(184f) Studying the Effect of Media Systems on Multiple Cell Types within an Advanced Allergy Tissue Model

Authors: 
Derakhshan, M., Oklahoma State University
Fahlenkamp, H. G., Oklahoma State University

Our goal is to create an advanced 3D allergy tissue model that can be used for testing and studying human allergic inflammatory responses.  The tissue model includes mast cells (MCs), which are considered the central effector cells in the early events associated with allergic inflammatory responses, fibroblast cells (FCs), which support the growth and development of MCs, and endothelial cells (ECs), which are involved in leukocyte recruitment during inflammation. For development of the tissue model, primary human cells are cultured in a complex 3D matrix-like environment, which has never before been integrated. MC progenitor cells (CD133+ cells isolated from adult human peripheral blood leukocytes) along with FCs are cultured within a collagen gel to mimic a tissue layer.  The top surface of the gel is seeded with ECs to form a single, confluent layer of cells to mimic the interior surface of a blood vessel in body.  The goal of this study was to determine the type of media system that would contribute to the growth and function of all cell types. CD133+ cells, FCs, and ECs were cultured separately in various media systems and characterized for their growth and function. FCs and ECs were labeled by CellTracker fluorescent probe or CellTrace CFSE dye to determine cell proliferation. The viability of the cells was monitored over time by the CellTiter-Blue assay. The function of FCs and ECs and their contribution to MC development were investigated by quantifying stem cell factor (SCF) and interleukin-6 (IL-6) release using the ELISA. CD133+ cells were cultured for seven weeks in a collagen gel in various media systems that were supplemented with IL-3 (first three weeks), SCF, IL-6, and serum (last week). The generation of MCs was monitored until characterization on the seventh week of culture under normal conditions. Cell viability was measured using the CellTiter-Blue assay.  The phenotype and maturation of the generated MCs was determined by immunocytochemical staining of the tryptase and chymase granules. Expression of FcεRI and c-kit receptors, which differentiates MC from other type of cells and is the characteristic of mature MC, was identified by immunocytochemical staining and flow cytometry. The MC function was examined after challenge with human myeloma IgE and anti-human IgE, which mimics an immediate type allergenic response, by quantifying the amount of histamine release using the ELISA. The overall results show that the media system that best supports MC growth and development resulted in good FC viability, but poor EC viability and proliferation.  The results indicate that the timing of the addition of ECs to the tissue model should be adjusted depending on the presence of serum in the media.