<figure><img src="image_1.png" /></figure>

HAL
open science

# Development and Characterisation of a New In Vitro Murine Mucosal Mast Cell Model

Louise Battut, Jasper Kamphuis, Nadine Serhan, Laurent Reber, Nicolas

Cenac, Gilles Dietrich, Eric Espinosa

► To cite this version:

Louise Battut, Jasper Kamphuis, Nadine Serhan, Laurent Reber, Nicolas Cenac, et al. Development and Characterisation of a New In Vitro Murine Mucosal Mast Cell Model. Allergy, 2025, 10.1111/all.70022. hal-05243935

HAL Id: hal-05243935

https://hal.inrae.fr/hal-05243935v1

Submitted on 8 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

LETTER

OPEN ACCESS

# Development and Characterisation of a New In Vitro Murine Mucosal Mast Cell Model

Louise Battut<fnref n="1" /> ID | Jasper Kamphuis² ID | Nadine Serhan² ID | Laurent Reber² ID | Nicolas Cenac<fnref n="1" /> ID | Gilles Dietrich<fnref n="1" /> ID | Eric Espinosa<fnref n="1" /> ID

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Correspondence:** Eric Espinosa (eric.espinosa@inserm.fr)

**Received:** 5 June 2025 | **Revised:** 16 July 2025 | **Accepted:** 15 August 2025

**Funding:** This work was supported by Institut National de la Santé et de la Recherche Médicale and Fondation pour la Recherche Médicale.

**Keywords:** allergy | mast cell | mucosal mast cell model

To the Editor,

Mouse mast cells (MCs) fall into two subpopulations with well-defined roles and characteristics: connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) [1, 2]. While in vitro models of CTMCs exist in humans and mice, a reliable and relevant model of MMCs is still lacking. While a few previously described protocols have used the addition of TGF-β1 and IL-9 alongside IL-3 and SCF from the onset of bone marrow cell culture to generate MMC-like cells, these approaches often vary in culture duration and in the nature of the resulting cells [3, 4]. Here, we propose a two-step protocol that more faithfully reflects the two major stages of MMC differentiation, enabling the generation of mouse bone marrow-derived mucosal mast cells (BM-MMCs) in vitro. Bone marrow cells were cultured in a complete Opti-MEM medium supplemented with IL-3 and SCF for 4 weeks to induce MC commitment before adding for one additional week both IL-9 and TGF-β1 to promote MMC proliferation and maturation [2, 3, 5] (Figures 1A and S1). This delayed addition of IL-9 and TGF-β1 resulted in a higher percentage of cells showing an MMC phenotype compared with its addition at the start of culture (Figure S2). BM-MMCs were phenotypically and functionally compared to the previously established CTMC model (PCMCs) [6]. In 5 weeks, this two-step differentiation protocol produces approximately 35 million MCs (36 ± 9 million, n = 7 mice) from 1 million bone marrow cells (Figure S3A). After 7 weeks in culture, the BM-MMC showed more than 95% viability (Figure S3B). BM-MMCs expressed the MC markers FcεRI, CD117 and ST2 (IL-33 receptor) together with the typical MMC markers CD103 and MCPT1 (Figure 1B–D) and showed IL-3

dependency (Figure S4). Furthermore, these cells did not stain positively for avidin (which binds to heparin contained in the granules of CTMCs) (Figure 1D) and exhibited a reduced granular mass, histamine, and MCPT6 contents as compared to their PCMC counterparts (Figure 1E–G). BM-MMCs degranulated in response to FcεRI aggregation but not to the 48/80 compound secretagogue, as expected for MMCs which do not express its receptor Mrgprb2 (Figure 1H–J).

Transcriptomic analysis showed clear differences in gene expression between the two cell types. Among the 40 most variable genes, the typical MMC genes *Mcpt1*, *Mcpt2*, *Mcpt8* and *Itgae*, and the typical CTMC genes *Mrgprb2* and *Mrgprb1* were clearly clustered in BM-MMCs and PCMCs, respectively (Figure S5A). We found 5174 differentially expressed genes (DEGs, Padj < 0.01 and Fold change > 2) (Figure 2A and Table S1) between BM-MMCs and PCMCs. Further analysis of the expression of the prototypical MC genes substantiated that BM-MMCs displayed MMC features, including a diminished ability to produce histamine and the expression of genes involved in chondroitin sulfate synthesis (Figure 2B–D) (5). This analysis also revealed that receptors relevant to MC biology were differentially regulated between BM-MMC and PCMC (Figure 2E).

We next investigated whether the genetic signature of BM-MMCs could be found in MMCs described in the literature. Based on a transcriptional analysis of β7 integrinHigh (MMCs) versus β7 integrinLow (CTMCs) lung MCs from HDM-challenged mice [2], gene set enrichment analysis (GSEA) indicated that genes

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Allergy* published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

<footnote n="1">¹Institut de Recherche en Santé Digestive (IRSD), INSERM U1220, INRA, INP-ENVT, University of Toulouse, Toulouse, France | ²Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), UMR 1291, INSERM, CNRS, University of Toulouse, Toulouse, France</footnote>
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_20.png" /></figure>

**FIGURE 1** | Legend on next page.
<figure><img src="image_21.png" /><figcaption>**FIGURE 2** | Legend on next page.</figcaption></figure>
**FIGURE 2** | BM-MMC transcriptome shows typical MMC characteristics. (A) Volcano plot of DEGs between BM-MMCs and PCMCs. Typical MMC and CTMC genes are highlighted. (B–D) Heatmaps showing the expression of MC genes related to granule protease content (B), biogenic amine biosynthesis (C), glycosaminoglycan biosynthesis (D) and the expression of relevant MC receptors (E). (F–G) Gene set enrichment analysis plots from integrin β7high lung MMC genes (black bars) described by Derakhshan et al. (F) and from intestinal MMCs identified by Tauber et al. (G) found in our dataset ranked genes (descending Log₂FC). Green line shows the enrichment score for each gene. (H–J) MC single cell RNA-Seq data from different organs were integrated by Tauber et al. (H). This dataset was used to compute an enrichment score of the 100 most upregulated genes found in our BM-MMCs (I) or PCMCs (J).

overexpressed in β7High MCs were significantly enriched in BM-MMCs (Figure 2F). Likewise, GSEA analysis of sc-RNA-Seq dataset from mouse MCs isolated from different tissues, which exhibited a clear clustering of MMCs (Mrgprb2⁻) and CTMCs (Mrgprb2⁺) [1] showed that the MMC gene signature was significantly enriched in BM-MMCs (Figure 2G). Reciprocally, the enrichment score of the 100 upregulated genes in BM-MMCs revealed a transcriptomic signature similar to that of mouse MMCs identified by Tauber et al. [1] (Figure 2H–J). Moreover, the RNA-Seq analysis of the genes coding pattern recognition receptors and antimicrobial molecules underlined the distinct functional roles of BM-MMCs and PCMCs and the obvious antimicrobial capacity of BM-MMCs (Figure S5B,C).

Thus, BM-MMCs, which have transcriptomic characteristics similar to MMCs, can be an effective tool for studying a significant number of viable MMCs during a 15-day period in vitro.

## Author Contributions

Experimental design: E.E., L.B.; Conducting experiments: L.B., N.S.; Statistical analysis: E.E., L.B.; Writing (original draft): E.E., L.B., G.D.; Writing (review and editing): all authors.

## Acknowledgements

We thank the INFINITy (UMR 1291) flow cytometry (F.E. L'Faqihi-Olive, V. Duplan-Eche, A-L. Iscache and H. Garnier) and cellular imaging (S. Allart, S. Lachambre and L. Lhobjois) core facilities connected to 'Toulouse Réseau Imagerie' network and the CRCT geonomics and transcriptomics platform UMR 1037 (Emeline SAROT, Carine Valle, Nathalie Saint Laurent) for technical assistance. We also thank L. Basso, L. Rey, S. Maurel, M. Berger, A. Le Franc, M. Bécret, C. Joly, L. Fauveau, H. Fries and C. Beuvry for their kind assistance during the course of this work. Louise Battut is supported by a fellowship from FRM, Fondation pour la Recherche Médicale, grants no.: ECO202106013677 and FDT202404018620; this study is supported by grants from Institut National de la Santé et de la Recherche Médicale, the Université de Toulouse and the French Agence Nationale de la Recherche (NOCICEPOP-ANR-22-CE14-0011-01, GD).

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

The data that support the findings of this study are openly available in Gene Expression Ombibus at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi, reference number GSE295774.

Louise Battut
Jasper Kamphuis
Nadine Serhan

Laurent Reber
Nicolas Cenac
Gilles Dietrich
Eric Espinosa

## References

1. M. Tauber, L. Basso, J. Martin, et al., “Landscape of Mast Cell Populations Across Organs in Mice and Humans,” *Journal of Experimental Medicine* 220, no. 10 (2023): e20230570.

2. T. Derakhshan, S. K. Samuchiwal, N. Hallen, et al., “Lineage-Specific Regulation of Inducible and Constitutive Mast Cells in Allergic Airway Inflammation,” *Journal of Experimental Medicine* 218, no. 1 (2021): e20200321.

3. H. R. Miller, S. H. Wright, P. A. Knight, and E. M. Thornton, “A Novel Function for Transforming Growth Factor-beta1: Upregulation of the Expression and the IgE-Independent Extracellular Release of a Mucosal Mast Cell Granule-Specific Beta-Chymase, Mouse Mast Cell Protease-1,” *Blood* 93, no. 10 (1999): 3473–3486.

4. A. Kakinoki, T. Kameo, S. Yamashita, K. Furuta, and S. Tanaka, “Establishment and Characterization of a Murine Mucosal Mast Cell Culture Model,” *International Journal of Molecular Sciences* 21, no. 1 (2019): 236.

5. L. Hultner, C. Druez, J. Moeller, et al., “Mast Cell Growth-Enhancing Activity (MEA) is Structurally Related and Functionally Identical to the Novel Mouse T Cell Growth Factor P40/TCGFIII (Interleukin 9),” *European Journal of Immunology* 20, no. 6 (1990): 1413–1416.

6. O. Malbec, K. Roget, C. Schiffer, et al., “Peritoneal Cell-Derived Mast Cells: An In Vitro Model of Mature Serosal-Type Mouse Mast Cells,” *Journal of Immunology* (Baltimore, Md.: 1950) 178, no. 10 (2007): 6465–6475.

## Supporting Information

Additional supporting information can be found online in the Supporting Information section. **Figure S1:** Determining the optimal timing for adding IL-9 and TGF-β1. Bone marrow cells were cultured in complete Opti-MEM medium with SCF and IL-3, and supplemented with IL-9 plus TGF-β1 either from Day 1 of culture or after 1, 2, 3 or 4 weeks. After 1 week in the IL-9 plus TGF-β1-supplemented medium, surface expression of CD117 and FcεR1 and intracellular expression of MCPT-1 were analysed by flow cytometry. (A) Diagram of experimental protocol. (B) Representative dotplots and histograms. (C) Pooled data from five independent experiments. **Figure S2:** Addition of IL-9 and TGF-β1 at the start of culture results in low MMC phenotype induction. Bone marrow cells were cultured in complete Opti-MEM medium supplemented with IL-3, SCF, IL-9 and TGF-β1 for a period of 3–7 weeks. Surface expression of CD117 and FcεR1 and intracellular expression of MCPT1 were analysed by flow cytometry. (A) Diagram of experimental protocol. (B) Representative dotplots and histograms. (C) Pooled data from three independent experiments. **Figure S3:** BM-MMC culture yield and cell viability. (A) BM-MMC number obtained per 10⁶ seeded cells (pooled data from two independent experiments, n=6 mice). (B) Flow cytometry analysis of BM-MMC viability after seven weeks in culture, representative experiment and pooled data from 3 independent
experiments, each data point represents an individual mouse, median ±95% confidence interval. **Figure S4:** BM-MMCs show IL-3 dependency. After five weeks in culture (Figure 1A), IL-3 concentration was reduced and cell viability was measured seven days later. **Figure S5:** RNA-Seq analysis of BM-MMCs compared with PCMCs. (A) Heatmap of the 40 most variable genes (with the highest variance across all samples), (B, C) heatmaps of genes encoding antimicrobial molecules (B) and pattern recognition receptors (C); gene lists were retrieved from the UniprotKB database and manually curated. Only genes with TPM (transcript per million) > 1 were shown. The Heatmaps clustered genes according to their relative expression. Log₂FoldChange and FDR (BM-MMCs vs. PCMCs), and TPM were also plotted. **Table S1:** Differentially expressed genes (|Log₂FoldChange| > 1 and FDR < 0.01) between BM-MMCs and PCMCs. Summary of statistical analysis with DESeq2 package and gene annotations.