Generation of Mast Cells in Co-Culture with Multiple Cell Types within an Advanced Allergy Tissue Model
Saturday, March 5, 2016
South Exhibit Hall H (Convention Center)
Tahereh Derakhshan, Rudra Bhowmick, James H. Meinkoth, Heather Gappa-Fahlenkamp
Rationale: An in vitro tissue model can be used to identify factors that regulate inflammation, leading to better diagnostic and therapeutic strategies. Since mast cells (MCs) play a major role in allergy development, their incorporation into the tissue model is critical. Due to difficulties in isolating MCs ex vivo, our objective was to generate functional MCs within a tissue model that mimics human physiology.

Methods: MC progenitors, isolated from leucocyte preparation of human donors, along with primary human fibroblasts were cultured in a collagen matrix to mimic connective tissue. Culture media was selected based on the support of the overall growth and function of the cell types. Samples were incubated for seven weeks to allow for MC generation and maturation. After six weeks, the matrix was coated with type IV collagen and fibronectin layer and primary human endothelial cells to represent a blood capillary. Formation of cytoplasmic granules was observed by metachromatic staining, indicative of MCs. Expression of MC phenotypic markers was determined by flow cytometry. MC function was examined after challenge with IgE and anti-IgE antibodies by quantifying the histamine release.

Results: After seven weeks, progenitor cells appeared as mononucleated (70%) and with surface projections, representing characteristics of MCs. The cells were positive for metachromatic granules and expressed MC phenotypic markers, 83±2% c-kit and 20±4% FcεRI. After activation with anti-IgE, generated MCs released 10-fold higher histamine content than unactivated cells (p<0.005).

Conclusions: Generated cells within the tissue model exhibited in vivo MC morphology, immunophenotype, and underwent degranulation in response to antigen receptor trigger.