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Human foreskin mast cell viability and functional activity is maintained ex vivo by coculture with fibroblasts
F Levi-Schaffer1, R Kelav-Appelbaum, E Rubinchik
1Department of Pharmacology, School of Pharmacy, Hebrew University Hadassah Medical School, Jerusalem, Israel.
Abstract:
The aim of the present study was to develop long-term culture conditions for foreskin-derived mast cells (HSMC) as we have previously done for rodent peritoneal mast cells (MC) and human lung-derived MC. HSMC were obtained by proteolytic treatment of foreskins from 8-day-old babies (4.8 +/- 2.0% purity) and seeded onto a confluent monolayer of human foreskin-derived fibroblasts (HF) in enriched culture medium. HSMC biochemical and functional properties were studied up to 8 days in these cocultures. Twenty-four hours after seeding the cell suspensions from the proteolytic-digested foreskins, all the cells which did not adhere to the HF were washed out. At this time point approximately 30% of the seeded HSMC were found to adhere to the HF monolayer. The only contaminating cells were endothelial cells (< 8%). Cocultured HSMC maintained their normal resting morphology and histamine content (3.1 +/- 0.7 pg/cell) up to 8 days in these cocultures. When challenged with compound 48/80 on Days 1, 2, 4, and 8 of coculture, HSMC released similar percentages of histamine which were comparable to the one released by freshly isolated HSMC in suspension (approximately 30%). Similarly, HSMC, sensitized with IgE antibodies and challenged at various time of coculture with anti-IgE antibodies, released throughout the experiment comparable percentages of this mediator (approximately 50%). Thus, coculture of HSMC with HF fibroblasts provides a suitable in vitro system for long-term studies on HSMC functional and biochemical properties in a microenvironment which mimics the physiologic one.
Insights
Researchers developed a new method for long-term culture of human foreskin-derived mast cells (HSMC). This technique uses fibroblast co-culture to maintain HSMC function and histamine release for up to 8 days.
Area of Science:
- Immunology
- Cell Biology
Background:
- Mast cells (MC) play crucial roles in allergic responses and inflammation.
- Establishing long-term in vitro culture systems for human mast cells is challenging.
- Previous studies established cultures for rodent peritoneal mast cells and human lung-derived mast cells.
Purpose of the Study:
- To develop and validate long-term culture conditions for human foreskin-derived mast cells (HSMC).
- To assess the biochemical and functional properties of HSMC in a co-culture system over time.
Main Methods:
- Human foreskin-derived mast cells (HSMC) were isolated via proteolytic treatment.
- HSMC were co-cultured with human foreskin-derived fibroblasts (HF) in enriched medium.
- Cell purity, morphology, histamine content, and degranulation responses were assessed up to 8 days.
Main Results:
- Co-culture achieved approximately 30% HSMC adherence to HF monolayers with <8% endothelial cell contamination.
- HSMC maintained normal morphology and histamine content (3.1 ± 0.7 pg/cell) for 8 days.
- Co-cultured HSMC exhibited robust histamine release upon stimulation with compound 48/80 and anti-IgE, comparable to freshly isolated cells.
Conclusions:
- Co-culture of HSMC with HF fibroblasts provides a suitable in vitro model for long-term studies.
- This system effectively mimics the physiological microenvironment of mast cells.
- The developed method supports sustained HSMC function, enabling detailed investigation of their properties.