Cell Biology

Inherited germline variants are now recognized as important contributors to hematologic myeloid malignancies, but their reliable detection depends on obtaining uncontaminated germline DNA. In solid tumors, peripheral blood remains free of tumor cells and therefore serves as a standard source for germline testing. In contrast, peripheral blood often contains neoplastic or clonally mutated cells in hematologic malignancies, making it impossible to distinguish somatic from germline variants. This unique challenge necessitates using an alternative, non-hematopoietic tissue source for accurate germline assessment in patients with hematologic myeloid malignancies. Cultured skin fibroblasts derived from punch biopsies have long been considered the gold standard for this purpose. Nevertheless, most existing protocols are optimized for research settings and lack detailed, patient-centric workflows for routine clinical use. Addressing this translational gap, we present a robust, enzyme-free protocol for culturing dermal fibroblasts from skin punch biopsies collected at the bedside during routine bone marrow procedures. The method details practical bedside collection, sterile transport, mechanical dissection without enzymatic digestion, plating strategy, culture expansion, and high-yield DNA isolation with validated purity. By integrating this standardized approach into routine hematopathology workflows, the protocol ensures reliable germline material with minimal patient discomfort and a turnaround time suitable for clinical diagnostics.

Crypts at the base of intestinal villi contain intestinal stem cells (ISCs) and Paneth cells, the latter of which work as niche cells for ISCs. When isolated and cultured in the presence of specific growth factors, crypts give rise to self-renewing 3D structures called organoids that are highly similar to the crypt-villus structure of the small intestine. However, the organoid culture from whole crypts does not allow investigators to determine the contribution of their individual components, namely ISCs and Paneth cells, to organoid formation efficiency. Here, we describe the method to isolate Paneth cells and ISCs by flow cytometry and co-culture them to form organoids. This approach allows the determination of the contribution of Paneth cells or ISCs to organoid formation and provides a novel tool to analyze the function of Paneth cells, the main component of the intestinal stem cell niche.

Regulatory T cells (Tregs) are essential for maintaining immune balance by controlling the activation and expansion of other immune cells. Conventional suppression assays often rely on co-culturing purified cell populations, which limits multiplexed phenotyping and physiological relevance. This protocol describes a high-dimensional, single-cell assay for profiling Treg-mediated suppression within a peripheral blood mononuclear cell (PBMC) system. Tregs are first isolated by cell sorting and then reintroduced into autologous PBMCs at defined ratios. A 52-marker mass cytometry (CyTOF) panel is used to quantify cell division and phenotypic responses across multiple immune subsets. This approach allows for integrated analysis of Treg function with broad compatibility for patient profiling and drug evaluation.

Adoptive immune cell therapy, especially chimeric antigen receptor T (CAR-T) cells, has emerged as a promising strategy in solid tumor treatment, owing to its unique ability to specifically recognize and effectively eliminate tumor cells. Patient-derived organoids (PDOs) offer a robust and physiologically relevant platform for assessing the safety and efficacy of CAR-T-cell-based therapies. We now describe a detailed protocol for an in vitro evaluation system based on the co-culture of PDOs and CAR-T cells. This system encompasses the establishment of tumor organoids from patient tumor specimens, the isolation of T cells from matched peripheral blood mononuclear cells (PBMCs), and the generation of antigen-specific CAR-T cells. Through the use of fluorescent labeling to visualize different cells and apoptosis-related events post-interaction, along with quantitative analyses of T-cell proliferation, tumor organoid apoptosis, and the secretion of immune effector molecules, this system enables a robust and multifaceted evaluation of CAR-T cell cytotoxicity in vitro. Collectively, this co-culture system provides a systematic and reproducible in vitro platform for evaluating the functional activity of CAR-T cells and advancing research in tumor immunology and immunotherapy.

Continuous and balanced bone remodeling is essential for maintaining mechanical integrity, mineral homeostasis, and hematopoiesis. Dysregulated bone metabolism develops pathological conditions, such as osteoporosis and bone metastasis. Functional and analytical recapitulation of bone remodeling in vitro is critical for advancing our understanding of bone mineral metabolism, disease mechanisms, and drug development. However, conventional models fail to replicate the essential complexity of the bone extracellular matrix (ECM) and the dynamic interplay between bone-forming osteoblasts and bone-resorbing osteoclasts. Recently, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone matrix. DBP supports osteoblastic mineral deposition and the subsequent transition to bone-lining cells. When co-cultured with bone marrow mononuclear cells under biochemical stimulation, osteoblasts shift their regulatory secretion profiles and effectively induce osteoclastogenesis. The semi-transparent nature of DBP, combined with primary osteogenic cells retrieved from DsRed and eGFP reporter mice, enables longitudinal fluorescent monitoring of these multicellular processes and quantitative analysis. In this protocol, we describe the methods for DBP generation, reconstituting mineralized bone tissue complexity with osteoblasts, and recapitulating the bone remodeling cycle through bone marrow monocytes co-culture under biochemical stimulation, offering a useful platform for the related and broader research community.

Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8⁺ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens.

Recurrent hormone receptor-positive (HR+) breast cancer is a leading cause of cancer mortality in women. Recurrence and resistance to targeted therapies have been difficult to study due to the long clinical course of the disease, the complex nature of resistance, and the lack of clinically relevant model systems. Existing models are limited to a few HR+ cell lines, organoid models, and patient-derived xenograft models, all lacking components of the human tumor microenvironment. Furthermore, the low take rate and loss of estrogen receptor (ER) expression in patient-derived organoids (PDOs) has been challenging. Our protocol allows simultaneous isolation of PDOs and matching cancer-associated fibroblasts (CAFs) from primary and metastatic HR+ breast cancers. Importantly, our protocol has a higher take rate and enables long-term culturing of PDOs that retain ER expression. Our matching PDOs and CAFs will provide researchers with a new resource to study the influence of the tumor microenvironment on various aspects of cancer biology such as cell growth and drug resistance in HR+ breast cancer.

The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures.

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3⁺ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4⁺ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4⁺ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4⁺ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4⁺ T cells in an antigen-specific context.

Key features

• This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022).

• This protocol requires transgenic mice, such as OTIIxRag2^-/- mice and the cognate peptide OVA_323–339, to assess mTEC antigen presentation to CD4⁺ T cells.

• This requires specific equipment such as a Miltenyi Biotec AutoMACS^® Pro Separator, a BD FACSAria^TM III cell sorter, and a BD^® LSR II flow cytometer.

Graphical overview

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.

Key features

• Ex vivo co-culture model of the alveolar niche.

• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.

Graphical overview
This protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).

This co-culture experiment is composed of three steps (Graphical overview):
1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively.
2. Culture of these ECs and bone marrow MOs for three days.
3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.