 million cells/cm².
- Positive staining for cytokeratins 6 and 18 confirmed mesothelial cell identity.
- Ultrastructural analysis showed microvilli, mitochondria, and pinocytotic vesicles, indicating active transport.
- NHMC growth correlated with serum concentration; epidermal growth factor significantly enhanced growth.
- Monolayer formation required seeding densities >3000 cells/cm².
- After passage 4, NHMC transformed into giant cells with irregular patterns and reduced cytokeratin expression.
Conclusions:
- Normal human mesothelial cells can be successfully isolated and cultured.
- Epidermal growth factor is a key stimulant for NHMC proliferation in vitro.
- Long-term serial passaging leads to significant morphological and phenotypic changes in NHMC, limiting their utility beyond passage 4.