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Optimal method for isolation of human peritoneal mesothelial cells from clinical samples of omentum
Manel Riera1, Peter McCulloch, Laszlo Pazmany
1School of Clinical Sciences, University of Liverpool, Liverpool L69 3BX.
Introduction:
Human peritoneal mesothelial cells (HPMC) are a valuable research tool for understanding the molecular biology of several pathologies, in both monolayer and three dimensional models. We compared different methods of HPMC isolation and assessed their outcome as well as fibroblast contamination, a common problem encountered during isolation.
Methods:
1-3cm(3) samples of omentum were collected from 40 consenting patients undergoing elective gastrointestinal surgery. A total of 11 samples were incubated in 0.05% trypsin solution for 20 minutes at 37 degrees C (group A) and 29 in 0.25% trypsin (15 samples for 10 minutes (group B) and 14 for 20 minutes (group C)). Following digestion cells were re-suspended and cultured in supplemented Ham's F-12 medium containing 10% foetal calf serum (FCS), penicillin-streptomycin, glutamine, insulin, transferrin and hydrocortisone. Positive outcomes were absence of fibroblast contamination and satisfactory HPMC growth to confluence in a characteristic cobblestone pattern. Cytokeratins 5, 8, 18, Vimentin, Ber-Ep4 and Factor VIII were used to characterise HPMC and fibroblasts by immunohistochemistry.
Results:
None of the 11 samples in group A yielded HPMC. 14 of 29 samples digested with 0.25% trypsin yielded HPMC: 10 of 14 yielded HPMC in group C versus four of 15 samples in group B (p = 0.02). Fibroblast contamination occurred in eight samples in group B versus three in group C.
Conclusion:
Optimal results are achieved with a 20 minute digestion in 0.25% trypsin. Fibroblast contamination could not be avoided completely. Other factors may minimise fibroblast contamination such as minimal tissue manipulation and early collection during surgery.
Insights
Optimal human peritoneal mesothelial cell (HPMC) isolation involves 20-minute digestion in 0.25% trypsin. While fibroblast contamination persists, careful tissue handling can minimize it.
Area of Science:
- Cell Biology
- Tissue Engineering
- Surgical Research
Background:
- Human peritoneal mesothelial cells (HPMC) are crucial for studying pathologies in 2D and 3D models.
- Fibroblast contamination is a significant challenge in HPMC isolation procedures.
Purpose of the Study:
- To compare different HPMC isolation methods.
- To assess the efficacy of various trypsin concentrations and incubation times.
- To evaluate the rate of fibroblast contamination in each isolation group.
Main Methods:
- Omental tissue samples (n=40) were digested using different trypsin concentrations (0.05% and 0.25%) and durations (10 and 20 minutes).
- Cells were cultured in Ham's F-12 medium with supplements.
- Immunohistochemistry was used to characterize HPMC and fibroblasts.
Main Results:
- 0.05% trypsin yielded no HPMC.
- 0.25% trypsin yielded HPMC in 14/29 samples.
- Digestion with 0.25% trypsin for 20 minutes (Group C) resulted in significantly higher HPMC yield (10/14) compared to 10 minutes (Group B, 4/15; p=0.02).
- Fibroblast contamination was observed in 3/14 samples in Group C versus 8/15 in Group B.
Conclusions:
- A 20-minute digestion in 0.25% trypsin is optimal for HPMC isolation.
- Fibroblast contamination cannot be entirely eliminated but may be reduced by minimizing tissue manipulation and early surgical collection.
