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Published on: December 31, 2015
Experience in primary culture of human peritoneal mesothelial cell
1Division of Nephrology, CGMH, Keelung 20401, Taiwan, Republic of China.
Abstract:
To compare the growth condition between different sources and different culture environments, mesothelial cells were isolated from omentum and peritoneal dialysate effluent (PDE), seeded at different densities (5 × 10⁵, 1 × 10⁵, 5 × 10⁴, 1 × 10⁴, 5 × 10³, 1 × 10³ and 5 × 10² cells/cm², respectively), supported with different fetal calf serum (FCS) concentrations (3%, 6%, 10% and 15%) and grown in dishes with and without gelatin pre-coating. Growth condition was evaluated by simple morphological observation. Cells phenotype was examined by immunofluorescent staining. The results showed that omentum-derived mesothelial cells generally showed a uniform growth pattern with good quality. Alternatively, there was a wide patient-to-patient variation in PDE-derived culture. Heterogeneous colonies composed of a mixture of large, small or abortive mesothelial colonies as well as fibroblastoid colonies were frequently observed. A minimum seeding density of 5 × 10³ cells/cm² is required for the omentum-derived mesothelial cells to grow to confluent monolayer (1-5 × 10⁴ cells/cm² for initial culture from fresh PDE). Appropriate seeding density is always associated with successful culture in omentumbased culture, but not in PDE-based culture. Mesothelial cells could grow to confluency regardless of FCS concentration and gelatin pre-coating. However, growth rate was slower in lower FCS concentrations and on dishes without gelatin coating. Most cells in culture expressed cytokeratin and vimentin, but not VWF. Alpha-smooth muscle actin frequently appeared in cytokeratin+ mesothelial cells, especially in higher FCS concentrations and in PDE-derived culture. Our data demonstrate that PDE, in contrast to omentum, provides a source of mesothelial cells with poor and unstable quality for primary culture. Healthy cell quality and sufficient seeding density seem to be the most important factors for successful culture of mesothelial cells. The frequent occurrence of epithelial-to-mesenchymal transition in cultured mesothelial cells indicates the feasibility of mesothelial cells to undergo phenotype change upon environment changes, especially following chronic exposure to uremic environment and dialysate in peritoneal dialysis patients.
Insights
Omentum-derived mesothelial cells offer superior quality and uniform growth compared to peritoneal dialysate effluent (PDE) cells. Optimal seeding density is crucial for successful omentum cell culture, while PDE cells exhibit variable and often poor quality.
Area of Science:
- Cell Biology
- Regenerative Medicine
- Tissue Engineering
Background:
- Mesothelial cells play a vital role in maintaining the peritoneal membrane's integrity.
- Primary mesothelial cell culture is essential for research and potential therapeutic applications.
- Peritoneal dialysate effluent (PDE) is a potential source of mesothelial cells, but their quality is uncertain.
Purpose of the Study:
- To compare the growth conditions and quality of mesothelial cells derived from omentum versus PDE.
- To determine optimal seeding densities, fetal calf serum (FCS) concentrations, and culture surface requirements.
- To assess the stability and phenotype of cultured mesothelial cells from different sources.
Main Methods:
- Mesothelial cells were isolated from omentum and PDE.
- Cells were cultured at various seeding densities, FCS concentrations, and on gelatin-coated or uncoated dishes.
- Growth morphology and cell phenotype (cytokeratin, vimentin, alpha-smooth muscle actin, VWF) were evaluated.
Main Results:
- Omentum-derived mesothelial cells exhibited uniform growth and good quality, unlike the variable quality of PDE-derived cells.
- A minimum seeding density of 5 × 10³ cells/cm² was required for omentum cells; PDE cells required 1-5 × 10⁴ cells/cm².
- Epithelial-to-mesenchymal transition markers were observed, particularly in PDE-derived cultures and at higher FCS concentrations.
Conclusions:
- PDE is an unreliable source for primary mesothelial cell culture due to poor and unstable cell quality.
- Healthy cell quality and appropriate seeding density are critical for successful mesothelial cell culture.
- Cultured mesothelial cells can undergo phenotype changes, suggesting adaptability to environmental shifts, relevant to peritoneal dialysis patients.
