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 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 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 IFNγ in comparison to healthy volunteers9.
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 authors have nothing to disclose.
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.
X.H. is also supported by NSFC 61263039 and NSFC 11101321.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Disposable Biopsy Punch | Kai Europe | BP-40F | 4 mm |
Disposable sterile scalpels | Dalhausen | 1100000510 | |
gentleMACS C tube | Miltenyi Biotech | 130-093-237 | Blue-capped, used as the dissociation tube. |
gentleMACS Dissociator | Miltenyi Biotech | 130-093-235 | Automated tissue dissociator. Using the "program spleen_01" for dissociation of the skin biopsy. |
Cell strainer | BD | 352350 | 70 µm nylon |
96-well U-bottom plate | Greiner Bio-One | 650180 | |
RPMI 1640 | Life Technologies | 22409-015 | |
Sodium pyruvate | Life Technologies | 11360-039 | |
GlutaMAX | Life Technologies | 35050-061 | |
Penicillin/Streptomycin | Life Technologies | 15140-122 | |
Human Pooled Serum (HPS) | in house prepared | ||
Collagenase I | Sigma-Aldrich | C2674 | Type 1-A, suitable for cell culture |
DNase I | Calbiochem | 260913 | |
PBS | B Braun | 3623140 | |
BSA | Sigma-Aldrich | A4503-500G | |
Fixable Viability Dye (FVD) APC-eFluo780 | eBioscience | 65-0865-18 | Stain dead cells prior to cell fixation; dilute with PBS at 1:1000 |
Fixation/Permeabilization Concentrate | eBioscience | 00-5123-43 | |
Fixation/Permeabilization Diluent | eBioscience | 00-5223-56 | |
Permeabilization Buffer (10x) | eBioscience | 00-8333-56 | |
BV421 Mouse anti-human CD45 | BD | 563879 | Clone: HI30; dilution factor 1:50 |
FITC Mouse anti-human CD14 | Dako | T0844 | Clone: TUK4; dilution factor 1:50 |
PE Mouse anti-human CD56 | Dako | R7127 | Clone: MOC-1; dilution factor 1:50 |
ECD Mouse anti-human CD3 | Beckman – Coulter | A07748 | Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining. |
PC5.5 Mouse anti-human CD4 | Beckman – Coulter | B16491 | Clone: 13B8.2; dilution factor 1:200 |
PeCy7 Mouse anti-human CD11c | Beckman – Coulter | A80249 | Clone: BU15; dilution factor 1:50 |
APC Mouse anti-human CD1c | Miltenyi Biotech | 130-090-903 | Clone: AD5-8E7; dilution factor 1:10 |
APC-AlexFluo700 Mouse anti-human CD8 | Beckman – Coulter | A66332 | Clone: B9.11; dilution factor 1:400 |
APC-AlexFluo750 Mouse anti-human CD19 | Beckman – Coulter | A94681 | Clone: J3-119; dilution factor 1:50 |
PeCy7 Mouse anti-human CD25 | eBioscience | AD5-8E7 | Clone: BC96; dilution factor 1:50 |
PE Rat anti-human CLA | Miltenyi Biotech | 130-091-635 | clone: HECA-452; dilution factor 1: |
eFluo450 Rat anti-human Foxp3 | eBIoscience | 48-4776-42 | Clone: PCH101; intracellular staining; dilution factor 1:50 |
AlexFluo488 Mouse anti-human IL-17A | eBioscience | 53-7179-42 | Clone: eBio64DEC17; intracellular staining; dilution factor 1:50 |
PeCy7 Mouse anti-human IFNg | eBioscience | 25-7319-41 | Clone: 4S.B3; intracellular staining; dilution factor 1:400 |
Flow-Count Fluorospheres | Beckman – Coulter | 7547053 | Counting beads, for flow cytometry |