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Conceived and designed the experiments: SKM RW. Performed the experiments: SKM MN SW PK CGT MK. Analyzed the data: SKM MH RW. Contributed reagents/materials/analysis tools: RW. Wrote the paper: RW SKM MH. Prepared all figures and drawings: SW.
Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.
Mast cells (MCs) are tissue-resident cells that are linked to the innate immune system. They are mostly known for their role in allergic and other inflammatory diseases [1,2]. Allergy is initiated by crosslinking of IgE-bound high-affinity receptors for IgE (FcεRI) by a specific antigen triggering MC degranulation . In addition, MCs have a strategic location at the host-environment interface that predisposes them as a critical gate-keeper for starting early host defense against intruders . On the other side, MCs are enriched in the tumour microenvironment of some carcinomas accelerating tumour progression, angiogenesis, epithelial-to-mesenchymal transition, and extracellular matrix degradation . During the last years several MC-deficient mouse strains were established that were generated either by targeted mutations in the Kit or the stem cell factor gene or by introducing inducible or constitutive deficiencies under the use of different manipulating strategies .
Many studies have shown that MCs are crucial for the maintenance of tissue function, tissue homeostasis, and during all steps of tissue repair from the initial inflammatory reaction and proliferation of connective cellular elements to final remodelling of the extracellular matrix [6,7]. However, some effects of MCs are controversial and frequently opposite most likely due to the phenotypic heterogeneity of MCs in different tissues .
During the past decades, the unravelling of MC functions in many laboratories has been in the focus of MC research. Nevertheless, one of the major limitations is the difficulty to obtain large quantities of primary MCs for in vitro (e.g. for sensitization and signaling studies) and in vivo (e.g. for adoptive transfer experiments) purposes. Therefore, many studies have been conducted in immortalized MC cell lines (e.g., L138.8A, HMC-1) resulting in findings that must be interpreted cautiously due to activating mutations in key signalling components like Kit/KIT. In addition, there is a coincident opinion that the wide experimental possibilities that could be addressed by the accessibility of large quantities of purified and homogeneous MCs would allow addressing key questions of MC biology. Fundamental insights into differentiation of murine BMMC from bone marrow precursors and in isolation of resident peritoneal MCs were already performed decades ago [9,10]. The proposed protocols of these pioneering studies are used in many laboratories to isolate immature BMMCs or mature PMCs.
In principle, MCs can be derived from multipotent progenitor cells that are matured in specialized culture media, or directly isolated as functional MC from diverse tissues that are classified as tissue MC. Murine progenitor MCs can be derived from bone marrow (i.e. bone-marrow derived MCs, BMMCs) or foetal tissue (e.g. skin, liver, spleen) with high MC content. However, the generation of mature MCs is a long-lasting process that requires IL-3 and stem cell factor (SCF) or even more complex cocktails of cytokines and often results in questionable mixtures of cells with incomplete maturation . Murine tissue MCs with a phenotype that is more consistent with connective tissue MCs can be isolated from the peritoneum and to a lesser amount from mucosa or skin . Both MC entities may significantly differ from each other in functional terms and each by itself might have limited functional explanatory power in estimating general properties of MCs. As a consequence, the simultaneous availability and comparative analysis of culture-maturated and tissue-derived MCs from one biological source would be helpful to conduct experiments with more functional significance.
First protocols for the isolation of primary MCs required wasteful and time-intensive gradient centrifugation steps utilizing media containing sucrose, albumin, Ficoll, Percoll or Nycodenz [12–15]. Unfortunately, the cells that were isolated by these protocols showed morphological and functional changes such as loss of histamine and alteration of responsiveness against histamine-releasing agents and antigens. Also the isolation of MCs from tissue by enzymatic digestion protocols using collagenase and elastase resulted in cells with variable viability and purity . Other protocols allowed the isolation of rat peritoneal MCs (PMCs) from a single peritoneal lavage resulting in high yield of MCs . In addition, time consuming multistep separation techniques that combined enzymatic treatment, elutriation, magnetic CD14+ cell depletion (monocytes, macrophages) and cell sorting techniques with monoclonal antibodies directed against CD117/KIT were suitable to isolate and enrich MCs from human lung, skin, and uterus .
Here, we describe a two-step simple protocol suitable to simultaneously isolate murine mature MCs from the peritoneum and immature MC progenitors from the bone marrow. The resulting cells are highly homogenous and show the typical MC phenotype and expression of marker genes and can be propagated in culture.
A complete material list of all surgical instruments, laboratory equipment and sterile preparation utensils (scissors, forceps), plastic ware (Falcon tubes, 10 mL syringes, plastic Pasteur pipettes) that we use in our laboratory is given in S1 Table. Further equipment necessary are pre-cooled centrifuges and a standard cell culture including a sterile hood and a humidified incubator.
There are several solutions that must be prepared prior beginning of the protocol. All these solution that are listed in the following should be prepared in sterile fashion.
35 g NH4Cl, 1.85 g KCl, 1.5 g Na2HPO4, 0.75 g K2PO4 x 2 H2O, 5.0 g D-glucose, 50 mg phenol red (all from Merck, Darmstadt, Germany) are added to 1 L of distilled H2O. Sterile filter and store in 500 mL aliquots at 4°C.
0.42 g MgCl2 x 6 H2O, 0.14 g MgSO4 x 7 H2O, 0.45 g CaCl2 x 2 H2O (all from Merck, Darmstadt, Germany) are added to 100 mL of distilled H2O. Autoclave and store in 50 mL aliquots at 4°C.
2.25 g NaHCO3 (from Merck, Darmstadt, Germany) is added to 100 mL of distilled H2O. Autoclave and store in 50 mL aliquots at 4°C.
To obtain the Gey’s working solution 70 mL of distilled H2O, 20 mL of Gey's solution A, 5 mL of Gey's solution B, and 5 mL of Gey's solution C are mixed and sterile filtered.
8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4 and 0.27 g KH2PO4 (all chemicals were obtained from Merck) are added to 1 L of distilled H2O. Sterile filter and store in 500 mL aliquots at 4°C.
700 mL absolute ethanol is mixed with 300 mL distilled H2O.
RPMI 1640 medium, including 2 mM L-glutamine, was supplemented with 15% FCS, 1 x Penicillin/streptomycin (all from Gibco, ThermoFisher Scientific, Darmstadt, Germany), 0.07% β-mercaptoethanol, 10 mM HEPES and 1% of X63-Ag8-653-conditioned medium as a source of IL-3 (final concentration: ~ 30 ng/mL) that is necessary for proper MC differentiation and growth . The myeloma cell line X63-Ag8-653 stably carries plasmids constitutively driving expression of large quantities of mouse IL-3 .
BMMC medium supplemented with supernatant derived from CHO cells that were transfected with an expression vector constitutively expressing murine SCF. The final concentration of SCF in the PMC medium as assessed by ELISA testing was adjusted to approximately 20 ng/mL.
All mice (C57BL/6J) were maintained under specific-pathogen-free conditions in the RWTH Aachen University Hospital Laboratory Animal Facility according to the guidelines of the Federation for Laboratory Animal Science Associations (FELASA). Animals were housed at 4–5 mice per cage in an animal room maintained at a constant temperature of 20°C, a relative humidity of 50%, and with a 12-hour on/off light cycle. The animals had free access to a standard mouse chow and trap water. For the performed experiments, female or male mice at age of 4–8 weeks that were bred in the RWTH Aachen University Hospital were taken. The protocol was approved by the author’s Institutional Animal Care and Use Committee (LANUV, Recklinghausen, Germany) and all experiments were conducted in accordance with the German federal law regarding the protection of animals and 'Guide for the Care and Use of Laboratory Animals' (National Institutes of Health publication 8th Edition, 2011 .
Schematically, the outlined protocol can be divided into two steps. In the first step, peritoneal cells are flushed out from the animal’s peritoneal cavity (Fig 1). This cell population contains a small fraction (~ 1–3%) of PMCs that are enriched and amplified during prolonged cell culture periods, while other cell populations do not survive the selected cell culture condition. In addition, PMCs may emerge from MC precursors.
In the second step, the long bones of the legs are exposed and stem cells and progenitor cells are purged out from the tibia and femur with repeated injections of media with a syringe (Fig 2).
In the following the different steps are successively described:
MCs have a highly characteristic morphology that is mainly characterized by granules that contain a plethora of preformed constituents including lysosomal enzymes, biogenic amines, cytokines, proteoglycans, MC-specific proteases, non-MC-specific proteases, and membrane associated proteins . The metachromatic granules in fixed MCs can be easily detected in the fast stain procedure (Fig 9).
In the staining protocol that we use in our laboratory, approximately 70,000 cells are first fixed on Shandon cytospins (#59910056/M, Thermo Scientific) by centrifugation following a standard procedure. After centrifugation, the cells were fixed in methanol, and subsequently stained with Haema fast staining solution for 5 min (#LT 003, LT-SYS Diagnostika, Berlin, Germany), and then stained for 10 min with solution LT 004 (LT-SYS Diagnostika). Thereafter, the stained cells were washed for 2 min in distilled water. Then the cells were air-dried and analyzed by light microscopy.
In our laboratory we use the Mast/stem cell growth factor receptor that is also known as proto-oncogene Kit (CD117) and the FcεRIα for identity verification. We use this double stain because the cell surface marker CD117 that mediates sensitivity towards SCF is also present on certain other types of hematopoietic progenitors, melanocytes, intestinal cells, some stem cells, and earliest lymphoid progenitors. On the other side, the high-affinity IgE receptor FcεRIα is not only found on MC but also detectable in eosinophils, basophils and epidermal Langerhans cells. The double stain, however, is only found in MCs and highly suitable to verify MC identity.
In our flow cytometry, PMCs and BMMCs that were cultured for 3–4 weeks were incubated with anti-mouse CD117/KIT-APC (1:400) (BD Pharmingen, 553356) and anti-mouse FcεRIα-FITC (1:400) (eBioscience, 11-5898-82) that is specific for the high-affinity IgE receptor, α subunit. The labelled cells were washed twice with PBS, resuspended in PBS at a cell density of 1 x 106/mL and analysed in a BD FACSAria II (Becton Dickinson, Heidelberg, Germany). This FACS machine is equipped with a laser with an excitation wavelength of 640 nm and 488 nm and bandpass filters (670/14 BP, 505 LP followed by 525/50 BP) that allows to detect the stained cells at the emission ranges of the fluorophores (Fig 10).
In principle, the expression of KIT and the FcεRI can also be proven by Western blot analysis. However, it should be mentioned that MCs and especially PMCs contain large quantities of diverse proteases that become released from sensitized or damaged MCs. In particular the large quantity of aggressive proteases in PMCs might hinder or even prevent the detection of individual proteins in Western blot analysis when samples are prepared by standard protein extraction methods .
Over the past few decades, the functional importance of MCs as important effector cells of the immune system has been convincingly established. However, some of the biological attributes of MCs described were only established in immortalized cell lines resulting in findings that must be interpreted cautiously. The outlined protocol provides a rational for reliable isolation of primary mature peritoneal MCs and bone marrow-derived MC precursors from mouse. Both MC entities can be effectively expanded in culture and maintained for weeks with a phenotype that is characteristic for MCs (e.g. positivity for KIT and FcεRIα, protease expression in secretory lysosomes, and ability to degranulate). The simultaneous availability and comparative analysis of culture-maturated and tissue-derived MCs from one biological source will be helpful to dissect molecular pathways that are in general necessary for MC differentiation or to identify specific features of MCs derived from different origin. Furthermore, the parallel analysis of both MC entities will allow to set up studies in which the biological effects during various inflammatory settings can be comparatively analyzed. In addition, it will be essential to analyze if MCs isolated from different sources have the same biological attributes. In this context it should be mentioned that most recently it was shown that in the skin acupuncture points have a higher density of MCs compared with non-acupoints and that further the primo nodes isolated from the surfaces of internal organs (e.g., liver, small and large intestines, and bladder) as well as from the lymph vessels of rats contain relative high concentrations of MCs reaching values up to 15–20% of the residential cells with respect to other cells .
RW is supported by grants from the German Research Foundation (SFB/TRR57, P13/Q3) and a grant from the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (IZKF Aachen, Project E7-6). MH is supported by a grant from the German Research Foundation (HU794/10-1).
This study was supported by the Deutsche Forschungsgemeinschaft, www.dfg.de, grants SFB/TRR57 (P13, Q3) and HU794/10-1, and IZKF Aachen, www.izkf-aachen.de/#, grant E7-6. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
All relevant data are within the paper and its Supporting Information files.