This work was supported by grants fromthe Belgian Fund for Scientific Research (FNRS), Les Amis de l&#8217;Institut Bordet, FNRS-Op&#233;ration T&#233;l&#233;vie, Plan Cancer of Belgium, Fonds Lambeau-Marteaux, Fonds J.C. Heuson and Fonds Barsy.

NOTE: All specimens were acquired using a protocol approved by the Medical Ethics Committee of the Institute Jules Bordet with written informed consent obtained from each patient.
1. Preparation of the Tissue Homogenate
<ol>
	<li>Dissect resected tissues (malignant and normal tissue resected from the operating room) are in the pathology lab by trained personnel for immediate pickup. Tumor, NANT (taken the furthest distance from the tumor as possible) and normal tissue fragments are routinely processed within 1 - 3 hr of surgical excision in a BSL2 laboratory using standard biosafety procedures for human tissues. A flow chart of the protocol is illustrated in Figure 1.</li>
	<li>Weigh all tissue fragments (normal, NANT and tumor) and measure the length, width, and height (length x width x height). This is an important step for the subsequent normalization of cell subpopulations, extracted RNA, etc. 
	NOTE: The range of sample size is 100 to 10,000 mm3 with no fat if possible.</li>
	<li>Imprint the tumor fragment on a glass slide for H&#38;E staining to verify that the tissue is actually part of the tumor.
	<ol>
		<li>Do this by pressing a glass microscope slide on the tumor fragment and applying gentle pressure with your fingers for a few seconds.</li>
		<li>Fix the slide with isopropanol for 2 min followed by a washing step in water. Counterstain the tissue for 30 sec with Mayer&#39;s hematoxylin.</li>
		<li>Wash the slide in six baths of water. Stain in Phloxine B 2% for 15 sec.</li>
		<li>Wash in one bath of water followed by four baths of isopropanol and finish with one bath of water.</li>
		<li>Incubate in isopropanol for 1 min and drain. Clear in two baths of xylene.</li>
		<li>Mount with xylene based mounting medium. Examine for tumor cellularity (Figure 2). 
		NOTE: Mainly, tumor cells stick to the imprinted slide - the stromal, lymphoid or adipose cells rarely remain, leaving spaces between the tumor cells (Figure 2).</li>
	</ol>
	</li>
	<li>Place the tissue fragment in a small culture dish containing 1 ml of the chemically defined, serum-free hematopoietic cell medium (hereafter referred to as medium) at room temperature and dice it into small pieces (~1 - 2 mm2) using a sterile scalpel.</li>
	<li>Transfer everything (tissue fragments + medium) to a mechanical dissociator C tube.</li>
	<li>Rinse the Petri dish and scalpel with 2 ml of medium using a Pasteur pipette and add this to the C tube (volume of medium for dissociation = 3 ml).</li>
	<li>Use the mechanical dissociator program A.01 for C tubes (the most gentle program) to homogenize the tissue fragments into a single cell suspension. Place the C tube in the apparatus and run the program twice in succession (one cycle = 25 sec). 
	NOTE: This homogenization procedure has been established and validated for human breast tissue, other tumor or tissue types may need to use a different program and should be tested first.</li>
	<li>Remove the C tube from the apparatus and decant the homogenate directly into a 40 &#956;m cell strainer seated on a 50 ml tube. Using the same Pasteur pipette as in step 1.6, transfer any liquid remaining in the C tube to the cell strainer.</li>
	<li>Transfer the filtered liquid into a 15 ml tube using a 1 ml micropipette tip. Temporarily keep the cell strainer and its 50 ml tube.</li>
	<li>Rinse the C tube with an additional 3 ml of medium and transfer this, again using the same Pasteur pipette as in step 1.6, to the cell strainer still seated on the 50 ml tube. Squeeze a maximum amount of the residual liquid trapped in the unhomogenized tissue into the 50 ml tube by gently moving it around the strainer with a clean Pasteur pipette or 1 ml tip that is subsequently thrown away to avoid contaminating the eluate.</li>
	<li>Place the cell strainer upside down on the original C tube and rinse with 3 ml of medium so that the unhomogenized tissue drops back into the C tube.</li>
	<li>Re-homogenize as in step 1.7 for two cycles of the A.01 program.</li>
	<li>Pour this second homogenate through the cell strainer seated on the 50 ml tube, rinse the C tube again with 3 ml medium (as in step 1.10) and transfer with the Pasteur pipette to the cell strainer seated on the 50 ml tube again squeezing a maximum amount of liquid from the residual connective tissue trapped in the cell strainer.</li>
	<li>At this point, a volume of ~2.5 ml is in the 15 ml tube and ~9 ml in the 50 ml tube.</li>
</ol>
2. Separation of the Tissue Supernatant and Cells
<ol>
	<li>Centrifuge the homogenates in the 15 ml and 50 ml tubes for 15 min at 600 x g at room temperature.</li>
	<li>Decant the SN from the 15 ml tube into a clean tube and temporarily store at 4 &#176;C. This supernatant = primary tumor, NANT or normal tissue SN (final volume 2.5 ml) is subsequently clarified and aliquoted prior to storage at -80 &#176;C for future analyses (see below).</li>
	<li>Discard the supernatant from the 50 ml tube.</li>
	<li>Gently resuspend both cell pellets in a final volume of 1 ml medium. Briefly, first gently break the cell pellet in both tubes (by tapping the tube on a hard surface). Resuspend the loose cell pellet in the 50 ml with 500 &#181;l of medium and transfer this cell suspension to the 15 ml tube to resuspend the second pellet. Repeat this step once with the second 500 &#181;l of medium for maximum recovery of cells.</li>
	<li>Transfer 10 &#181;l of the cell suspension to a small tube, mix with 10 &#181;l of trypan blue (dilution 1:1) and count the number of viable cells using a hemocytometer. 
	NOTE: At this point a fraction of the cell suspension can also be analyzed by flow cytometry to evaluate cell size, granularity and if desired a limited number of subpopulation markers for more precise assessment of the relative cell distribution in the homogenate prior to extensive analysis or experimentation. All analyses by flow cytometry incorporate CD45 labeling for normalization of subpopulations.</li>
	<li>Pellet the cells by centrifugation at 300 x g for 10 min at room temperature. The cells from the tumor, NANT, or normal tissue are now ready for further purification or analysis. These additional steps are best when performed on the same day as surgery. 
	NOTE: For flow cytometric analysis but not cell sorting the residual red blood cells should be lysed after antibody labeling by adding 0.4 ml of red blood cell lysis buffer to the cell pellet, immediately vortexing for 1 sec and incubating a minimum of 10 min at room temperature (protected from light) before analysis.</li>
</ol>
3. Clarification of the Tissue Supernatant
<ol>
	<li>Centrifuge the 1.5 ml tubes with tissue SN at 15,000 x g for 15 min at 4 &#176;C.</li>
	<li>Carefully remove the supernatant without touching or disturbing the pellet. Transfer to a clean tube (or tubes) depending upon the number and volume of aliquots desired.</li>
	<li>Store the supernatant at -80 &#176;C for future use.</li>
</ol>
4. Patient Blood
<ol>
	<li>Collect a blood sample from each patient as a control by venipuncture into heparinized tubes the day prior to surgery. Centrifuge the blood at 400 x g for 10 min at 20 &#186;C with the brake off to obtain the plasma and a buffy coat.</li>
	<li>Remove the plasma and clarify it by centrifugation at 10,000 x g for 15 min at 20 &#186;C. Aliquot and store plasma at -80 &#176;C for future use. Dilute the buffy coat in medium</li>
	<li>Separate the mononuclear cells using standard ficoll-hypaque gradient centrifugation prior to immediate analysis, subpopulation isolation, DNA/RNA/protein extraction or cryopreservation.</li>
</ol>
5. Flow Cytometry
<ol>
	<li>Label cells according to manufacturer&#8217;s instructions at 4 &#176;C, protected from light. Lyse red blood cells in cell suspensions from tissue fragments after antibody labeling by adding 0.4 ml of cell lysis buffer to the cell pellet.</li>
	<li>Vortex immediately for 1 sec and incubate a minimum of 10 min at room temperature, protected from light. Pass the labeled cells in a flow cytometer for data acquisition without washing.</li>
</ol>

<ol>
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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+cells+in+the+tumor+microenvironment.">Innate and adaptive immune cells in the tumor microenvironment.</a> Nat Immunol. 14 (10), 1014-1022 (2013).</li><li>Quezada, S. 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A., Marits, P., Sherif, A., Winqvist, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+B+cell+characterization+in+blood,+lymph+nodes,+and+tumors+from+patients+with+malignancies.">Multiplex B cell characterization in blood, lymph nodes, and tumors from patients with malignancies.</a> J Immunol. 190 (11), 5847-5855 (2013).</li><li>Gu-Trantien, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+follicular+helper+T+cell+infiltration+predicts+breast+cancer+survival.">CD4(+) follicular helper T cell infiltration predicts breast cancer survival.</a> J Clin Invest. 123 (7), 2873-2892 (2013).</li><li>Buisseret, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytes+Infiltrating+Breast+Cancer+:+Density,+Composition+And+Organization.">Lymphocytes Infiltrating Breast Cancer : Density, Composition And Organization.</a> Annals of Oncology. 25 (1), 17 (2014).</li><li>Garaud, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+B+Cells+Infiltrating+Human+Breast+Cancer.">Characterization of B Cells Infiltrating Human Breast Cancer.</a> Annals of Oncology. 25 (1), 18 (2014).</li><li>Gu-Trantien, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cxcl13-Producing+Follicular+Helper+T+Cells+In+Human+Breast+Cancer.">Cxcl13-Producing Follicular Helper T Cells In Human Breast Cancer.</a> Annals of Oncology. 25 (1), 17 (2014).</li><li>Yee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+endogenous+T+cells+for+adoptive+transfer.">The use of endogenous T cells for adoptive transfer.</a> Immunol Rev. 257 (1), 250-263 (2014).</li><li>Butler, M. 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The authors declare that no conflict of interest exists.

This study describes an optimized method for the rapid preparation of normal and malignant breast tissue homogenates without enzymatic digestion for subsequent cell sorting, extraction, cryopreservation and/or phenotypic analysis of CD45+ subpopulations. The goal of this experimental approach is to produce images of the TIL that closely reflect their in vivo state and compare them to normal tissues with minimal manipulation of the tissues fresh from the operating room. To date, our laboratory has used this protocol to analyze &#62;250 fresh BC tissues (tumor and NANT), &#62;35 normal breast tissues from mammary reductions. In principal, this protocol should be directly applicable to other tumor or tissue types; however, some optimization may be necessary (i.e., the mechanical dissociation program, number of cycles, media volume, etc.). For tissues with low lymphocyte infiltration, using a larger tissue fragment may be necessary, which is what we do for normal tissues where the lymphocyte count is low. Alternatively, a density gradient centrifugation step could be added to enrich the leukocyte suspension. This analysis of human T and B cell subpopulations infiltrating breast tumors includes an assessment of &#62;100 markers by flow cytometry (flow cytometry data for 74 markers is available in the supplementary data of Gu-Trantien, et al.9) and purification of specific lymphocyte subpopulations for subsequent immunological and molecular analyses. Further, we have evaluated cytokines, chemokines, immunoglobulins and tumor antigens in the primary tumor SN (compared to NANT and normal tissue SN)10,11 as a reflection of these molecular mediators in the tumor microenvironment. We have also tested the effect of treating peripheral blood lymphocytes from healthy donors with tumor SN and shown that many of the gene expression changes detected in the TIL can be specifically reproduced9.
In their microenvironment, the tumor and stromal cells are more tightly held in the tissue matrix than the immune cells, which are characteristically migratory. The method described here provides a simple and rapid means for isolating and analyzing TIL without enzymatic digestion. Our numerous attempts to join this mechanical homogenization approach with a short enzymatic digestion to produce viable and receptor positive lymphoid and epithelial cells from the same tumor fragment have been unsuccessful. Tissue homogenization efficiently releases lymphocytes but results in a significant loss of tumor cell viability. A short enzymatic digestion of the tissue liberates viable tumor cells (the total number increases as a function of digestion time) but has the consequence of a parallel decrease in the expression of many surface receptors, particularly apparent on lymphocytes. Thus, for the comparative analysis of tumor cells and TIL from the same tumor it is still necessary to separately process two sequential tumor fragments.
Currently, the majority of studies designed to characterize TIL have employed enzymatic digestion (hours to O/N) frequently coupled with some form of mechanical dissection4-8. Expansion of TIL for further analysis or therapy generally involves the subsequent ex vivo cultivation of TIL or tumor tissue fragments with stimulatory agents (days to weeks)13-15. The rapid, non-enzymatic method for tissue dissociation described here provides a simple and reproducible means for extracting intact CD45+ cells from normal and abnormal tissue fragments prior to their expansion or analysis. This speedy acquisition of CD45+ cells from patients undergoing tumorectomy could possibly be developed for use in a prognostic biomarker assay. In addition, it may yield TIL with greater potential for ex vivo expansion prior to adoptive immunotherapy.

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table><tbody><tr><td>GentleMacs Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>BD Medimachine is somewhat equivalent</td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td></td><td>or other standard table top centrifuge</td></tr><tr><td>Centrifuge 5417 R</td><td>Eppendorf</td><td></td><td>or other standard microcentrifuge</td></tr><tr><td>Esco Class II A2 Biosafety Cabinet</td><td>ESCO global</td><td></td><td>or other standard BSL2 hood</td></tr><tr><td>Inverted Microscope</td><td>Nikon eclipse TS100</td><td></td><td>or other microscope compatible for a hemacytometer</td></tr><tr><td>B&amp;uuml;rker Chamber</td><td>Marienfield&amp;nbsp;</td><td>640210</td><td>or other standard hemacytometer</td></tr><tr><td>Navios Flow Cytometer</td><td>Beckman Coulter</td><td></td><td>or other flow cytometer (8-10 color recommended)</td></tr><tr><td>GentleMacs C-Tube</td><td>Miltenyi Biotec</td><td>130-096-344</td><td>BD Medimachine uses Filcon</td></tr><tr><td>Cell Culture Dish</td><td>Sarstedt</td><td>72,710</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Disposable Scalpel</td><td>Swann-Morton</td><td>0510</td><td>or standard single use sterile scalpel</td></tr><tr><td>BD Cell Strainer 40 &amp;micro;m</td><td>Becton Dickinson</td><td>734-0002</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD Falcon Tube 50 ml</td><td>Becton Dickinson</td><td>352070</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD Falcon Tube 15 ml</td><td>Becton Dickinson</td><td>352097</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD FACS Tube 5 ml</td><td>Becton Dickinson</td><td>352008</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Sterile Pasteur Pipette 5 ml</td><td>VWR</td><td>612-1685</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Microfuge Tube 1.5 ml</td><td>Eppendorf</td><td>7805-00</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>X-Vivo 20</td><td>Lonza</td><td>BE04-448Q</td><td>serum-free medium recommended</td></tr><tr><td>Phosphate buffered saline</td><td>Lonza</td><td>BE17-516F</td><td>standard physiological PBS</td></tr><tr><td>Trypan blue</td><td>VWR</td><td>17942E</td><td>or other vital stain</td></tr><tr><td>VersaLyse</td><td>Beckman Coulter</td><td>A09777</td><td>for flow cytometry experiments</td></tr><tr><td>Fixable viability Dye eFluor 780</td><td>eBioscience</td><td>65-0865-14</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD3 FITC</td><td>BD Biosciences</td><td>345763</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD3 Vio Blue</td><td>Miltenyi Biotec</td><td>130-094-363</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD4 PE</td><td>BD Biosciences</td><td>345769</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD4 APC</td><td>Miltenyi Biotec</td><td>130-091-232</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD8 ECD</td><td>Beckman Coulter</td><td>737659</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD8 PerCP</td><td>BD Biosciences</td><td>345774</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD19 APC-Vio770</td><td>Miltenyi Biotec</td><td>130-096-643</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD45 VioGreen</td><td>Miltenyi Biotec</td><td>130-096-906</td><td>for flow cytometry experiments</td></tr></tbody></table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Enzymatic digestion of tissue fragments with either commercially available tissue dissociation solutions or various laboratory mixtures of collagenase, DNase and/or hyaluronidase inhibitors, cleave a wide variety of receptors on the surface of cells. Our studies, initially focused on CD4+ T cells infiltrating breast tumors, were quickly presented with a major technical problem due to cleavage of surface CD4 receptors using standard enzymatic digestion protocols4-8. We tested a variety of collagenase enzymes with or without DNase and hyaluronidase inhibitors, finding that collagenase I and II completely removed CD4 from the cell surface, despite high lymphocyte viability (Figure 3A). A moderate effect on CD4 was observed with collagenase IV at short incubation times (1 - 2 hr) with lower CD4 antibody labeling detected in digested lymph node and tumor tissue compared to undigested blood and bone marrow from the same patient (Figure 3B; note that due to limitations on the tissue we receive we were unable to compare digested and undigested lymph node and tumor tissue). This loss of CD4 was confirmed to be a technical byproduct of collagenase IV digestion by comparing undigested and digested CD4+ T cells isolated from healthy donor blood (data not shown). While these CD4lo T cells can be isolated from collagenase IV-digested tissue fragments using magnetic beads, the purified populations frequently contain varying amounts of contamination by other cells from the tumor microenvironment and thus were suboptimal for our experimental purposes.
In a continuous search for a better approach, in 2008 we tested a new mechanical dissociator without using enzymes. This apparatus produced breast tissue homogenates rapidly and with surface receptor integrity maintained9. Representative flow cytometry dot plots of the tumor cell homogenates following dissociation and labeling with specific antibodies against epithelial cells (EpCAM) and leukocytes (CD45) are shown in Figure 3C. These images are typical routine observations for breast tumor homogenates. This protocol does not significantly alter the viability of CD45+ cells (4% dead cells); however, there is a considerable loss of viable EpCAM+ cells (38% dead cells in tumor shown; varies between tumors). Despite this loss, the viable EpCAM+ and CD45+ subsets can be segregated on the basis of size and structure into large epithelial cells (= tumor cells), small epithelial cells and large CD45+ cells and small CD45+ cells (the majority of cells, which are principally lymphocytes; Figure 3C).
The significant increase in viable CD45+ cells obtained using this approach permits the acquisition of reproducible multicolor flow cytometry data (using up to ten colors) to more fully characterize the TIL subsets in breast cancer. Representative flow cytometry dot plots of the major TIL subsets are shown in Figure 4, with our recent experiments demonstrating that it is also possible to detect and quantify minor T and B cells subsets infiltrating the tumor10-12, including intracellular staining for canonical T and B cell transcription factors12. Gating strategies for lymphocytes are based on viable CD45+ cells identified with a viability stain (note: tumors can vary greatly in the amount of cellular debris and dead epithelial cells depending on the BC subtype, grade, etc.). This approach has been successfully used to purify lymphocyte subpopulations from the homogenate with magnetic beads or cell sorting for gene expression analysis using microarrays or qRT-PCR9. Soluble mediators in the SN, including cytokines and immunoglobulins, have been quantified using a bead-based immunoassay with the resulting data normalized to the size of the tissue fragment. A comparison of cytokine expression (a Th1/Th2/Th7/Th9/Th17 pool) or total immunoglobulins (IgA, IgE, IgG, IgM) in BC SN with SN from normal breast tissue reveals increases in both associated with the tumor tissue (Figure 5). These primary tissue SN&#8217;s have also been effectively analyzed for antigens10-12 and used to experimentally treat lymphocytes from healthy donors, thereby reproducing the TIL phenotype in normal cells9.
<img alt="Figure 1" src="/files/ftp_upload/52392/52392fig1highres.jpg" width="700" /> 
Figure 1. Protocol flow chart. Our procedure for processing fresh human tissues and some analytical approaches that can subsequently be used to assess lymphocytes infiltrating human tissues are shown.
<img alt="Figure 2" src="/files/ftp_upload/52392/52392fig2highres.jpg" width="700" /> 
Figure 2. H&#38;E stained image of a breast tumor tissue imprint. This imprint, taken from a fresh breast tumor tissue fragment, shows the presence of tumor cells with the open spaces reflecting areas that were not transferred. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52392/52392fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" src="/files/ftp_upload/52392/52392fig3highres.jpg" width="700" /> 
Figure 3. Optimization of breast tissue dissociation for lymphocyte analysis. Flow cytometric analysis of: (A) CD4 surface expression on lymphocytes from undigested and Collagenase I/II digested tumor tissue; (B) CD4 expression on lymphocytes from lymph node and tumor tissue digested with Collagenase IV and compared to undigested blood and bone marrow cells from the same patient; and (C) analysis of total leukocytes (CD45+) and epithelial cells (EpCAM+) from tumor tissue rapidly mechanically dissociated using the protocol described here. The viability of epithelial cells and CD45+ cells using our protocol is assessed via the incorporation of fixable viability dye. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52392/52392fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52392/52392fig4highres.jpg" width="700" /> 
Figure 4. Tumor infiltrating lymphocyte subsets in the homogenate. Multicolor flow cytometric analysis was performed on the day of surgery after mechanical dissociation using the protocol described here. The viable cells and major lymphocyte subpopulations are shown: CD3 is a pan T cell marker, CD4 and CD8 are major T cell subset markers and CD19 is a pan B cell marker. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52392/52392fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52392/52392fig5highres.jpg" width="350" /> 
Figure 5. Soluble mediators in the supernatant. A panel of cytokines (Th1/Th2/Th7/Th9/Th17) and immunoglobulins (IgA, IgE, IgG, IgM) were assessed using bead-based immunoassays to analyze SN derived from normal and BC tissues using the protocol described here.

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

19.9K Views.  Université Libre de Bruxelles. This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Video: A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Cancer Research

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Medicine

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Bioengineering

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue

Source: Garaud et. al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/52392/a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human">A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes.</a> J. Vis. Exp. (2014).
This video describes the non-enzymatic dissociation of fresh human tissue to obtain viable cells. The technique is used for qualitative and quantitative analysis of CD45 positive cells (lymphocytes/leukocytes) present in various normal and malignant human tissues.

Source: Garaud et. al., A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes. J. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

Summary

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Chapters in this video

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Isolation of Murine Lymph Node Stromal Cells

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

Mouse Wound Models and Preparation of Single-Cell Suspensions

Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue