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Skin Deep: the secrets of skin tissue dissociation
Time:2022-03-01 15:23:22  

The skin is the organ with the largest area in an organism, and acts as a physical barrier and an acquired/innate immune interface to protect the body from the external environment. The skin is composed of epidermis, dermis, and subcutaneous layers, and contains accessory organs (sweat glands, sebaceous glands, fingernails, toenails) as well as blood vessels, lymphatic vessels, nerves, and muscles.

 

When the physiological function of the skin is disturbed, it can cause various disorders. Single-cell RNA sequencing (scRNA-seq), which detects gene expression patterns of individual cells and deciphers cellular interaction network, could provide a new approach to define cellular composition in the skin, including the discovery of new or rare cell populations, in order to better illustrate the roles of skin in health and diseases. 

 

Singleron’s service lab has been optimizing skin tissue dissociation protocols to obtain single cell suspension with high viability and minimal aggregates, which is required for optimal single cell sequencing results. We found that the skin thickness, number of hair follicles, and cellular contents can vary significantly in different species. Here we would like to share with you our skin tissue protocols and experience for human, mouse, and rat skins.

 

 

Human skin dissociation

Protocol

 

1. Wash the human skin tissue (freshly obtained or stored in sCeLiVE® tissue preservation solution for up to 72h) 3 times with pre-cooled 1×HBSS buffer. Remove the visible fat parts, and cut the full thickness of the skin into pieces to facilitate subsequent epidermal peeling;

2. Transfer the cut tissue to a 15mL low adsorption centrifuge tube and add sCeLiVE® Skin A Dissociation Solution;

3. Place the centrifuge tube horizontally at 37°C, swirling on a 180rpm shaker, and generally digest for 1 hour (the digestion time may vary depending on the type of disease);

4. After the preliminary digestion, use two pointed tweezers to peel off the epidermis in one piece;

5. Chop the peeled epidermis, weigh it, and place in sCeLiVE® Skin B Dissociation Solution. Shake at 37°C, 160rpm for 20-30min, until the tissue is completely digested;

6. Cut the remaining dermis and subcutaneous tissue into pieces and place them in sCeLiVE® Skin C Dissociation Solution. shake at 37°C, 140rpm for 30-40min, until the tissue is completely digested;

7. Filter the digested cell suspension with a 40µm filter, collect by centrifugation at 350g, discard the supernatant, and resuspend the cell pellet in medium;

8. Perform a quality control of the cell suspension with a fluorescence cell counter.

 

Results

 

Epidermal cells: 72mg

[Fluorescence cell counting: about  0.9M cells, 93.8% viability]

 

Dermal cells:593mg

[Fluorescence counting: about 0.7M cells, 92.5% viability]

 

Fig1: UMAP plot of human skin scRNA-seq results, colored by cell types.

 

 

Mouse skin dissociation

Protocol

 

1. After the mice are sacrificed, depilate the back skin of the mouse to remove excess hair as much as possible;

2. Wash the freshly obtained mouse skin tissue (or stored in sCeLiVE® tissue preservation solution within 72h after ex vivo) with pre-cooled 1×HBSS buffer 3-4 times to remove the depilatory agent from the tissue. After spreading, cut the full thickness of the skin into chunks;

3. Transfer the cut tissue to a 15mL low adsorption centrifuge tube, and add sCeLiVE® Skin A dissociation solution;

4.Place the centrifuge tube horizontally at 37°C, swirling on a shaker at 180rpm, and digest for 1h;

5. After the digestion, collect the cell suspension digested with sCeLiVE® Skin A dissociation solution, terminate the digestion with medium, and temporarily store it on ice;

6. Take out the tissue and cut it into pieces for secondary digestion. At this time, add it to sCeLiVE® Skin C Dissociation Solution, shake at 37°C, and vortex at 160rpm for 20-30min until the tissue is completely digested;

7. Filter the digested cell suspension with a 40µm filter, collect by centrifugation at 350g, discard the supernatant, and resuspend the cell pellet in medium;

8. Perform quality control on the cell suspension with a fluorescence counter.

 

Results

 

Dermal cells:312mg

[Fluorescence cell counting: about 1.6M cells, 88.3% viability]

 

Fig2: UMAP plot of mouse skin scRNA-seq results, colored by cell types.

 

 

Rat skin dissociation

Protocol

 

1. After the rats are sacrificed, depilate the skin of the back of the rats to remove excess hair as much as possible;

2. Wash the freshly obtained rat skin tissue (or preserved in sCeLiVE® tissue preservation solution for less than 72 hours after ex vivo) 3-4 times with pre-cooled 1×HBSS buffer, and remove the depilatory agent. Remove the fat part and cut the full thickness of the skin into chunks;

3. Transfer the tissue to a sterile petri dish, add sCeLiVE® skin A dissociation solution, and incubate at 4°C overnight;

4. Transfer to a 15mL low-adsorption centrifuge tube the next day, place it at 37°C, 180rpm shaker for about 1h, and collect the digested cell suspension;

5. Chop the remaining tissue and transfer to a new 15mL low adsorption centrifuge tube for secondary digestion, add sCeLiVE® Skin B dissociation solution, and place at 37°C, 180rpm shaker for 15-20min;

6. Filter the digested cell suspension with a 40µm filter, collect by centrifugation at 350g, discard the supernatant, and resuspend the cell pellet in medium;

7. Use a fluorescence counter to control the quality of the cell suspension.

 

Results

 

Dermal cells:905mg

[Fluorescence cell counting: about 4M cells, 85.6% viability]

 

Fig3: UMAP plot of rat skin scRNA-seq, colored by cell types.

 

 

References

Daniels, J., Doukas, P.G., Escala, M.E.M. et al. Cellular origins and genetic landscape of cutaneous gamma delta T cell lymphomas. Nat Commun 11, 1806 (2020). 

Vorstandlechner V, Laggner M, Kalinina P, Haslik W, Radtke C, Shaw L, Lichtenberger BM, Tschachler E, Ankersmit HJ, Mildner M. Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing. FASEB J. 2020 Mar;34(3):3677-3692. 

Sawaya, A.P., Stone, R.C., Brooks, S.R. et al. Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing. Nat Commun 11, 4678 (2020).

Foster, D.S., Marshall, C.D., Gulati, G.S. et al. Elucidating the fundamental fibrotic processes driving abdominal adhesion formation. Nat Commun 11, 4061 (2020).

Wang, S., Drummond, M.L., Guerrero-Juarez, C.F. et al. Single cell transcriptomics of human epidermis identifies basal stem cell transition states. Nat Commun 11, 4239 (2020).

Guerrero-Juarez, C.F., Dedhia, P.H., Jin, S. et al. Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds. Nat Commun 10, 650 (2019).

 

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