Name: Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture (Video) | JoVE Journal
Uploaded: 2016-04-08T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Learn lymphocyte isolation from human skin biopsies for phenotypic analysis and ex vivo cell culture. This method enables flow cytometry and immunostaining of viable skin resident T cells for immunology research.

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5 minutes

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/pubmed?term=((The+vast+majority+of+CLA%2B+T+cells+are+resident+in+normal+skin%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Normal+human+dermis+contains+distinct+populations+of+CD11c%2BBDCA-1%2B+dendritic+cells+and+CD163%2BFXIIIA%2B+macrophages%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus%5BTitle%5D)+AND+%22Nature+Immunol%22%5BJournal%5D)">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/pubmed?term=((Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">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/pubmed?term=((The+inflammatory+infiltrate+in+psoriasis%5BTitle%5D)+AND+%22Clin+Dermatol%22%5BJournal%5D)">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/pubmed?term=((Skin+infection+generates+non-migratory+memory+CD8%2B+T%28RM%29+cells+providing+global+skin+immunity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1%2B+dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((CCR7+expression+and+memory+T+cell+diversity+in+humans%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Targeting+PKC+in+human+T+cells+using+sotrastaurin+%28AEB071%29+preserves+regulatory+T+cells+and+prevents+IL-17+production%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">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/pubmed?term=((A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">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/pubmed?term=((IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">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/pubmed?term=((Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation%5BTitle%5D)+AND+%22Experimental+dermatology%22%5BJournal%5D)">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/pubmed?term=((Skin-infiltrating+CD8%2B+T+cells+initiate+atopic+dermatitis+lesions%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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

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 ± 615 (mean ± SEM, skin of healthy volunteers) up to 178,000 ± 760 (mean ± 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%), whereas few B cells (C...

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 ...

The overall goal of this procedure is to efficiently isolate skin resident T cells from human skin biopsies. This is accomplished by first obtaining human skin biopsies using a round biopsy punch. Next, each skin biopsy is cut into four smaller pieces in a petri dish.Then, the skin tissue is mechanically and enzymatically dissociated to produce a single cell suspension. Finally, the skin resident lymphocytes are analyzed by flowing cytometry and cultured ex vivo. Ultimately, sufficient numbers of viable human skin resident lymphocytes can be isolated and analyzed by immunostaining and FACS analysis.This method will help to answer key questions in dermatology and skin tumor immunology. As for example, how resident T cells in the skin contribute to the disease. Demonstrating the procedure will be Xuehui He, a PhD student from our lab.After preparing culture medium, according to the text protocol, add two milliliters of complete medium to three wells of a sterile six well culture plate. Then, use a four millimeter round biopsy punch to obtain a skin biopsy. Place it in RPMI 1640 complete culture medium and leave them on the bench for up to four hours, or at four degrees Celsius over night.While the biopsy is incubating, label a blue capped dissociation tube and add five milliliters of complete culture medium. Next, use sterile tools to place the biopsy into the first well to rinse it. Transfer the tissue to the second well, rinse, and then repeat the process one more time in the third well.Transfer the well rinsed skin biopsy to a sterile petri dish and pipette 100 micro-liters of complete culture medium on top of the sample. Then, use a stainless steal disposable sterile scalpel to carefully scrape off the subcutaneous fat tissue. In a fresh sterile petri dish, cut each skin biopsy into four smaller pieces.Then transfer the samples to the prepared dissociation tube containing five milliliters of complete medium. Tightly cap the tube 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.Next, start the dissociation process by running program M spleen zero one. To dissociate the biopsy at the appropriate rotating speed for 56 seconds. 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.Add 150 micro-liters of collagenous one A to the dissociation tube and incubate the sample in a shaking water bath at 37 degrees Celsius for 60 minutes. Then, add 100 micro-liters of DNase into the dissociation tube and mix well. Attach the dissociation tube to the sleeve of the automated tissue dissociator and run program M spleen zero one to dissociate the biopsy one more time.Then, place a 70 micro-molar nylon cell strainer on the top of a 50 milliliter falcon tube and apply the dissociated sample materials to the cell strainer to remove cell clumps and tissue debris. Use five milliliters of complete medium to wash the cell strainer once. Then, centrifuge the filtrate at 450 G and 20 degrees Celsius, plus or minus two degrees for 10 minutes before aspirating the supernatant.After repeating the wash step one more time, use 300 micro-liters of complete culture medium to re-suspend the cell pallets. Single cell suspensions are now ready for further analysis. To carry out intracellular cytokine staining, use PMA, ionomysin, and brefeldin A to stimulate the cells and incubate at 37 degrees Celsius and five percent carbon dioxide for four hours before performing flow cytometry analysis.To perform polyclonal activation of skin resident T cells, aliquot 100 micro-liters of single cell suspension into a round bottom 96 well plate. Add anti-CD3, anti-CD28 mass antibody coated microbeads, recombinant human cytokine, IL-2 and IL-1B. With a well labeled weight lid, cover the culture plate and incubate at 37 degrees Celsius, five percent carbon dioxide, and 100%humidity.Then, when the color of the medium turns yellow, change it. A clear cell colony can be observed on day eight in cell culture. To carry out FACS analysis, transfer cells of interest into a V bottom 96 well plate.Centrifuge at 450 G and 20 degrees Celsius, plus or minus two degrees for two minutes. Then, aspirate the supernatant. Use 100 micro-liters of prepared fluorescent 780 conjugated fixable viability to dye to stain the cells by incubating at four degrees Celsius for 30 minutes.Then, add 100 micro-liters of FACS buffer and centrifuge before aspirating the supernatant. Next, after selecting the cell surface marker monoclonal antibodies from the list provided in the text protocol, use FACS buffer to prepare the antibody solutions. Use isotope control of the antibodies together with a non-stained sample to define gate settings.Now, add 25 micro-liters of prepared mouse antibodies mixture into each well and protect from light. Incubate at 20 degrees Celsius for 20 minutes. Add 100 micro-liters of FACS buffer, centrifuge, and aspirate the supernatant.Using buffers prepared for foxp3 staining according to the text protocol, add 100 micro-liters of fixation and permeabilization buffer to re-suspend the pallets. Mix well and incubate at four degrees Celsius for 30 minutes. Then, add 100 micro-liters of permeabilization buffer to each well and centrifuge before aspirating the supernatant.After washing the cells one more time and choosing the correct antibodies, add 20 micro-liters of prepared monoclonal antibody mixture into each well and protect from light before incubating at four degrees Celsius for 30 minutes. Using permeabilization buffer, wash the cells one more time. Then, use 110 micro-liters of FACS buffer to re-suspend the pallets and transfer the cell suspensions into mico FACS tubes.Finally, if the exact cell number is required, at 10 micro-liters of a well mixed uniform suspension of flurospheres to each sample immediately before taking FACS measurements. As shown here, different types of CD45 positive leukocytes were identified in single cell suspensions derived from skin of healthy individuals. Including CD4 positive T cells, CD8 positive T cells, and CD11c positive dendritic cells.Whereas few B cells, NK cells, or micro phages were observed. In addition, the methodology allows for the analysis of CD4 positive CD25 positive foxp3 positive cells in human skin biopsies. For example, by using intracellular staining after fixation and permeabilization, it is possible to demonstrate expression of the transcription factor foxp3 in CD25 positive T cells.As demonstrated here, to avoid background auto fluorescence and to avoid the auto fluorescent cell population, the anti-human CD45 mouse antibody conjugated to a brilliant violent 421 fluorochrome is recommended for gating of the lymphocyte population. This figure illustrates that the majority of resident cells in human skin were CD3 positive T cells. For cell viability analysis, a fixable viability dye is recommended to distinguish viable from dead or dying cells.For further functional analysis, by using a single four millimeter skin biopsy of lesional skin from psoriasis patients, it was shown that biopsy derived T cells produced IL-17a and inteferon gamma. Following a four hour stimulation with PMA ionomycin in the presence of brefeldin A.Finally, this figure shows that T cells from psyoriatic individuals have a much higher capacity to produce the psoriasis associated cytokines IL-17a and interferon gamma. Don't forget that working with human tissue and laboratory agents can be hazardous.Always take safety measures into account.

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Research

JoVE Journal

Immunology and Infection

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.

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

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.

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

This video describes the isolation of keratinocytes—the most prominent cells of human skin's epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

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.

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

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.

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

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.

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

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.

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

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.

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

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer'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.

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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.

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

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.

Murine Dermal Lymphatic Endothelial Cell Isolation

Learn lymphocyte isolation from human skin biopsies for phenotypic analysis and ex vivo cell culture. This method enables flow cytometry and immunostaining of viable skin resident T cells for immunology research.

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

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