Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

This protocol facilitates the isolation of functionally distinct fibroblast subsets from human skin by FAC sorting based on the cell surface marker expression. For the first time, papillary and reticular fibroblasts can be isolated directly from the skin without in vitro manipulation. This is a huge advancement compared to previously established isolation methods using explant cultures.

Dermal fibroblast subsets harbor distinct functions under homeostatic conditions. With this tool, it is possible to investigate functional differences in skin pathologies, including cancer or inflammatory skin diseases. Since handling of the human tissue requires some practice to attain satisfactory cell yields, we would like to provide a visual demonstration to facilitate and accelerate this process.

To prepare full-thickness dermis, place a piece of human skin on a thick filter paper with the epidermis facing downwards. Hold the epidermis tightly with forceps, and scrape off the subcutaneous fat layer with a scalpel. Then, slice the tissue into five-millimeter-wide strips before putting them into a Petri dish.

To section the dermis into papillary and reticular layers, place a piece of skin on a thick filter paper with the epidermis facing upwards. Hold the skin tightly on its edges with forceps, and slice out a section of 300-micrometer thickness with an electric dermatome. Transfer the first layer consisting of epidermis and papillary dermis to a Petri dish.

Then, proceed immediately to separate the epidermis and dermis, or add 1x PBS to keep the tissue from drying out. Next, adjust the dermatome to a cutting thickness of 700 micrometers, and slice the remaining dermis. Place the upper slice, which is defined as the upper reticular dermis, in a separate Petri dish.

Subsequently, scrape away the subcutaneous fat layer with a scalpel from the residual lower reticular dermal layer, and discard it. Collect the lower reticular dermis in another Petri dish. Proceed immediately to enzymatic digestion procedure, or add 1x PBS to keep the tissue from drying out.

To perform enzymatic digestion, place the five-millimeter skin strips or the epidermis/papillary dermis with the epidermis facing upwards in a Petri dish with 10 milliliters of the dissociating enzyme solution. Then, incubate the Petri dish at 37 degrees Celsius for one hour. After incubation, transfer the epidermis/papillary dermis to the Petri dish's lid.

Separate the epidermis from the upper dermis with two forceps, each holding the edge of the dermis. Subsequently, mince each dermal layer as thoroughly as possible. The smaller the pieces, the better the tissue digestion.

Then, prepare the digestion mix. Transfer the minced tissue with 10 millimeters of the prepared digestion mix into a 50-millimeter tube. Incubate the tissue at 37 degrees Celsius for one hour under agitation.

Invert the tube several times during digestion. To prepare a single-cell suspension, stop enzymatic tissue digestion by adding 10 milliliters of fibroblast medium to the digested tissue on ice. Next, pour the contents of each tube through a regular sterile stainless tea strainer, and collect the cell suspension in a clean Petri dish.

Wash the strainer with medium, and mash the undigested tissue pieces with the edge of a syringe plunger. Afterwards, pipette the collected cell suspension through a 70-micrometer cell strainer into a 50-milliliter tube. Rinse the cell strainer with medium, and collect flow through the same tube.

Then, centrifuge the tube at 500 times g at four degrees Celsius for 10 minutes. Remove the supernatant, and wash the cell pellet with five milliliters of fibroblast medium. Repeat the centrifugation step again.

Afterward, remove the supernatant, and resuspend the pellet in one milliliter of ACK erythrocyte lysis buffer. Leave the cells in lysis buffer for approximately one minute at ambient temperature. Then, stop lysis by adding nine milliliters of 1x PBS with 10%FCS.

Centrifuge the tube again at 500 times g at four degrees Celsius for five minutes, and discard the supernatant subsequently. After the cells have been stained for FAC sorting following standard protocols, sort three fibroblast subpopulations into separate microcentrifuge tubes filled either with 350 microliters of lysis buffer for RNA isolation or 350 microliters of fibroblast growth medium for cell culture. Invert the tubes immediately, and either put the lysis buffer tubes into liquid nitrogen, or put the fibroblast growth medium tubes on ice.

Following FACS, spin the cells down at 500 times g for three minutes at four degrees Celsius. Then, plate an equal amount of cells in fibroblast growth medium in a 24-well cell culture dish, and leave them to grow until they reach 70%confluency. Next, replace the cultured cell medium with adipocyte differentiation medium, and let the cells differentiate for 14 days.

At differentiation day 14, fix the cells with 4%PFA for 20 minutes. Wash the wells with 60%isopropanol, and let it evaporate completely. Then, stain the cells with five millimolars of filtered Oil Red O for 20 minutes.

After 20 minutes, wash the stained cells four times with distilled water. Image the cells immersed in water with an inverted microscope at 10x magnification with transmitted light. This protocol enables the identification of three fibroblast populations in human skin, which presents with different intradermal localization, gene expression profiles, and functions.

FAP-positive CD90-negative are enriched in the papillary dermis, whereas FAP-positive CD90-positive and FAP-negative CD90-positive are more abundant in the reticular dermis. All three sorted subpopulations display typical fibroblast morphology upon culture for seven days. Interestingly, they behave differently in an adipogenesis assay.

After 14 days in culture, FAP-positive CD90-negative do not differentiate into adipocytes, while FAP-positive CD90-positive and FAP-negative CD90-positive readily undergo adipogenesis. Gene expression profiling shows that FAP-positive CD90-negative cells express high levels of markers commonly attributed to papillary fibroblasts, such as CD26, NTN, and PDPN, while CD90-positive cells express the known reticular markers, such as CD36, smooth muscle actin, and PPAR-gamma at high levels. We conclude that FAP-positive CD90-negative cells belong to the papillary and CD90-positive cells belong to the reticular lineage.

Digestion works best if the dermal pieces are thoroughly minced and incubated in a sufficient amount of digestion medium. Dermal fibroblast subsets are functionally diverse. Exploring their function in the context of disease pathogenesis will pave the way for new diagnostic and therapeutic interventions.