Research paperAn optimized protocol for isolating lymphoid stromal cells from the intestinal lamina propria
Introduction
The microenvironment of lymphoid organs is formed by blood and lymphatic vasculature, as well as distinct subsets of mesenchymal stromal cells collectively termed lymphoid stromal cells (LSCs). LSCs play essential roles in the migration and survival of leukocytes, and thus in the generation of efficient immune responses. Several subsets of LSCs have been characterized. Follicular dendritic cells (FDCs) within the lymph node cortex support B cell responses while fibroblastic reticular cells (FRCs) within the paracortex support T cell responses (Bajénoff et al., 2006, Link et al., 2007). During inflammation, FRCs promote LN expansion by enhancing the recruitment of naïve lymphocytes and antigen-presenting cells (Malhotra et al., 2012, Yang et al., 2014). FRCs also regulate immune responses by restricting the proliferation of activated T cells and eliminating auto-reactive T cells from the repertoire (Fletcher et al., 2010, Lukacs-Kornek et al., 2011). FRCs are commonly defined by expression of the surface glycoprotein podoplanin (gp38+) in the non-hematopoietic (CD45−) non-endothelial (CD31−) population, a phenotype distinct from blood endothelial cells (BECs) (gp38−CD31+) and lymphatic endothelial cells (LECs) (gp38+CD31+). In addition to lymphoid organs, an abundant population of gp38+ LSCs with characteristics of FRCs is present constitutively in the intestinal lamina propria, and is readily induced by inflammation in other organs (Peduto et al., 2009). To which extent these cells are related to other subsets of intestinal stromal cells, such as subepithelial myofibroblasts (Mifflin et al., 2011), is still unclear.
The large number of gp38+ LSCs in the intestinal lamina propria suggests that these cells may have important roles in intestinal homeostasis and defense. However, protocols to isolate immune cells from the lamina propria (Sawa et al., 2011, Bargalló et al., 2014) are not optimal for the recovery of LSCs, and therefore these cells remain poorly characterized. Here, we describe a novel protocol for the efficient isolation of viable LSCs, as well as BECs and LECs, from the intestinal lamina propria.
Section snippets
Mice
We obtained C57BL/6J wild-type mice from Charles Rivers and housed them in a specific pathogen-free (SPF) condition at the Institut Pasteur. The study was performed in accordance to institutional guidelines for animal care and use.
Isolation of intestinal stromal cells
To isolate intestinal stromal cells, we excised and washed in PBS the whole colon and a 15 cm-long piece of terminal ileum of 8 week old C57BL/6J mice. After removal of Peyer's patches from the ileum, we opened gut fragments longitudinally, cut them into 2 cm pieces and
Main steps for intestinal LSC isolation
The main steps to isolate intestinal stromal cells are described in Fig. 1. The first step is similar to protocols used for isolation of immune cells from the lamina propria (Sawa et al., 2010, Bargalló et al., 2014) and aims at eliminating epithelial cells by EDTA treatment. The second step consists in tissue dissociation of the lamina propria by a mixture of enzymes combining collagenases I and II and a low amount of neutral protease (thermolysin), which disrupts the extracellular matrix to
Discussion
Intestinal LSCs are a major component of the intestinal lamina propria, and may have roles in intestinal homeostasis and defense. However, the lack of an efficient isolation protocol has limited the characterization of these cells. Here, we describe an optimized protocol for the efficient isolation of intestinal LSCs cells that preserves cellular viability. This multi-step protocol (as described in Fig. 1), aims at i) eliminating epithelial cells by EDTA treatment, a step shared with protocols
Acknowledgements
This work was funded by the Institut Pasteur and the Agence Nationale de la Recherche. I.S. was funded by the Ministère de l'Enseignement Supérieur et de la Recherche, Université Paris 7, and the Fondation pour la Recherche Medicale en France (FDT20130928338).
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