Isolation of five different primary cell types from a single sample of human skin

Abstract

We have developed a technique to isolate primary keratinocytes, melanocytes, fibroblasts, preadipocytes, and microvascular endothelial cells from an individual sample of human skin. The protocol describes step-by-step instructions for processing, cells isolation, and culture of neonatal foreskin, with adaptation for more demanding adult tissues. The availability of multiple isogenic cell types derived from individual skin samples offers the ability to investigate various areas of biology, in the context of cell-type specificity without potential confounding influence of inter-individual or genetic differences. For complete details on the use and execution of this protocol, please refer to Holliman et al. (2017), Horvath et al. (2019), Horvath et al. (2018), Kabacik et al. (2018), Lowe et al. (2020), Lu et al. (2019), and Lu et al. (2018).

Keywords: Cell Biology; Cell culture; Cell isolation; Clinical Protocol; Health Sciences.

Graphical abstract

Figure 1

Intra- and inter-individual variability in protein expression in response to ionizing radiation Cells were exposed to sham dose or 4 Gy of X-ray and collected for western blot analysis 2 h after exposure. (A) Western blot analysis of 5 different cell types isolated from the same neonatal donor; K – keratinocytes, M – melanocytes, F –Fibroblasts, EC – microvascular endothelial cells, A – preadipocytes. (B) Western blot analysis of 6 different donors of neonatal keratinocytes; donor F is the same one as in (A). The following antibodies were used for western blotting: Ataxia Telangiectasia Mutated (ATM), Abcam (ab32420, dilution 1:5,000); pATM ser 1981, Abcam (ab81292, dilution 1:5,000); Tumor Protein P53 (TP53), Santa Cruz (sc-126, dilution 1:10,000); Cyclin Dependent Kinase Inhibitor 1A (CDKN1A), Cell Signalling (2947, dilution 1:30,000); Lamin B1 (LMNB1), Abcam (ab16048, dilution 1:5,000); Tubulin Alpha 1b (TUBA1B), Proteintech (66031-1-AP, dilution 1:100,000); Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Proteintech (60004-1-Ig, dilution 1:100,000); Actin beta (ACTB), Proteintech (20536-1-AP, dilution 1:5,000).

Figure 2

Initial tissue processing (A) Neonatal foreskin tissue in 10 cm dish. (B) Adult facial skin tissue samples from 92 and 63 years old male donors in 10 cm dishes and skin sample from 85 years old male in 6 cm dish. (C) Neonatal foreskin cut open into a single sheet. (D) Neonatal tissue cut into small pieces for liberase digestion.

Figure 3

Tissue processing after liberase digestion (A) Neonatal foreskin tissue in 6 cm dish after 16–20 h digestion in liberase. Please note that the tissue pieces are less compact now. (B) Epidermis and dermis separated, please note that dermis pieces have pale, “top” side and vascularized “bottom” side. (C) Mechanically disintegrated epidermis, epidermis pieces are no longer floating on the surface of the solution. (D) Pieces of dermis prepared for fibroblast outgrowth, the vascularized side of dermis should be facing up. (E) Minced dermis prepared for collagenase digestion. (F) Digested epidermis repeatedly pipetted up and down to release cells from the tissue. Note that solution became cloudy. It is normal to see pieces of epidermis still remaining in the plate.

Figure 4

Expression of cell-type-specific markers in cells isolated from skin samples (A) Immunofluorescence detection of cell type specific markers. Cells were seeded on coverslips, fixed, permeabilized and stained with appropriate antibodies. Each coverslip was incubated with two different antibodies raised in different hosts and then the signal was pseudocolored green. The scale bar represents 100 μm. The following antibodies were used for immunofluorescence: Keratin 14 (KRT14), Abcam (ab7800, dilution 1:100); Tyrosinase Related Protein 1 (TYRP1), Covance (SIG-38150, dilution 1:500); Vimentin (VIM), Santa Cruz (sc-6260, dilution 1:100); Platelet Derived Growth Factor Receptor Alpha (PDGFRα), Abcam (ab203491, dilution 1:500); Cadherin 5 (CDH5), Santa Cruz (sc-9989, dilution 1:100); Platelet And Endothelial Cell Adhesion Molecule 1 (PECAM1), Millipore (04-1074, dilution 1:100); CD34 Molecule (CD34), Abcam (ab81289, dilution 1:100). (B) FACS analysis of CD34 expression (CD34 antibody, BD Biosciences, cat. no. 550760, dilution 1:50). Cells were trypsinized and fixed without permeabilization to detect membrane bound CD34 protein. Blue color represents control with only secondary antibody, pink shows CD34 expression.

Figure 5

Functional analysis of different cell types (A) Western blot analysis of keratinocytes differentiation by treatment with 1.5 mM CaCl₂. Neonatal primary keratinocytes were sham-treated or treated with 1.5 mM CaCl₂ for 5 days. Samples for western blot analysis were collected before treatment, on day 2 and 5. The following antibodies were used for western blotting: Keratin 14 (KRT14), Abcam (ab7800, dilution 1:20,000); Keratin 10 (KRT10), Abcam (ab76318, dilution 1:5,000); Actin beta (ACTB), Proteintech (20536-1-AP, dilution 1:5,000); Histone H3, Novus Biologicals (NB500-171, dilution 1:100,000). (B) Spectroscopic analysis of melanin release into media by melanocytes after 48 h treatment with 10 μM PMA (pink line with PMA and green line without PMA, showing peak absorption differences in the UVB spectrum), picture below shows collected media and pelleted melanocytes without PMA and with PMA displaying dark melanin in both cells and released into the media; right shows media in cuvettes compared to fresh melanocyte media. (C) Acetylated LDL uptake by endothelial cells. Microvascular endothelial cells were seeded on collagen fibronectin coated coverslip, next day Dil-Ac-LDL was added to the media at concentration 10 μg/mL and incubated for 4 h at 37°C. After incubation cells were washed in HBSS, fixed in formalin and imaged. The scale bar represents 200 μm. (D) Differentiation of preadipocytes into mature adipocytes. Preadipocytes were seeded into gelatin coated coverslips and differentiated into matured adipocytes by using StemPro™ Adipogenesis Differentiation Kit according to manufacturer’s protocol. After 14 days of differentiation, cells were fixed, stained with Oil Red O and imaged. The scale bar represents 100 μm.