quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses

Accepted Manuscript Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses R.R.C.E Schreurs, A Drewniak, R Bakx, W.E Corpeleijn, T.H.B Geijtenbeek, J.B van Goudoever, M.J Bunders PII: DOI: Reference: S0022-1759(16)30277-0 doi: 10.1016/j.jim.2017.03.006 JIM 12282 To appear in: Journal of Immunological Methods Received date: Revised date: Accepted date: 21 October 2016 15 February 2017 March 2017 Please cite this article as: R.R.C.E Schreurs, A Drewniak, R Bakx, W.E Corpeleijn, T.H.B Geijtenbeek, J.B van Goudoever, M.J Bunders , Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses The address for the corresponding author was captured as affiliation for all authors Please check if appropriate Jim(2017), doi: 10.1016/j.jim.2017.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses R.R.C.E Schreursa,b, A Drewniakb, R Bakxc, W.E Corpeleijna, T.H.B Geijtenbeekb, J.B van Goudoevera, M.J Bundersa,b* a Department of Pediatrics, Emma Children’s Hospital, Academic Medical Center (AMC), University of PT Amsterdam (UvA), Amsterdam, The Netherlands Department of Experimental Immunology, AMC, UvA c Department of Pediatric Surgery, Pediatric Surgical Center, Emma Children’s Hospital, AMC & Free RI b SC University Medical Center Amsterdam, Amsterdam, The Netherlands * Correspondence should be addressed to M.J.B (m.j.bunders@amc.nl), department of Experimental NU Immunology, AMC, UvA, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands Keywords: MA Human Lymphocytes Monocytes D Isolation method PT E Intestine ABSTRACT Studies on immune cells derived from the human intestine are needed to understand the CE pathogenesis of gastrointestinal diseases and to develop novel treatment strategies Isolation techniques to extract these immune cells from intestinal tissue are largely based on murine studies AC and comparative data on isolation from human intestine is scarce In this study we evaluated cell yield, viability, and surface-molecule expression on mononuclear leukocytes, comparing three techniques to obtain a single immune cell suspension from human intestine; low concentrations of either the enzymes Collagenase D or Liberase TL, and enzyme-free mechanical dissociation with the Medimachine Both enzymatic isolation techniques provided a higher cell yield than mechanical dissociation Expression of surface molecules remained intact after Collagenase D treatment, while Liberase TL digestion resulted in a strong decrease in the expression of the CD4 receptor on T cells Monocyte-derived cell surface molecules were not differentially affected by either enzyme Taken together, Collagenase D digestion provides the highest yield of T cells while keeping surface molecule ACCEPTED MANUSCRIPT expression intact Both Collagenase D and Liberase TL result in a good yield without differentially altering the expression of cell surface molecules when investigating monocyte-derived cells Introduction The human intestinal mucosa is a dynamic interface between the body and the environment The intestinal lumen provides an extensive surface area for the digestion and absorption of essential metabolites (Mowat & Agace, 2014) At the same time, adequate barrier function is required to PT prevent microbial translocation (Brown, Sadarangani, & Finlay, 2013; Cebra, 1999) To maintain homeostasis under continuous microbial exposure, tissue-resident intestinal immune cells such as RI lymphocytes and monocyte-derived cells play a crucial role However, protective immune responses (Siddiqui & Powrie, 2008; Veenbergen & Samsom, 2012) SC need to be tightly regulated in order to prevent excess inflammation and collateral tissue damage NU Dysregulation of intestinal immune cells leads to increased susceptibility to severe infections, while deficient tolerant responses of lymphocytes and monocyte-derived cells provide the underlying conditions for intestinal inflammatory diseases such as ulcerative colitis (Troncone, Marafini, Pallone, MA & Monteleone, 2013) In order to understand the pathogenesis of intestinal diseases, in-depth analyses of immune responses at the tissue level are essential as populations of immune cells in the D blood and the intestinal compartments differ substantially (Kunkel & Butcher, 2002) PT E The number of studies investigating tissue-resident immune cells is rapidly increasing (Annunziato et al., 2007; Bunders et al., 2012; Sathaliyawala et al., 2013; Thome et al., 2016) Throughout those studies, various techniques are applied to isolate the mononuclear leukocyte CE fraction from the human intestine, first pioneered by Wahl and Smith (1991) However, these techniques differ in terms of cell yield and expression of the molecules of interest on the cell surface AC (Chen et al., 2014; Shen et al., 2015) Mucosal lymphoid and monocyte-derived cells are present in several intestinal compartments; the epithelium, the underlying lamina propria, the Peyer’s patches (PPs) embedded in the small intestine, and the isolated lymphoid follicles (ILFs) embedded in the colorectum These compartments contain cell types with a phenotype and functionality unique to their anatomical location (Mowat & Agace, 2014) In this study we focus on the isolation of mononuclear leukocytes from the epithelium and from the lamina propria, excluding PPs and ILFs on macroscopic visual inspection of the tissues The isolation of the intraepithelial mononuclear leukocytes (IELs) is homogenous among protocols; dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) are used to remove ACCEPTED MANUSCRIPT mucus and to detach the epithelial cells from the tissue Subsequently, enrichment of the cell suspension for IELs is commonly achieved with gradient centrifugation, which separates cells based on their buoyant density (Fuss, Kanof, Smith, & Zola, 2009) However, layer gradients vary between protocols from 60%-67.5% Percoll (Braunstein, Qiao, Autschbach, Schürmann, & Meuer, 1997; Ebert & Roberts, 1995; Lundqvist, Hammarström, Athlin, & Hammarström, 1992) to using standard FicollHypaque (Comer, Ramey, Kotler, & Holt, 1986) PT After detachment of the epithelial layer, the mononuclear leukocytes from the lamina propria (LPLs) can be obtained via mechanic or enzymatic disaggregation of the tissue The described RI isolation techniques to obtain this cell population vary greatly with regards to cell yield (Shacklett et al., 2003) Furthermore, the expression of molecules of interest on the cell surface (Chen et al., 2014; SC Shen et al., 2015) can be differentially altered by the techniques thereby impacting the feasibility of the study as well as the interpretation of the obtained results An isolation technique that harvests NU the maximal number of cells while limiting alterations to cell surface marker expression is particularly relevant to pediatric studies as collection of sufficient tissue is often more challenging than in adults MA Taken together, comparative data of cell numbers and surface marker consistency of the most frequently used techniques to isolate mononuclear leukocytes from the epithelium and lamina propria is needed to design the appropriate methodology for performing intestinal immune cell D analyses In this study, we compared three frequently used methods of lamina propria disaggregation PT E to obtain a single mononuclear leukocyte suspension: Medimachine (enzyme-free mechanical disaggregation), and low concentrations of the enzymes Collagenase D and Liberase TL Furthermore, we investigated the efficiency of using different density gradients to enrich for IELs and LPLs The CE different isolation and enrichment techniques of leukocytes (T lymphocytes and monocyte-derived cells) from intestinal mucosal tissue were compared regarding cell yield, viability, and surface marker AC expression Materials and methods 2.1 Tissue samples Human tissues were collected after the donors or their guardians provided informed consent All experiments were performed on fresh large bowel tissue (colon) from ten individuals obtained during surgery (Supplementary Table 1); four were infants (median age months, interquartile range (IQR) 4-11 months), of which three were surgically treated for Hirschsprung’s disease and one for anorectal malformation; six were adults (median known age 62 years, IQR 44-63 years), of which four were surgically treated for ulcerative colitis and two for colorectal cancer All ACCEPTED MANUSCRIPT tissue was derived from the surgical margin and based on visual assessment all tissues, except from one adult patient with ulcerative colitis, were presumed to be not severely affected Every tissue was processed within six hours after resection Because of the variation in donor age and diagnosis, direct comparisons were only made within individual donors, not between them The study was approved by the medical ethical committee of our institute, the Academic Medical Center (University of Amsterdam) and in accordance with the Declaration of Helsinki PT 2.2 Tissue preparation The tissue was rinsed by manually shaking 20 seconds in 20 ml sterile PBS in a 50 ml RI canonical centrifuge tube (Greiner, Sigma-Aldrich), followed by removal of the muscular layer using scissors (Fig 1) To determine cell yield per cm2, the size of the mucosal tissue was measured after PT E D MA NU SC removal of the muscular layer The tissue was divided into pieces with equal surface area (0.5 cm2) Figure Removal of the muscular layer (bottom) from the mucosa (top) CE 2.3 Epithelial layer detachment To remove the remaining mucus and detach the epithelial layer containing the IELs, the AC intestinal fragments were incubated for 20 minutes in a shaking water bath (approximately 100 strokes/min) at 37°C in 10 ml Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza, Verviers, Belgium) containing mM DTT, mM EDTA, 5% fetal calf serum (FCS; Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Gibco, Life Technologies) in a 50 ml canonical centrifuge tube The suspension including the tissue was then vortexed (IKA; MS3 basic Lab Shaker) at maximum speed (3000 rpm for this model) for 15 seconds after which the cell suspension without tissue was passed through a 70 µm single-cell strainer (Falcon, Corning, USA), rinsed with 4°C PBS and centrifuged at 400 G at 4°C for 10 minutes A second incubation with the above described DTT/EDTA solution followed for the remaining tissue using the same procedure and ACCEPTED MANUSCRIPT resulting in a second single cell suspension The two single cell suspensions were pooled after washing them with 4°C PBS and stored in PBS on ice until density gradient centrifugation 2.4 Lamina propria disaggregation 2.4.1 Mechanical disaggregation using the Medimachine The BD Medimachine System (BD Biosciences) was used as described before (Bunders et al., PT 2012) Briefly, it is an automated mechanical disaggregation system of solid human tissues without enzymes The Medimachine spins the tissue in the Medicon filter (BD Biosciences, San Jose, USA) to RI disaggregate tissue fragments into single cells After detachment of the epithelial layer, one tissue fragment at a time (0.5 cm2) was placed in the Medicon filter with ml PBS The single-cell SC suspension was obtained by placing a ml syringe on the out-port of the Medimachine and extracting the cell suspension This procedure was repeated until each fragment was dissolved NU completely Cells were passed through a 70 µm single-cell strainer, rinsed with 4°C PBS, and centrifuged at 400 G (4°C) for 10 minutes The obtained single cell suspension was stored in PBS on ice until density gradient isolation Medimachine isolation was always compared with Collagenase D MA isolation; half of the tissue of one donor was mechanically disaggregated with Medimachine while the other half was enzymatically digested with Collagenase D as described in the next paragraph (and D in Fig 2) PT E 2.4.2 Enzymatic disaggregation using Collagenase D After detachment of the epithelial layer, tissue fragments were placed in a 50 ml tube with 10 ml disaggregation solution, which contains IMDM with mg/ml (0.15 U/mg) Collagenase D CE (Roche, Mannheim, Germany), 1% FCS, and 1000 U/ml DNAse type I (Roche) The fragments were incubated in the medium in a shaking water bath at 37°C for 30 minutes (approximately 100 AC strokes/min) The supernatant containing the cell suspension was collected and passed through a 70 µm single-cell strainer, rinsed with 4°C PBS, and centrifuged at 400 G at 4°C for 10 minutes The remaining fragments were incubated in fresh disaggregation solution containing Collagenase D for another 30 minutes after which the remaining cell suspension was filtered through a 70 µm singlecell strainer The filtered single cell suspension was rinsed with 4°C PBS, centrifuged at 400 G at 4°C for 10 minutes and stored in PBS on ice till density gradient isolation 2.4.3 Enzymatic digestion using Liberase TL Analogous to the above described isolation technique of lamina propria with collagenase D, the tissue fragments (after detachment of the epithelial cells) were placed in a 50 ml canonical ACCEPTED MANUSCRIPT centrifuge tube with 10 ml disaggregation solution containing IMDM with 125 μg/ml (0.65 U/ml) Liberase TL (Roche, Mannheim, Germany), 1% FCS, and 1000 U/ml DNAse type I The fragments were incubated in the medium in a shaking water bath at 37°C for 30 minutes (approximately 100 strokes/min) The cell suspension was removed and passed through a 70 µm single-cell strainer, rinsed with 4°C PBS and centrifuged at 400 G at 4°C for 10 minutes in a 50 ml canonical centrifuge tube The remaining fragments were incubated in fresh enzymatic solution containing Liberase TL for another 30 minutes after which the remaining cell suspension was filtered with a 70 µm single-cell PT strainer The filtered single cell suspension was rinsed with 4°C PBS, centrifuged at 400 G at 4°C for 10 minutes in a 50 ml canonical centrifuge tube and stored in PBS on ice till density gradient RI isolation Isolation with Liberase TL was always compared with Collagenase D isolation; half of the D as described in the previous paragraph (and in Fig 2) SC tissue of one donor was digested with Liberase TL while the other half was digested with Collagenase NU 2.5 Mononuclear leukocyte cell enrichment by density centrifugation The single cell suspensions isolated from the epithelium and lamina propria were divided into MA equal volumes (to be able to compare different gradients; Fig 2) and enriched for mononuclear leukocytes by density gradient centrifugation using either standard Lymphoprep (Axis-Shield, Oslo, Norway) or a 60% Standard Isotonic Percoll solution (SIP; GE Healthcare, Uppsala, Sweden); the SIP D solution was prepared by supplementing 100% Percoll with 10% 10X PBS after which it was diluted to PT E 60% with 1X PBS The cell suspensions were pelleted and then resuspended in 10 ml IMDM after which they were layered on top of ml Lymphoprep or ml 60% SIP solution in a 15 ml canonical centrifuge tube The gradient tubes were centrifuged at 1000 G for 22 minutes with no break and at CE room temperature The interphases containing mononuclear leukocytes were collected by careful aspiration with sterile polystyrene pipettes (Falcon, Corning, USA) The single cell suspension was washed with 4°C PBS and centrifuged at 400 G for 10 minutes Cell yield and viability were AC determined by Trypan blue (Sigma-Aldrich) exclusion; samples (10 µl) were mixed with an equal amount of 4% Trypan blue, placed in a Bürker chamber (Blau, Wertheim, Germany) and only live cells were counted Concentrations of 10·106 cells/ml in 1X PBS were prepared for immune phenotyping and kept on ice 2.6 PBMC isolation from whole blood and treatment with enzymes Whole blood (5 ml) from three healthy adult donors was collected in heparine vacuettes (Greiner) The blood was diluted 1:1 with IMDM and enriched for peripheral blood mononuclear cells (PBMC) by density gradient centrifugation over ml Lymphoprep (as described in section 2.5) After washing the PBMC twice (with 4°C PBS and centrifugation at 400 G for 10 minutes), each PBMC suspension ACCEPTED MANUSCRIPT was divided equally over three 15 ml canonical centrifuge tubes and treated with either Collagenase D (section 2.4.2), Liberase TL (section 2.4.3), or left untreated (IMDM with 1% FCS, and 1000 U/ml DNAse type I) for hour in a shaking water bath at 37°C The PBMC samples were washed twice more and concentrations of 10·106 cells/ml in 1X PBS were prepared for immune phenotyping and SC RI PT kept on ice NU Figure Workflow of protocol comparisons Tissues of individual donors were divided in half and treated with different disaggregation protocols (Collagenase D, Liberase TL, MA or enzyme-free Medimachine) Resulting cell suspension were divided in half and enriched for mononuclear leukocytes with density gradients (Lymphoprep of 60% Standard Isotonic Percoll (SIP)) Within donor comparisons of disaggregation PT E D method or density gradient were made (indicated in bold) 2.7 Cell staining and flow cytometry Samples of 1·106 cells were placed in 96-well plates and stained immediately with a cocktail of CE monoclonal antibodies for 30 minutes at 4°C Cells were stained with previously optimized antibodyfluorochrome combinations to the following markers (all anti-human) to identify T cells, monocyte- AC derived, and dendritic cell sub populations: LIVE/DEAD Fixable Red (Invitrogen), CD45-FITC, CD3-FITC, CD3-AF700, CD27-APC-eFluor780, CD4-PerCP-Cy5.5, CD103-PerCP-eFluor710, CD28-PE, CD19-FITC, CD20-FITC, CD56-FITC, HLA-DR-APC-eFluor780, CD83-PE-Cy7, CD56-PE-Cy7 (all eBioscience), CD45V500, CD3-V500, CD4-BUV737, CCR7-BUV395, CD69-BV421, CD14-BUV737, CD11c-BUV395, CD141BV711, CD86-BV650 (all BD Horizon), CD4-BV570, CD8a-BV785, CD11b-BV785 (Biolegend), CCR7-PE, CD27-FITC, CD123-PE (all BD Pharmingen) After staining, the cells were washed with PBS and resuspended in 1X stabilizing fixative (BD Biosciences, San Jose, USA) Flow cytometric analyses were performed on an LSR Fortessa (BD) within 24 hours after fixation ACCEPTED MANUSCRIPT 2.8 Statistical analysis The flow cytometry data were analyzed with FlowJo vX.0.7 software (TreeStar) Due to variability in the age and diagnosis of the donors, which can affect the frequency of cell populations as well as cell surface molecule expression, comparisons between isolation and enrichment techniques were always made within individual donors Subsequently, in order to compare results between donors, differences in results per donor were expressed in individual fold changes; PT Lymphoprep was divided by 60% SIP; Collagenase D was divided by Medimachine, Liberase TL, or Untreated Statistical significance of differences was then assessed using one sample (compared to a RI value of ‘1’ indicating no change), paired T tests (Wilcoxon where appropriate) or ANOVAs The software package GraphPad Prism 7.02 (GraphPad Software, San Diego, California) was used for data SC and statistical analyses Values of p