Name: lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture
Uploaded: 2016-04-08T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

Skin biopsies from psoriasis patients were kindly provided by Dr. Andreas Koerber (Dermatology department at University of Essen, Germany) after oral or written informed consent for scientific use.&#160;
X.H. is also supported by NSFC 61263039 and NSFC 11101321.

NOTE: Skin biopsies from healthy individuals were obtained from abdominal skin leftover of individuals undergoing elective plastic surgery after oral or written informed consent for scientific use. The use of human skin was approved and in accordance with the regulations set by the Medical Ethical Committees for human research of the Radboud university medical centre, Nijmegen, the Netherlands and University of Essen, Germany.
1. Preparation of Single Cell Suspensions from Human Skin (Work Sterile in a Flow-cabinet if Subsequent Cell Culture is Required)
<ol>
	<li>Prepare cell culture medium: RPMI 1640 + penicillin/streptomycin (final concentrations 100 units/ml and 100 &#181;g/ml, respectively) + pyruvate (0.02 mM) and glutamax (0.02 mM), with no serum added.</li>
	<li>Prepare complete culture medium: culture medium prepared in step 1.1 + 10% human pooled serum (HPS); store at 4&#176;C. Bring medium to 20 &#176;C &#177; 2 before using.</li>
	<li>Obtain the skin biopsy using a 4mm round biopsy punch instrument and keep it in RPMI1640 complete culture medium at 20 &#176;C &#177; 2 for up to 4 hr&#160;or at 4 &#176;C ON. Process the biopsy as soon as possible upon arrival in the laboratory. 
	NOTE: Longer storage of skin will influence the cell yield and cell viability.</li>
	<li>Label a blue-capped dissociation tube and add 5 ml complete culture medium into the labelled tube.</li>
	<li>Add 2 ml of complete culture medium into each well (in total 3 wells) of a sterile 6-well culture plate. Use sterile tools to place the biopsy into a single well, rinse, move it over to a second well and repeat this step one more time, thus achieving a total of three rinses.</li>
	<li>Transfer the well rinsed skin biopsy to a sterile Petri dish, add 100 &#181;l of complete culture medium on the top of biopsy, and carefully scrape off the subcutaneous fat tissue using a stainless steel disposable sterile scalpel. 
	NOTE: This is a critical step.</li>
	<li>Cut each skin biopsy into 4 smaller pieces on a sterile Petri dish. Transfer samples (up to four of 4 mm biopsies per tube) to the prepared dissociation tube containing 5 ml of complete culture medium.</li>
	<li>Tightly close the tubes with the cap, and attach upside down to the sleeve of the automated tissue dissociator. Make sure that all sample material is located in the area of the rotor.</li>
	<li>Start the dissociation process by running the &#8220;program m_spleen _01&#8221; (a pre-defined program provided by the instrument&#8217;s internal memory or by the accompanied program card) to dissociate the biopsy at the appropriate rotating speed for 56 sec.</li>
	<li>After processing, detach the dissociation tube from the dissociator and make sure that all the dissociated material is collected at the bottom of the tube.</li>
	<li>Add 150 &#181;l collagenase I-A (80 mg/ml) into the dissociation tube and incubate the sample in a shaking water bath at 37 &#176;C for 60 min. Add 100 &#181;l of DNase I (5 MU/ml) into thedissociation tube, mix well. 
	NOTE: Higher concentration of collagenase or longer incubation time will alter cell viability.</li>
	<li>Attach the dissociation tube to the sleeve of the automated tissue dissociator and run the &#8220;program m_ spleen _01&#8221; to dissociate the biopsy one more time.</li>
	<li>Place a 70 &#181;M nylon cell strainer on the top of a 50 ml Falcon tube. Apply dissociated sample materials to this cell strainer to remove cell clumps/tissue debris.</li>
	<li>Wash cell strainer once with 5 ml of complete culture medium. Centrifuge at 20 &#176;C &#177; 2, 450 x g for 10 min and aspirate supernatant.</li>
	<li>Repeat the washing step one more time. Resuspend cell pellets in 300 &#181;l of complete culture medium. Single-cell suspensions are ready for further analysis; continue with protocols for ex vivo cell culture or flow cytometry analysis.</li>
	<li>In case of further intracellular cytokine staining, stimulate cells with PMA (12.5 ng/ml), Ionomycin (500 ng/ml) and Brefeldine A (5 &#181;g/ml) for 4 hr&#160;at 37 &#176;C, 5% CO2 incubator for 4 hr&#160;before performing flow cytometry analysis.</li>
</ol>
2. Polyclonal Activation of Skin-Resident T Cells (Ex Vivo Cell Culture)
<ol>
	<li>Aliquot 100 &#181;l single-cell suspensions (prepared in step 1.15) into a round-bottom 96-well plate. 
	NOTE: For each of 4 mm skin biopsy, the acquired single-cell suspensions can be split into at least two wells of a 96-well plate.</li>
	<li>Add anti-CD3/anti-CD28 mAb-coated microbeads (25,000 beads per well), recombinant human cytokine rIL-2 (final concentration 25 U/ml) and rIL-1&#946; (final concentration 50 ng/ml).</li>
	<li>Cover the culture plate with a well-labelled plate lid, incubate in 5% CO2, 100% humidity, 37 &#176;C incubator. Change medium when the medium colour turns to yellow. 
	NOTE: A clear cell colony can be observed from day 8 of cell culture.</li>
</ol>
3. Flow Cytometry Analysis of Primary/Cultured Skin-Resident T Cells
<ol>
	<li>Prepare FACS buffer: PBS + 0.2% BSA.</li>
	<li>Transfer cells of interest into a v-bottom 96-well plate. Centrifuge at 20 &#176;C &#177; 2, 450 x g for 2 min, and aspirate supernatant.</li>
	<li>Resuspend the pellet in 100 &#181;l of PBS. Centrifuge at 20 &#176;C &#177; 2, 450 x g for 2 min, and aspirate supernatant.</li>
	<li>Stain cells with 100 &#181;l of prepared eFluorescence780 conjugated fixable viability dye (1:1,000 dilution using PBS) at 4 &#176;C for 30 min. Add 100 &#181;l of FACS buffer, centrifuge at 20 &#176;C &#177; 2, 450 x g for 2 min, and aspirate supernatant.</li>
	<li>Select required cell surface marker mAbs, for example: CD45-BV421 (HI30, 1:20), CD3-ECD(UCHT1, 1:50), CD4-PC5.5(1388.2, 1:200), CD8-APC-AlexFluo700(B9.11, 1:400), CD14-FITC(TUK4, 1:50), CD19-APC-AlexFluo750(13-119, 1:50), CD56-PE(MEM-188, 1:50), CD25-PeCy7(BC96, 1:50), CD11c-PeCy7(BU15, 1:10), and CD1c-APC(AD5-8E7, 1:10), prepare the mAbs-mixture using FACS buffer according to each mAb dilution factor tested. Define gate settings using isotype control of antibody together with non-stain sample.</li>
	<li>Add 25 &#181;l of prepared mAbs-mixture into each well. Incubate 20 min at 20 &#176;C &#177; 2, protect from light.</li>
	<li>Add 100 &#181;l of FACS buffer, centrifuge at 20 &#176;C &#177; 2, 450 x g for 2 min, and aspirate supernatant.</li>
	<li>For samples that only require cell surface staining, continue with step 3.16.</li>
	<li>In case of intra-cellular Foxp3 staining:
	<ol>
		<li>Prepare fixation and permeabilization buffer by mixing one part of concentrate with 3 parts of diluents.</li>
		<li>Prepare 1x permeabilization buffer: 1 part of 10x permeabilization buffer + 9 parts of sterilized H2O.</li>
	</ol>
	</li>
	<li>Resuspend pellets in 100 &#181;l of fixation and permeabilization buffer, mix well, and incubate at 4 &#176;C for 30 min.</li>
	<li>Add 100 &#181;l permeabilization buffer to each well, centrifuge at 450 x g at RT for 2 min, and aspirate supernatant.</li>
	<li>Wash cells with permeabilization buffer one more time.</li>
	<li>Select required intracellular mAbs, for example, Foxp3-eFluo450(PCH101, 1:50), IL-17A-AlexFluo488(eBio64DEC1, 1:50) and IFN&#947;-PeCy7(4S.B3, 1:400). Prepare the mAbs mixture using permeabilization buffer containing 2% normal rat serum according to each mAb dilution factor tested. Define gate settings using isotype control of antibody together with non-stain sample.</li>
	<li>Add 20 &#181;l of prepared mAb-mixture into each well. Incubate at 4 &#176;C for 30 min, protect from light.</li>
	<li>After 30 min incubation, add 100 &#181;l of permeabilization buffer into each well. Centrifuge at 20 &#176;C &#177; 2, 450 x g for 2 min, and aspirate supernatant.</li>
	<li>Wash cells with permeabilization buffer one more time. Resuspend pellet in 110 &#181;l of FACS buffer, and transfer cell suspensions into microFACS tube. 
	NOTE: Sample is ready for measurement using 10-colour flow cytometry.</li>
	<li>In case of exact cell number required, add 10 &#181;l of well-mixed uniform suspension of fluorospheres into each sample immediately before performing measurements using flow cytometry.</li>
</ol>

<ol>
	<li>Clark, R. A., 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=The+vast+majority+of+CLA++T+cells+are+resident+in+normal+skin.">The vast majority of CLA+ T cells are resident in normal skin.</a> J Immunol. 176, 4431-4439 (2006).</li><li>Zaba, L. C., Fuentes-Duculan, J., Steinman, R. M., Krueger, J. G., Lowes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+dermis+contains+distinct+populations+of+CD11c+BDCA-1++dendritic+cells+and+CD163+FXIIIA++macrophages.">Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.</a> J Clin Invest. 117, 2517-2525 (2007).</li><li>Gebhardt, T., 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=Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus.">Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.</a> Nature Immunol. 10, 524-530 (2009).</li><li>Boyman, O., 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=Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha.">Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha.</a> J Exp Med. 199, 731-736 (2004).</li><li>Christophers, E., Mrowietz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+infiltrate+in+psoriasis.">The inflammatory infiltrate in psoriasis.</a> Clin Dermatol. 13, 131-135 (1995).</li><li>Jiang, X., 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=Skin+infection+generates+non-migratory+memory+CD8++T(RM)+cells+providing+global+skin+immunity.">Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.</a> Nature. 483, 227-231 (2012).</li><li>Havran, W. 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=Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1++dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody.">Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.</a> Proc Natl Acad Sci U S A. 86, 4185-4189 (1989).</li><li>Campbell, J. J., 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=CCR7+expression+and+memory+T+cell+diversity+in+humans.">CCR7 expression and memory T cell diversity in humans.</a> J Immunol. 166, 877-884 (2001).</li><li>He, X., 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=Targeting+PKC+in+human+T+cells+using+sotrastaurin+(AEB071)+preserves+regulatory+T+cells+and+prevents+IL-17+production.">Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.</a> J Invest Dermatol. 134, 975-983 (2014).</li><li>Clark, R. A., 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=A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin.">A novel method for the isolation of skin resident T cells from normal and diseased human skin.</a> J Invest Dermatol. 126, 1059-1070 (2006).</li><li>Clark, R. A., Kupper, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin.">IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.</a> Blood. 109, 194-202 (2007).</li><li>Keijsers, R. R., 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=In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17.">In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORgammat and IL-17.</a> J Invest Dermatol. 134, 1276-1284 (2014).</li><li>Hybbinette, S., Bostrom, M., Lindberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation.">Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.</a> Experimental dermatology. 8, 30-38 (1999).</li><li>Hennino, A., 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=Skin-infiltrating+CD8++T+cells+initiate+atopic+dermatitis+lesions.">Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions.</a> J Immunol. 178, 5571-5577 (2007).</li></ol>

The authors have nothing to disclose.

Here, we present a protocol to efficiently isolate skin resident T cells from human skin biopsies. The advantage of this protocol is the isolation of relatively high numbers of viable lymphocytes, and expressing relevant surface markers. The cell subsets identified were: CD11c+ DCs, CD4+ and CD8+ T cells and Foxp3+CD25+ cells. Importantly, ex vivo culture of isolated skin resident T cells was very well feasible and allowed for subsequent functional analysis....

The skin, as the primary interface between the body and the environment, provides the first line of defence against external physical, chemical and biological insults such as wounding, ultraviolet radiation and micro-organisms. Skin comprises two main compartments, the epidermis and the dermis, and contains a variety of immune cells including Langerhans cells, macrophages, dendritic cells (DCs), and about 20 billion memory T cells, nearly twice the number present in the entire blood volume1,2. A growing body of data supports the notion that the skin has essential immunological functions, both during tissue homeostasis and in various pathological conditions. Immune cells resident in normal skin are thought to conduct immunosurveillance3 and have been shown to play a role in the development of inflammatory disorders such as psoriasis4. In psoriatic lesional skin, both CD4+ and CD8+ infiltrated T cells were observed and it was shown that the ratio of the CD4 and CD8 varies depending on the disease status5. However, these populations of cells are difficult to study because existing techniques allow the isolation of only few cells.
The currently widely used techniques for T cell isolation from human skin combine mechanical skin dissociation with enzymatic treatment. Human skin biopsies are extensively minced and incubated with enzymes like trypsin, collagenase and/or EDTA6-8. Considering that skin is a barrier tissue which is highly resistant to tensile forces and mechanical disaggregation, the established methods of T cell isolation produced very few cells, and even lower numbers of viable cells, which makes ex vivo cell culture of these cell populations difficult and challenging.
Here, we report a modified method to isolate lymphocytes from both healthy and involved psoriatic human skin by combining mechanical dissociation of the skin using an automated tissue dissociator instead of the established method of extensively mincing, together with enzymatic digestion using collagenase. Various viable immune cell subsets including DCs and T cells were observed after preparation of a single-cell suspension. Importantly the expression of the surface markers CD3, CD4 and CD8 was well preserved. Cells thus prepared, are ready for use in ex vivo cell cultures or flow cytometric analysis. This protocol has been successfully employed for the analysis of single skin biopsies (4 mm) derived from lesional skin of psoriasis patients. Results showed that skin resident patient T cells produced more inflammatory cytokines like IL-17 and IFN&#947; in comparison to healthy volunteers9.

<table><tbody><tr><td>Disposable Biopsy Punch</td><td>Kai Europe</td><td>BP-40F</td><td>4 mm</td></tr><tr><td>Disposable sterile scalpels</td><td>Dalhausen</td><td>1100000510</td><td></td></tr><tr><td>gentleMACS C tube</td><td>Miltenyi Biotech</td><td>130-093-237</td><td>Blue-capped, used as the dissociation tube.</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Automated tissue dissociator. Using the &quot;program spleen_01&quot; for dissociation of the skin biopsy.</td></tr><tr><td>Cell strainer&amp;nbsp;</td><td>BD</td><td>352350</td><td>70 &amp;micro;m nylon</td></tr><tr><td>96-well U-bottom plate</td><td>Greiner Bio-One</td><td>650180</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>22409-015</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Human Pooled Serum (HPS)</td><td>in house prepared</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Sigma-Aldrich</td><td>C2674</td><td>Type 1-A, suitable for cell culture</td></tr><tr><td>DNase I</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>PBS</td><td>B Braun</td><td>3623140</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503-500G</td><td></td></tr><tr><td>Fixable Viability Dye (FVD) APC-eFluo780</td><td>eBioscience</td><td>65-0865-18</td><td>Stain dead cells prior to cell fixation; dilute with PBS at 1:1,000</td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td></td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Permeabilization Buffer (10x)</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>BV421 Mouse anti-human CD45</td><td>BD</td><td>563879</td><td>Clone: HI30; dilution factor 1:50</td></tr><tr><td>FITC Mouse anti-human CD14</td><td>Dako</td><td>T0844</td><td>Clone: TUK4; dilution factor 1:50</td></tr><tr><td>PE Mouse anti-human CD56</td><td>Dako</td><td>R7127</td><td>Clone: MOC-1; dilution factor 1:50</td></tr><tr><td>ECD Mouse anti-human CD3</td><td>Beckman - Coulter</td><td>A07748</td><td>Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td></tr><tr><td>PC5.5 Mouse anti-human CD4</td><td>Beckman - Coulter</td><td>B16491</td><td>Clone: 13B8.2; dilution factor 1:200</td></tr><tr><td>PeCy7 Mouse anti-human CD11c</td><td>Beckman - Coulter</td><td>A80249</td><td>Clone: BU15; dilution factor 1:50</td></tr><tr><td>APC Mouse anti-human CD1c</td><td>Miltenyi Biotech</td><td>130-090-903</td><td>Clone: AD5-8E7; dilution factor 1:10</td></tr><tr><td>APC-AlexFluo700 Mouse anti-human CD8</td><td>Beckman - Coulter</td><td>A66332</td><td>Clone: B9.11; dilution factor 1:400</td></tr><tr><td>APC-AlexFluo750 Mouse anti-human CD19</td><td>Beckman - Coulter</td><td>A94681</td><td>Clone: J3-119; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human CD25</td><td>eBioscience</td><td>AD5-8E7</td><td>Clone: BC96; dilution factor 1:50</td></tr><tr><td>&amp;nbsp;PE Rat anti-human CLA</td><td>Miltenyi Biotech</td><td>130-091-635</td><td>clone: HECA-452; dilution factor 1:</td></tr><tr><td>eFluo450 Rat anti-human Foxp3</td><td>eBIoscience</td><td>48-4776-42</td><td>Clone: PCH101; intracellular staining; dilution factor 1:50</td></tr><tr><td>AlexFluo488 Mouse anti-human IL-17A</td><td>eBioscience</td><td>53-7179-42</td><td>Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human IFNg</td><td>eBioscience</td><td>25-7319-41</td><td>Clone: 4S.B3; intracellular staining; dilution factor 1:400</td></tr><tr><td>Flow-Count Fluorospheres</td><td>Beckman - Coulter</td><td>7547053</td><td>Counting beads, for flow cytometry</td></tr></tbody></table>

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.

The protocol presented here will yield between 2,200 &#177; 615 (mean &#177; SEM, skin of healthy volunteers) up to 178,000 &#177; 760 (mean &#177; SEM, lesional skin of psoriasis patients) viable lymphocytes from human skin when using a single 4 mm skin biopsy.
Different types of CD45+ cells were identified in single-cell suspensions derived from skin of healthy individuals including CD4+ T-cells (~45%), CD8+ T-cells (~30%), and CD11c+ DCs (~5%), wh...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

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.

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 ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Research

JoVE Journal

Immunology and Infection

14.5K Views.  Radboud University Medical Centre. 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.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetics

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.

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 ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Biology

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

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

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Chapters in this video

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

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

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

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

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Murine Dermal Lymphatic Endothelial Cell Isolation