Isolation and feeder-free primary culture of four cell types from a single human skin sample

Abstract

Four types of primary cells-dermal fibroblasts, dermal microvascular endothelial cells, epidermal keratinocytes, and epidermal melanocytes-can be isolated simultaneously from a single human skin sample, without the use of xenogeneic murine feeder cells. This protocol describes the procedures for isolation of these cells from adult full-thickness skin obtained from surgical discard tissue. The cells isolated using this protocol contain stem cell populations and are competent to form functional skin tissue in three-dimensional reconstructed skin models. For complete details on the use and execution of this profile, please refer to Supp et al. (2002), Boyce et al. (2015), Boyce et al. (2017a), Boyce et al. (2017b), and Supp et al. (2019).

Keywords: Cell Biology; Cell culture; Cell isolation; Stem Cells; Tissue Engineering.

Graphical abstract

Figure 1

Trimming human skin in preparation for primary cell isolation (A) Abdominal skin with subcutaneous fat, collected from elective panniculectomy. (B) Appearance of skin after initial trimming from surgical discard tissue, showing subcutaneous fat layer. (C) Technique for removing subcutaneous fat using curved scissors. (D) Skin after removal of subcutaneous fat layer, showing whitish appearance of dermal tissue. Size after trimming is ∼20 cm².

Figure 2

Illustration of steps involved in establishing primary cell cultures from human skin (A) Dishes arranged in tissue culture hood for decontamination and rinsing of skin tissue, showing dishes containing 5% Dettol (whitish solution) and HBS rinses (pinkish solution). (B) Cutting skin into strips measuring ∼2 cm × 0.25 cm after trimming and decontaminating, and prior to Dispase digestion. (C) Separation of epidermis from dermis following overnight (∼16 h) Dispase digestion. (D) Strips of dermis (left) and epidermis (right) after Dispase digestion and separation. (E) Extrusion of HDMEC from dermal strips. (F) Mincing of dermal strips prior to collagenase digestion. (G) Suspension of minced dermis in collagenase. (H) T150 flask containing dermal fibroblasts and dermal “pieces” following collagenase digestion, rinsing, and inoculation. (I) Filtration of digested epidermal tissue through cell strainer following trypsin treatment.

Figure 3

Images of P0-P1 primary human keratinocytes in culture (A) Image of P0 keratinocytes in hemacytometer, viewed through phase-contrast microscope. The 16-square area for counting is indicated by white arrows. Note that cell morphology is heterogeneous and only refractory (bright) cells of roughly uniform size should be counted. Dark looking cells and debris (black arrows) should not be counted. (B) Human P0 keratinocytes at day 1, ∼24 h after inoculation into collagen-coated flask. Note that the cell population appears heterogeneous at this stage, with many cells appearing bright and raised from culture surface. Cells have irregular shapes, and some 2-cell colonies or dividing cells may be observed (white arrows). (C) Human P0 keratinocytes at day 2 of culture. The cells exhibit diverse morphologies: rounded-up cells, which may be in the process of cell division (white arrow), can be seen, in addition to more flattened cells. This appearance is normal at P0 in KGM-LC. In KGM-LC, P0 keratinocytes may appear large and flat, and form loosely-associated colonies. Occasional elongated cells, most likely melanocytes, may be observed (black arrow). (D) By culture day 6, P0 keratinocyte morphology appears more uniform, with numerous flattened, widely separated cells. A melanocyte is identified by its dendritic morphology (black arrow). (E) P0 keratinocytes at day 7, immediately prior to melanocyte removal. Melanocytes are indicated by black arrows. (F) Nearly confluent P0 keratinocytes at the time of harvesting for passage. (G) P1 keratinocytes shown 48 h after inoculation. (H) P1 keratinocytes 6 days after inoculation, at time of harvest (∼80%–85% confluent). Note tighter colonies and more uniform morphology after passage and culture in “regular” calcium (0.2 mM) KGM. Original image magnification: 10× (B–D); 4× (A, E–H).

Figure 4

Images of P0-P1 primary human dermal microvascular endothelial cells (HDMECs) in culture (A) Image of HDMEC P0 culture on day 1 after initiation. Note the appearance of cells associated with residual pieces of tissue. HDMECs are relatively compact at this stage and tightly associated with each other. (B) Image of the same HDMEC P0 culture on day 4 after initiation. Note that the HDMECs now appear elongated but remain tightly associated in colonies. A contaminating fibroblast is observed (arrow). (C) HDMEC at P0 culture day 6. (D) HDMEC at P0 culture day 8, at time of harvest for passaging. White arrows indicate contaminating fibroblasts. (E) P1 HDMEC, one day after passaging. (F) P1 HDMEC at day 5. Contaminating fibroblasts are occasionally observed (white arrows in B, D, E, F). These tend to have more irregular shapes than HDMEC and are usually present as individual cells rather than colonies. Original image magnifications: 10× (A, B); 4× (C-F).

Figure 5

Images of P0-P1 primary human fibroblasts in culture (A) P0 fibroblasts in #1 flask at culture day 2, after removal of tissue “pieces.” (B) P0 fibroblasts in #1 flasks at culture day 4. (C) P0 fibroblasts in #2 flask on culture day 4, 3 days after removal from #1 flasks. (D) P0 fibroblasts in #2 flasks on day 5, 4 days after removal from #1 flasks, at time of harvest for cryopreservation and/or passaging. (E) P1 fibroblasts, 1 day after passage and inoculation. (F) P1 fibroblasts, 5 days after inoculation, one day prior to planned harvest. Original image magnifications: 10× (A, B); 4× (C–F).

Figure 6

Images of P1 primary human melanocytes in culture (A) P1 melanocytes at culture day 2, 2 days after removal from P0 keratinocytes, photographed at 10× magnification. (B) P1 melanocytes at culture day 5, photographed at 10× magnification. (C) P1 melanocytes at culture day 5, photographed at 4× magnification. A small cluster of cells resembling keratinocytes is present (white arrow). Note that using a diluted trypsin solution when harvesting melanocytes, and trypsinizing for the minimum length of time required, will eliminate keratinocytes from successive passages. (D) P1 melanocytes at day 8, at time of harvest for subculture. Some contaminating cells resembling keratinocytes are present (white arrows).

Figure 7

Immunofluorescent staining of primary skin cells using cell type-specific markers Immunofluorescence was used to validate the identify of individual primary cell types. (A) Primary human dermal fibroblasts were labeled using a primary antibody against vimentin (1:200) followed by Alexa Fluor 594-labeled donkey anti-rabbit secondary antibody (1:400). (B) Primary human keratinocytes were labeled using a primary antibody against keratin 14 (1:100) followed by Alexa Fluor 594-labeled donkey anti-rabbit secondary antibody (1:400). (C) Primary human dermal microvascular endothelial cells (HDMEC) were labeled using a primary antibody against PECAM1/CD31 (1:100), followed by Alexa Fluor 594-labeled donkey anti-mouse secondary antibody (1:400). (D) Primary human melanocytes were labeled with a primary antibody against tyrosinase-related protein 1 (1:100) followed by Alexa Fluor 594-labeled donkey anti-mouse secondary antibody (1:400). Cells were stained using standard immunofluorescence methods as described elsewhere (Hahn et al., 2021). For all cell types, specific staining is indicated by red fluorescence; nuclei were counterstained blue using mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Note that the cell morphologies may vary slightly from the phase-contrast images shown in Figures 3, 4, 5, and 6 because the cells were not cultured on tissue culture-treated plastic for staining. Fibroblasts and keratinocytes were cultured for immunofluorescence using chamber slides without coating; HDMEC and melanocytes were cultured using glass coverslips coated with 0.1% gelatin or FBS, respectively. The scale bar shown in A is the same for all panels.