Journal of Immunological Methods 421 (2015) 14–19
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
An optimized protocol for isolating lymphoid stromal cells from the intestinal lamina propria Igor Stzepourginski, Gérard Eberl, Lucie Peduto ⁎ Institut Pasteur, Lymphoid Tissue Development Unit, Paris, France
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 8 November 2014 Accepted 8 November 2014 Available online 17 January 2015 Keywords: Mucosal immunity Intestinal lymphoid stromal cells Tissue dissociation Stromal cell viability/yield Flow cytometry
a b s t r a c t Mesenchymal stromal cells in lymphoid organs, also called lymphoid stromal cells (LSCs), play a pivotal role in immunity by forming specialized microenvironments that provide signals for leukocyte migration, positioning, and survival. Best characterized in lymphoid organs, LSCs are also abundant in the intestinal mucosa, which harbors a rich repertoire of immune cells. However, the lack of efficient procedures for isolation and purification of LSCs from the intestine has been a major limitation to their characterization. Here we report a new method to efficiently isolate, in addition to immune cells, viable lymphoid stromal cells and other stromal subsets from the intestinal lamina propria for subsequent phenotypic and functional analysis. © 2015 Elsevier B.V. All rights reserved.
1. 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
⁎ Corresponding author at: Institut Pasteur, Lymphoid Tissue Development Unit, Paris, France. E-mail address: [email protected] (L. Peduto).
http://dx.doi.org/10.1016/j.jim.2014.11.013 0022-1759/© 2015 Elsevier B.V. All rights reserved.
(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−) nonendothelial (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.
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2. Material and methods
2.5. Antibodies
2.1. Mice
Antibodies purchased from eBiosciences include: V500conjugated CD45.2 (104; 1:100) and biotin or eFluor 660conjugated CD31 (390; 1:200). Syrian hamster antibody to gp38 (1:50 dilution) was a gift from A. Farr (University of Washington, Seattle). Cy3-conjugated anti-Syrian hamster was purchased from Jackson ImmunoResearch. Cy3-conjugated streptavidin was purchased from Sigma.
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. 2.2. Isolation of intestinal stromal cells
2.6. Immunofluorescence histology
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 incubated for 20 min at 37 °C in calcium- and magnesium-free DMEM (Gibco) containing 10 mM EDTA under gentle agitation (80 rpm). We washed tissue pieces by vortexing four times with calcium-free PBS until obtaining a clear supernatant devoid of epithelial cells. We subsequently cut gut pieces into 1 mm fragments and incubated for 20 min at 37 °C in a dissociation mix composed of 5 mL DMEM (Gibco), Liberase TL (1 Wünsch unit/mL; Roche) and DNase I (1 U/mL; Invitrogen). To help dissociation, we gently mixed tissue pieces by pipetting up and down every 10 min, using a truncated pipet tip. After 20 min, we harvested supernatants and added 1 volume of DMEM containing 10% bovine serum (BS, Sigma Aldrich) while adding 5 mL of fresh dissociation mix to the remaining tissue pieces. We repeated this step 3 times for a total time of 60 min. After completion of the 3 cycles, we mechanically disaggregated the remaining intestine fragments on a 100 μm mesh using a syringe plunger. We washed the cellular suspension twice with DMEM in order to eliminate floating debris and filtered twice through a 40 μm mesh. After centrifugation, we resuspended cells in PBS containing 2% BS and 2 mM EDTA and proceeded with antibody staining for FACS analysis . The protocol is summarized in Fig. 1.
We previously described procedures for tissue processing and immunofluorescence staining (Dulauroy et al., 2012). Briefly, we washed tissues in PBS, fixed overnight at 4 °C in 4% paraformaldehyde (Sigma-Aldrich), washed for 24 h in PBS, and incubated for 2–4 h in a solution of 30% sucrose (Sigma-Aldrich) in PBS until the samples sank. We then embedded tissues in OCT compound (VWR Chemicals), and frozed them in a bath of isopentane cooled with liquid nitrogen. We processed 8 μmthickness collected sections on Superfrost Plus slides (VWR International) for antibody staining: after blocking with 10% bovine serum in PBS containing 1% Triton (PBSXG) for 1 h at room temperature, we incubated slides with primary antibodies in PBSXG overnight at 4 °C, washed three times for 5 min with PBSXG, incubated with secondary antibodies or streptavidin for 1 h at room temperature, washed once, incubated with DAPI (Sigma) for 5 min, washed three times for 5 min and mounted with FluoromountG (Southern Biotechnology Associates). We examined slides with an AxioImager M1 fluorescence microscope (Zeiss) equipped with a CCD camera and processed images with AxioVision software (Zeiss).
2.3. Counting We estimated cell numbers in samples using a scepter cell counter (Millipore), with 60 μm sensors. 2.4. Flow cytometry For FACS staining, we first incubated cells with monoclonal antibody 2.4G2 (93; BD Pharmingen) to block Fcγ receptors, and then with the appropriate antibody for 40 min in a total volume of 100 μL of PBS containing 2 mM EDTA and 2% bovine serum (FACS buffer), followed by appropriate secondary antibodies for 30 min when necessary. After centrifugation, we resuspended cells in 200 μL of FACS buffer for analysis. We incubated cells for 1 min with DAPI (Sigma) before analysis to exclude dead cells, and systematically excluded cell doublets during analysis. We performed cell analysis with Fortessa (BD Biosciences) and Flowjo software (Tristar). Fluorescence intensity is expressed in arbitrary units on a logarithmic scale, and forward scatter and side scatter are represented on a linear scale.
3. Results 3.1. 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 allow the release of individual stromal cells from the tissue. Finally, the third step allows for the removal of debris generated during the procedure to obtain single cell suspension for subsequent FACS analysis or sorting. 3.2. Identification of intestinal LSCs The three main non-hematopoietic components of the lamina propria, namely BECs (blood endothelial cells), LECs (lymphatic endothelial cells) and LSCs (lymphoid stromal cells) can be visualized by immunofluorescence staining of gp38 and CD31. While LSCs and BECs are single positive for gp38+ or CD31+ respectively, lymphatics are gp38+CD31+ (Fig. 2A). Here we show by FACS that our protocol for cellular dissociation of the lamina propria is efficient in isolating these
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Fig. 1. Main steps to isolate lymphoid stromal cells from the intestinal lamina propria.
three main non-hematopoietic components of the colonic and ileal lamina propria, in proportions that appear coherent with results obtained by histology. Accordingly, by FACS, LSCs were the main component of the non-hematopoietic fraction (up to 70% and 51% of total CD45− cells in the colon and the ileum lamina propria, respectively, Fig. 2B). Importantly, we could not detect E-cadherin expression in the non-hematopoietic fraction of the lamina propria obtained after step I (Fig. 2C), indicating that the epithelium elimination step is efficient in avoiding epithelial cell contamination in the preparation. 3.3. Cell yield and viability Our protocol allowed us to efficiently purify LSCs from the lamina propria of the colon (106 cells/sample representing about 68 ± 2.3% of non-hematopoietic cells) and ileum (300 × 103
cells/sample representing 46 ± 5.3% of non-hematopoietic cells) while preserving a good cellular viability (89–90%) (Table 1). Such viability is comparable with published protocols for isolation of immune cells from the lamina propria (Bargalló et al., 2014) and is a prerequisite for subsequent cellular or molecular analysis. 3.4. Intestinal LSCs versus immune cells It is noteworthy that identification of LSCs of the intestinal lamina propria by FACS requires an adjustment of the forward/ side scatter gate, as compared to the lymphocyte gate. As shown in Fig. 3B, LSCs are higher in side-scattered light (SSC) than lymphocytes, indicating higher cellular granularity. Moreover, LSCs are highly heterogeneous in forward-scattered light (FSC), thus in size. These results are consistent with previously
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Fig. 2. Identification of intestinal lymphoid stromal cells (LSCs) in the lamina propria of B6 mice. (A) Immunofluorescence analysis of gp38 (red) and CD31 (green) expression by lamina propria cells in the colon or ileum, which identifies LSCs (red), BECs (green) and LECs (orange). (B) FACS plots and percentage of total live (DAPI−) non-hematopoietic (CD45−) cells expressing gp38 and CD31 in the colon or ileum. (C) FACS plots showing E-cadherin (ECad) expression on live (DAPI−) nonhematopoietic cells (CD45−) from the lamina propria fraction (left) or epithelial fraction (right) of the ileum. In A–C, a representative experiment from three to ten independent experiments is shown. LSCs: lymphoid stromal cells (gp38+CD31−), LECs: lymphatic endothelial cells (gp38+CD31+), BECs: blood endothelial cells (gp38−CD31+), DNCs: double negative cells (gp38−CD31−).
reported data describing LSCs in the LN (Fletcher et al., 2010). Therefore, an FSC/SSC gate adapted to the analysis of LSCs should be applied during FACS analysis. Importantly, we could detect all major intestinal lymphocyte populations (B and T lymphocytes, NK cells, and ILCs) as well as DC populations (CD103+ and CD103− DCs) (Fig. 3A) indicating that this protocol is efficient in isolating immune cells as well.
4. 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
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Table 1 Percentage of live cells (DAPI−) and LSC cells (in the DAPI−CD45− gate) obtained by FACS after dissociation of the colon and ileum lamina propria of 8 week old B6 mice. The far right column indicates total LSC numbers. Data are mean ± SD of 4 mice. Viability −
Colon Ileum
Yield
DAPI cells (%)
LSCs among CD45− cells (%)
LSC numbers (×103)
90.1 ± 1.36 89.12 ± 1.81
68.8 ± 2.31 46.4 ± 5.39
1040 ± 188 303 ± 57
intestinal LSCs cells that preserves cellular viability. This multistep protocol (as described in Fig. 1), aims at i) eliminating epithelial cells by EDTA treatment, a step shared with protocols for isolation of immune cells from the lamina propria (Sawa et al., 2011; Bargalló et al., 2014), ii) disrupting the extracellular matrix of the lamina propria using a standardized premix of collagenases in order to obtain single cell suspensions of LSCs, and iii) removing the cellular debris by several cycles of centrifugation and filtration. High variability in yield and poor viability are common issues in isolating stromal cells from tissues, as these cells are
typically embedded within the extracellular matrix and form a complex reticulum. To isolate intestinal LSCs efficiently, we therefore tested several combinations of enzymes, including collagenase crude extract preparations (collagenases A, P, D…) as well as standardized collagenase premixes with defined protease activity. The best yields were obtained with a standardized premix of collagenases I and II + thermolysin (Liberase TL, see Material and methods), which provided excellent cell viability as well as good reproducibility among experiments. We obtained similar results when isolating stromal cells from inflamed colon, suggesting that this protocol might be used to process stromal cells in gut pathologies as well. As liberase TL preparations have reduced unspecific proteolytic activity, which is one of the major causes of decreased cellular viability, time for digestion might be slightly increased if needed when processing stiffer intestines found in aged mice or mice suffering from fibrotic pathologies. To further improve cell viability during the dissociation step, we gently homogenized the digestion mix by pipetting up and down slowly using a truncated tip, thus applying minimal mechanical stress to the tissue. We observed that stronger dissociation methods commonly used to harvest immune cells
Fig. 3. Isolation of intestinal immune cells in the lamina propria of B6 mice. (A) FACS plots and percentages of the major immune cell populations isolated from the ileum lamina propria. (B) FACS plots showing the forward-scattered light (FSC) and side-scattered light (SSC) of lymphocytes (blue) and gp38+ LSCs (red) isolated from the colon and ileum. In A–B, a representative experiment from three independent experiments is shown. LSCs: lymphoid stromal cells (gp38+CD31−); ILCs: innate lymphoid cells; NK: natural killer; non-lymphoid cells refers to CD3−CD19−NK1.1−CD90− cells.
I. Stzepourginski et al. / Journal of Immunological Methods 421 (2015) 14–19
from the lamina propria (shaking, GentleMacs…) (Sawa et al., 2011; Bargalló et al., 2014) significantly decreased stromal cell viability. FACS analysis and cell sorting requires single cell suspensions free of cellular debris and other dissociation products from the matrix, which are abundant upon dissociation of the intestinal lamina propria. We found that several cycles of washing and filtration through a 40 μm filter (see Material and methods) are more efficient to remove debris while preserving a good cellular yield than the Percoll gradient, commonly performed when isolating lymphocytes from the lamina propria (Sawa et al., 2011). Altogether, we describe here a novel and optimized protocol for the efficient isolation of both LSCs and immune cells from the intestinal lamina propria, as well as other nonhematopoietic cells like BECs and LECs. The relative yields of LSCs obtained were consistent with histological data of mouse colon and ileum, and were combined with excellent cellular viability. Interestingly, the heterogeneous FSC profile of intestinal LSCs suggests that there are distinct subpopulations, possibly with distinct functions. Isolation and characterization of intestinal LSCs may shed new light on their function(s) in intestinal homeostasis and gut immunity. 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). References Bajénoff, M., Egen, J.G., Koo, L.Y., Laugier, J.P., Brau, F., Glaichenhaus, N., Germain, R.N., 2006. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25 (6), 989.
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Bargalló, A., Abad, L., Òdena, G., Planas, R., Bartolí, R., 2014. New method for isolation of rat lamina propria macrophages in colonic tissue. J. Immunol. Methods 408, 132. Dulauroy, S., Di Carlo, S.E., Langa, F., Eberl, G., Peduto, L., 2012. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat. Med. 18 (8), 1262. Fletcher, A.L., Lukacs-Kornek, V., Reynoso, E.D., Pinner, S.E., Bellemare-Pelletier, A., Curry, M.S., Collier, A.-R., Boyd, R.L., Turley, S.J., 2010. Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions. J. Exp. Med. 207 (4), 689. Link, A., Vogt, T.K., Favre, S., Britschgi, M.R., Acha-Orbea, H., Hinz, B., Cyster, J.G., Luther, S.A., 2007. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8 (11), 1255. Lukacs-Kornek, V., Malhotra, D., Fletcher, A.L., Acton, S.E., Elpek, K.G., Tayalia, P., Collier, A.-R., Turley, S.J., 2011. Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes. Nat. Immunol. 12 (11), 1096. Malhotra, D., Fletcher, A.L., Astarita, J., Lukacs-Kornek, V., Tayalia, P., Gonzalez, S.F., Elpek, K.G., Chang, S.K., Knoblich, K., Hemler, M.E., Brenner, M.B., Carroll, M.C., Mooney, D.J., Turley, S.J., Immunological Genome Project Consortium, 2012. Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks. Nat. Immunol. 13 (5), 499. Mifflin, R.C., Pinchuk, I.V., Saada, J.I., Powell, D.W., 2011. Intestinal myofibroblasts: targets for stem cell therapy. Am. J. Physiol. Gastrointest. Liver Physiol. 300 (5), G684. Peduto, L., Dulauroy, S., Lochner, M., Späth, G.F., Morales, M.A., Cumano, A., Eberl, G., 2009. Inflammation recapitulates the ontogeny of lymphoid stromal cells. J. Immunol. 182 (9), 5789. Sawa, S., Lochner, M., Satoh-Takayama, N., Langa, F., Di Santo, J.P., Eberl, G., 2010. Lineage relationship analysis of RORgammat + innate lymphoid cells. Science 330 (6004), 665. Sawa, S., Lochner, M., Satoh-Takayama, N., Dulauroy, S., Bérard, M., Kleinschek, M., Cua, D., Di Santo, J.P., Eberl, G., 2011. ROR gamma t(+) innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. Nat. Immunol. 12 (4) (320-U371). Yang, C.-Y., Vogt, T.K., Favre, S., Scarpellino, L., Huang, H.-Y., Tacchini-Cottier, F., Luther, S.A., 2014. Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes. PNAS 111 (1), E109.
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
An optimized protocol for isolating lymphoid stromal cells from the intestinal lamina propria Igor Stzepourginski, Gérard Eberl, Lucie Peduto ⁎ Institut Pasteur, Lymphoid Tissue Development Unit, Paris, France
a r t i c l e
i n f o
Article history: Received 16 September 2014 Received in revised form 8 November 2014 Accepted 8 November 2014 Available online 17 January 2015 Keywords: Mucosal immunity Intestinal lymphoid stromal cells Tissue dissociation Stromal cell viability/yield Flow cytometry
a b s t r a c t Mesenchymal stromal cells in lymphoid organs, also called lymphoid stromal cells (LSCs), play a pivotal role in immunity by forming specialized microenvironments that provide signals for leukocyte migration, positioning, and survival. Best characterized in lymphoid organs, LSCs are also abundant in the intestinal mucosa, which harbors a rich repertoire of immune cells. However, the lack of efficient procedures for isolation and purification of LSCs from the intestine has been a major limitation to their characterization. Here we report a new method to efficiently isolate, in addition to immune cells, viable lymphoid stromal cells and other stromal subsets from the intestinal lamina propria for subsequent phenotypic and functional analysis. © 2015 Elsevier B.V. All rights reserved.
1. 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
⁎ Corresponding author at: Institut Pasteur, Lymphoid Tissue Development Unit, Paris, France. E-mail address: [email protected] (L. Peduto).
http://dx.doi.org/10.1016/j.jim.2014.11.013 0022-1759/© 2015 Elsevier B.V. All rights reserved.
(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−) nonendothelial (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.
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2. Material and methods
2.5. Antibodies
2.1. Mice
Antibodies purchased from eBiosciences include: V500conjugated CD45.2 (104; 1:100) and biotin or eFluor 660conjugated CD31 (390; 1:200). Syrian hamster antibody to gp38 (1:50 dilution) was a gift from A. Farr (University of Washington, Seattle). Cy3-conjugated anti-Syrian hamster was purchased from Jackson ImmunoResearch. Cy3-conjugated streptavidin was purchased from Sigma.
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. 2.2. Isolation of intestinal stromal cells
2.6. Immunofluorescence histology
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 incubated for 20 min at 37 °C in calcium- and magnesium-free DMEM (Gibco) containing 10 mM EDTA under gentle agitation (80 rpm). We washed tissue pieces by vortexing four times with calcium-free PBS until obtaining a clear supernatant devoid of epithelial cells. We subsequently cut gut pieces into 1 mm fragments and incubated for 20 min at 37 °C in a dissociation mix composed of 5 mL DMEM (Gibco), Liberase TL (1 Wünsch unit/mL; Roche) and DNase I (1 U/mL; Invitrogen). To help dissociation, we gently mixed tissue pieces by pipetting up and down every 10 min, using a truncated pipet tip. After 20 min, we harvested supernatants and added 1 volume of DMEM containing 10% bovine serum (BS, Sigma Aldrich) while adding 5 mL of fresh dissociation mix to the remaining tissue pieces. We repeated this step 3 times for a total time of 60 min. After completion of the 3 cycles, we mechanically disaggregated the remaining intestine fragments on a 100 μm mesh using a syringe plunger. We washed the cellular suspension twice with DMEM in order to eliminate floating debris and filtered twice through a 40 μm mesh. After centrifugation, we resuspended cells in PBS containing 2% BS and 2 mM EDTA and proceeded with antibody staining for FACS analysis . The protocol is summarized in Fig. 1.
We previously described procedures for tissue processing and immunofluorescence staining (Dulauroy et al., 2012). Briefly, we washed tissues in PBS, fixed overnight at 4 °C in 4% paraformaldehyde (Sigma-Aldrich), washed for 24 h in PBS, and incubated for 2–4 h in a solution of 30% sucrose (Sigma-Aldrich) in PBS until the samples sank. We then embedded tissues in OCT compound (VWR Chemicals), and frozed them in a bath of isopentane cooled with liquid nitrogen. We processed 8 μmthickness collected sections on Superfrost Plus slides (VWR International) for antibody staining: after blocking with 10% bovine serum in PBS containing 1% Triton (PBSXG) for 1 h at room temperature, we incubated slides with primary antibodies in PBSXG overnight at 4 °C, washed three times for 5 min with PBSXG, incubated with secondary antibodies or streptavidin for 1 h at room temperature, washed once, incubated with DAPI (Sigma) for 5 min, washed three times for 5 min and mounted with FluoromountG (Southern Biotechnology Associates). We examined slides with an AxioImager M1 fluorescence microscope (Zeiss) equipped with a CCD camera and processed images with AxioVision software (Zeiss).
2.3. Counting We estimated cell numbers in samples using a scepter cell counter (Millipore), with 60 μm sensors. 2.4. Flow cytometry For FACS staining, we first incubated cells with monoclonal antibody 2.4G2 (93; BD Pharmingen) to block Fcγ receptors, and then with the appropriate antibody for 40 min in a total volume of 100 μL of PBS containing 2 mM EDTA and 2% bovine serum (FACS buffer), followed by appropriate secondary antibodies for 30 min when necessary. After centrifugation, we resuspended cells in 200 μL of FACS buffer for analysis. We incubated cells for 1 min with DAPI (Sigma) before analysis to exclude dead cells, and systematically excluded cell doublets during analysis. We performed cell analysis with Fortessa (BD Biosciences) and Flowjo software (Tristar). Fluorescence intensity is expressed in arbitrary units on a logarithmic scale, and forward scatter and side scatter are represented on a linear scale.
3. Results 3.1. 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 allow the release of individual stromal cells from the tissue. Finally, the third step allows for the removal of debris generated during the procedure to obtain single cell suspension for subsequent FACS analysis or sorting. 3.2. Identification of intestinal LSCs The three main non-hematopoietic components of the lamina propria, namely BECs (blood endothelial cells), LECs (lymphatic endothelial cells) and LSCs (lymphoid stromal cells) can be visualized by immunofluorescence staining of gp38 and CD31. While LSCs and BECs are single positive for gp38+ or CD31+ respectively, lymphatics are gp38+CD31+ (Fig. 2A). Here we show by FACS that our protocol for cellular dissociation of the lamina propria is efficient in isolating these
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Fig. 1. Main steps to isolate lymphoid stromal cells from the intestinal lamina propria.
three main non-hematopoietic components of the colonic and ileal lamina propria, in proportions that appear coherent with results obtained by histology. Accordingly, by FACS, LSCs were the main component of the non-hematopoietic fraction (up to 70% and 51% of total CD45− cells in the colon and the ileum lamina propria, respectively, Fig. 2B). Importantly, we could not detect E-cadherin expression in the non-hematopoietic fraction of the lamina propria obtained after step I (Fig. 2C), indicating that the epithelium elimination step is efficient in avoiding epithelial cell contamination in the preparation. 3.3. Cell yield and viability Our protocol allowed us to efficiently purify LSCs from the lamina propria of the colon (106 cells/sample representing about 68 ± 2.3% of non-hematopoietic cells) and ileum (300 × 103
cells/sample representing 46 ± 5.3% of non-hematopoietic cells) while preserving a good cellular viability (89–90%) (Table 1). Such viability is comparable with published protocols for isolation of immune cells from the lamina propria (Bargalló et al., 2014) and is a prerequisite for subsequent cellular or molecular analysis. 3.4. Intestinal LSCs versus immune cells It is noteworthy that identification of LSCs of the intestinal lamina propria by FACS requires an adjustment of the forward/ side scatter gate, as compared to the lymphocyte gate. As shown in Fig. 3B, LSCs are higher in side-scattered light (SSC) than lymphocytes, indicating higher cellular granularity. Moreover, LSCs are highly heterogeneous in forward-scattered light (FSC), thus in size. These results are consistent with previously
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Fig. 2. Identification of intestinal lymphoid stromal cells (LSCs) in the lamina propria of B6 mice. (A) Immunofluorescence analysis of gp38 (red) and CD31 (green) expression by lamina propria cells in the colon or ileum, which identifies LSCs (red), BECs (green) and LECs (orange). (B) FACS plots and percentage of total live (DAPI−) non-hematopoietic (CD45−) cells expressing gp38 and CD31 in the colon or ileum. (C) FACS plots showing E-cadherin (ECad) expression on live (DAPI−) nonhematopoietic cells (CD45−) from the lamina propria fraction (left) or epithelial fraction (right) of the ileum. In A–C, a representative experiment from three to ten independent experiments is shown. LSCs: lymphoid stromal cells (gp38+CD31−), LECs: lymphatic endothelial cells (gp38+CD31+), BECs: blood endothelial cells (gp38−CD31+), DNCs: double negative cells (gp38−CD31−).
reported data describing LSCs in the LN (Fletcher et al., 2010). Therefore, an FSC/SSC gate adapted to the analysis of LSCs should be applied during FACS analysis. Importantly, we could detect all major intestinal lymphocyte populations (B and T lymphocytes, NK cells, and ILCs) as well as DC populations (CD103+ and CD103− DCs) (Fig. 3A) indicating that this protocol is efficient in isolating immune cells as well.
4. 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
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Table 1 Percentage of live cells (DAPI−) and LSC cells (in the DAPI−CD45− gate) obtained by FACS after dissociation of the colon and ileum lamina propria of 8 week old B6 mice. The far right column indicates total LSC numbers. Data are mean ± SD of 4 mice. Viability −
Colon Ileum
Yield
DAPI cells (%)
LSCs among CD45− cells (%)
LSC numbers (×103)
90.1 ± 1.36 89.12 ± 1.81
68.8 ± 2.31 46.4 ± 5.39
1040 ± 188 303 ± 57
intestinal LSCs cells that preserves cellular viability. This multistep protocol (as described in Fig. 1), aims at i) eliminating epithelial cells by EDTA treatment, a step shared with protocols for isolation of immune cells from the lamina propria (Sawa et al., 2011; Bargalló et al., 2014), ii) disrupting the extracellular matrix of the lamina propria using a standardized premix of collagenases in order to obtain single cell suspensions of LSCs, and iii) removing the cellular debris by several cycles of centrifugation and filtration. High variability in yield and poor viability are common issues in isolating stromal cells from tissues, as these cells are
typically embedded within the extracellular matrix and form a complex reticulum. To isolate intestinal LSCs efficiently, we therefore tested several combinations of enzymes, including collagenase crude extract preparations (collagenases A, P, D…) as well as standardized collagenase premixes with defined protease activity. The best yields were obtained with a standardized premix of collagenases I and II + thermolysin (Liberase TL, see Material and methods), which provided excellent cell viability as well as good reproducibility among experiments. We obtained similar results when isolating stromal cells from inflamed colon, suggesting that this protocol might be used to process stromal cells in gut pathologies as well. As liberase TL preparations have reduced unspecific proteolytic activity, which is one of the major causes of decreased cellular viability, time for digestion might be slightly increased if needed when processing stiffer intestines found in aged mice or mice suffering from fibrotic pathologies. To further improve cell viability during the dissociation step, we gently homogenized the digestion mix by pipetting up and down slowly using a truncated tip, thus applying minimal mechanical stress to the tissue. We observed that stronger dissociation methods commonly used to harvest immune cells
Fig. 3. Isolation of intestinal immune cells in the lamina propria of B6 mice. (A) FACS plots and percentages of the major immune cell populations isolated from the ileum lamina propria. (B) FACS plots showing the forward-scattered light (FSC) and side-scattered light (SSC) of lymphocytes (blue) and gp38+ LSCs (red) isolated from the colon and ileum. In A–B, a representative experiment from three independent experiments is shown. LSCs: lymphoid stromal cells (gp38+CD31−); ILCs: innate lymphoid cells; NK: natural killer; non-lymphoid cells refers to CD3−CD19−NK1.1−CD90− cells.
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from the lamina propria (shaking, GentleMacs…) (Sawa et al., 2011; Bargalló et al., 2014) significantly decreased stromal cell viability. FACS analysis and cell sorting requires single cell suspensions free of cellular debris and other dissociation products from the matrix, which are abundant upon dissociation of the intestinal lamina propria. We found that several cycles of washing and filtration through a 40 μm filter (see Material and methods) are more efficient to remove debris while preserving a good cellular yield than the Percoll gradient, commonly performed when isolating lymphocytes from the lamina propria (Sawa et al., 2011). Altogether, we describe here a novel and optimized protocol for the efficient isolation of both LSCs and immune cells from the intestinal lamina propria, as well as other nonhematopoietic cells like BECs and LECs. The relative yields of LSCs obtained were consistent with histological data of mouse colon and ileum, and were combined with excellent cellular viability. Interestingly, the heterogeneous FSC profile of intestinal LSCs suggests that there are distinct subpopulations, possibly with distinct functions. Isolation and characterization of intestinal LSCs may shed new light on their function(s) in intestinal homeostasis and gut immunity. 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). References Bajénoff, M., Egen, J.G., Koo, L.Y., Laugier, J.P., Brau, F., Glaichenhaus, N., Germain, R.N., 2006. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25 (6), 989.
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Bargalló, A., Abad, L., Òdena, G., Planas, R., Bartolí, R., 2014. New method for isolation of rat lamina propria macrophages in colonic tissue. J. Immunol. Methods 408, 132. Dulauroy, S., Di Carlo, S.E., Langa, F., Eberl, G., Peduto, L., 2012. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat. Med. 18 (8), 1262. Fletcher, A.L., Lukacs-Kornek, V., Reynoso, E.D., Pinner, S.E., Bellemare-Pelletier, A., Curry, M.S., Collier, A.-R., Boyd, R.L., Turley, S.J., 2010. Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions. J. Exp. Med. 207 (4), 689. Link, A., Vogt, T.K., Favre, S., Britschgi, M.R., Acha-Orbea, H., Hinz, B., Cyster, J.G., Luther, S.A., 2007. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8 (11), 1255. Lukacs-Kornek, V., Malhotra, D., Fletcher, A.L., Acton, S.E., Elpek, K.G., Tayalia, P., Collier, A.-R., Turley, S.J., 2011. Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes. Nat. Immunol. 12 (11), 1096. Malhotra, D., Fletcher, A.L., Astarita, J., Lukacs-Kornek, V., Tayalia, P., Gonzalez, S.F., Elpek, K.G., Chang, S.K., Knoblich, K., Hemler, M.E., Brenner, M.B., Carroll, M.C., Mooney, D.J., Turley, S.J., Immunological Genome Project Consortium, 2012. Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks. Nat. Immunol. 13 (5), 499. Mifflin, R.C., Pinchuk, I.V., Saada, J.I., Powell, D.W., 2011. Intestinal myofibroblasts: targets for stem cell therapy. Am. J. Physiol. Gastrointest. Liver Physiol. 300 (5), G684. Peduto, L., Dulauroy, S., Lochner, M., Späth, G.F., Morales, M.A., Cumano, A., Eberl, G., 2009. Inflammation recapitulates the ontogeny of lymphoid stromal cells. J. Immunol. 182 (9), 5789. Sawa, S., Lochner, M., Satoh-Takayama, N., Langa, F., Di Santo, J.P., Eberl, G., 2010. Lineage relationship analysis of RORgammat + innate lymphoid cells. Science 330 (6004), 665. Sawa, S., Lochner, M., Satoh-Takayama, N., Dulauroy, S., Bérard, M., Kleinschek, M., Cua, D., Di Santo, J.P., Eberl, G., 2011. ROR gamma t(+) innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. Nat. Immunol. 12 (4) (320-U371). Yang, C.-Y., Vogt, T.K., Favre, S., Scarpellino, L., Huang, H.-Y., Tacchini-Cottier, F., Luther, S.A., 2014. Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes. PNAS 111 (1), E109.
