Name: lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture
Uploaded: 2016-04-08T13:00:00
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 Steri...

<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>

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.

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			<td height="21" style="height:21px;width:281px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch</td>
			<td>Kai Europe</td>
			<td>BP-40F</td>
			<td>4 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpels</td>
			<td>Dalhausen</td>
			<td>1100000510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td>Blue-capped, used as the dissociation tube.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">gentleMACS Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td style="width:299px;">Automated tissue dissociator. Using the &#34;program spleen_01&#34; for dissociation of the skin biopsy.</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer&#160;</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">352350</td>
			<td>70 &#181;m nylon</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96-well U-bottom plate</td>
			<td style="width:145px;">Greiner Bio-One</td>
			<td style="width:119px;">650180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>22409-015</td>
			<td></td>
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			<td height="21" style="height:21px;width:281px;">Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">GlutaMAX</td>
			<td>Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:281px;">Human Pooled Serum (HPS)</td>
			<td style="width:145px;">in house prepared</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Collagenase I</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C2674</td>
			<td>Type 1-A, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">DNase I</td>
			<td style="width:145px;">Calbiochem</td>
			<td style="width:119px;">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PBS</td>
			<td style="width:145px;">B Braun</td>
			<td style="width:119px;">3623140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">A4503-500G</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;">Fixable Viability Dye (FVD) APC-eFluo780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td style="width:299px;">Stain dead cells prior to cell fixation; dilute with PBS at 1:1000</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Buffer (10x)</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
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			<td height="21" style="height:21px;width:281px;">BV421 Mouse anti-human CD45</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">563879</td>
			<td>Clone: HI30; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">FITC Mouse anti-human CD14</td>
			<td>Dako</td>
			<td style="width:119px;">T0844</td>
			<td>Clone: TUK4; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PE Mouse anti-human CD56</td>
			<td style="width:145px;">Dako</td>
			<td style="width:119px;">R7127</td>
			<td>Clone: MOC-1; dilution factor 1:50</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:281px;">ECD Mouse anti-human CD3</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A07748</td>
			<td style="width:299px;">Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PC5.5 Mouse anti-human CD4</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">B16491</td>
			<td>Clone: 13B8.2; dilution factor 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD11c</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A80249</td>
			<td>Clone: BU15; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC Mouse anti-human CD1c</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-903</td>
			<td>Clone: AD5-8E7; dilution factor 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC-AlexFluo700 Mouse anti-human CD8</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A66332</td>
			<td>Clone: B9.11; dilution factor 1:400</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:281px;">APC-AlexFluo750 Mouse anti-human CD19</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A94681</td>
			<td>Clone: J3-119; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD25</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">AD5-8E7</td>
			<td>Clone: BC96; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">&#160;PE Rat anti-human CLA</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-091-635</td>
			<td>clone: HECA-452; dilution factor 1:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">eFluo450 Rat anti-human Foxp3</td>
			<td>eBIoscience</td>
			<td>48-4776-42</td>
			<td style="width:299px;">Clone: PCH101; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">AlexFluo488 Mouse anti-human IL-17A</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">53-7179-42</td>
			<td style="width:299px;">Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PeCy7 Mouse anti-human IFNg</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">25-7319-41</td>
			<td style="width:299px;">Clone: 4S.B3; intracellular staining; dilution factor 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Flow-Count Fluorospheres</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td align="right" style="width:119px;">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 (Video) | JoVE

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 ...

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