An Optimized Protocol for the Isolation and Functional Analysis of Human Lung Mast Cells

Abstract

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MC_T (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MC_TC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45⁺ immune cells and an even more pronounced increase in the yield of CD117⁺ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Keywords: enzymatic digestion protocols; human mast cells; lung; mast cell isolation; mast cells.

Figure 1

Human lung tissue processing: pictures taken during different steps of WEMP protocol. (A–C) Washing tissue, removing blood pockets. (D–I) Cutting tissue into thin strips and then into small pieces. (J–O) Washing and filtering uniformly cut pieces with PBS. (P) Processing tissue pieces with scalpel. (Q,R) Enzymatic digestion of tissue pieces at 37°C with magnetic stirrer. (S–V) Mechanical disruption of digested tissue using syringe. (W,X) Percoll gradient centrifugation and RBC lysis.

Figure 2

Gating strategy for mast cells from human lung tissue. (A) CD117 high mast cells from human lung single cell suspension was identified using flow cytometry. (B) CD117 high mast cells and CD14+ cells were analyzed for intracellular Tryptase using flow cytometry. (C) Human lung mast cells cultured for 4 days, activated by FcεRI crosslinking and CD63 expression analyzed by flow cytometry. FSC, forward scatter; SSC, side scatter.

Figure 3

Mast cell yield in Conventional and WEMP protocol. CD45+ CD117 high expressing FcεRI+ mast cell yield compared between conventional and WEMP protocol. **P < 0.01.

Figure 4

Human lung mast cell yield during different steps of protocol. (A) Cells isolated and frequency, number of mast cell analyzed from different steps of the protocol—WEMP. (B) Total number of cells from different steps of the protocol counted using microscope. Cells were collected from different steps of the protocol—wash, enzymatic digest, cell pellet and supernatant after mechanichal disruption followed by gradient centrifugation (Percoll purification). (C) Mast cells of CD45+ population from different steps of the protocol analyzed by flowcytomtery. (D) Number of mast cells per gram of tissue from different steps of protocol counted using microscope. (E) Number of mast cells per gram of tissue compared between conventional and WEMP protocol counted using microscope (by combining different steps of protocol). (F) Percentage of viable mast cells during different steps of the protocol analyzed by flowcytometry. *P < 0.05.

Figure 5

Frequency of ILC2 from human lung tissue. (A) Single cell suspension from human lung tissue was stained with markers for lineage, CD45, CD127, CD161, NKG2A, and CRTH2. ILC2 were identified by flow cytometry analysis. (B) ILC2 yield was compared between conventional and WEMP protocol.

Figure 6

Analysis of single cell RNA quality. (A) Gating strategy for sorting mast cells, ILC. (B) Gating strategy explained as flowchart. (C) RNA quality of single cell sorted mast cells and ILC shown as FU.