An optimized protocol for the generation and functional analysis of human mast cells from CD34 + enriched cell populations

Abstract

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30–50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46 ± 0.63 × 106 vs. 2.4 ± 0.28 × 106, respectively, from 1.0 × 108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to previous method (2.36 ± 0.70 × 106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT

“…The characteristics of these patients are specified in Table S1 in Supplementary Material. Primary HuMC cultures were derived from CD34 + progenitors as described ( 32 , 33 ); and mononuclear cells from marrow aspirates were separated in a Ficoll gradient and cultured for 5 days in StemPro media supplemented with 100 ng/mL SCF ( 34 ).…”

Advances in Proteomic Techniques for Cytokine Analysis: Focus on Melanoma Research

“…The most frequently studied cytokine producing cells are T-lymphocytes [ 190 ] but also other cell populations, including basophils [ 191 ], neutrophils [ 192 , 193 ], NK cells [ 194 ], monocytes [ 192 ] and mast cells [ 195 ] are investigated. In melanoma, flow cytometry was applied to study mechanisms of effects of immunotherapies, such as a PD-1 protein blockade by monoclonal antibodies pembrolizumab or nivolumab [ 196 , 197 , 198 ], CTLA-4 blockade with ipilimumab [ 198 , 199 , 200 , 201 ] or high-dose IL-2 therapy [ 202 , 203 ].…”

Reply

“…We had reported that IL-1 stimulates human mast cells to secrete IL-6 without tryptase. 5 This finding is important given that serum levels of IL-6 are elevated and correlate with disease severity in patients with systemic mastocytosis. 6,7 That mast cells can secrete mediators without tryptase (Table I) and that IL-33 augments nonallergic stimuli such as SP is critical for understanding the role of mast cells in diseases such as mastocytosis and mast cell activation syndrome in which most symptoms are neither due to allergic triggers nor due to tryptase.…”

“…The understanding of mast cell biology has lagged somewhat behind studies on other effector cells because mast cells do not normally occur in blood, there is no human condition characterized by the absence of mast cells in all tissues, and there is no pharmacologic therapy that specifically and only silences mast cell function. However, the relatively recent availability of human mast cell lines, including LAD2 cells developed in our laboratory, 3,4 along with improved techniques to more easily culture human mast cells 5 and to more easily obtain mast cells from human tissues such as skin and marrow, 6,7 have facilitated research into this interesting cell.…”