Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies

- Colony formation assay determines the ability of individual cells to proliferate and form colonies. Begin with an excised mouse-dorsal skin in a culture plate. Scrape off the subcutaneous tissue and fat from the ventral side of the skin. Treat the tissue with a digestion solution. The proteolytic enzymes in the solution degrade the extracellular matrix, inducing epidermal cell dissociation.

Gently scrape the epidermal layer to release the dissociating cells and hairs into a harvesting media. Filter the suspension through an appropriate strainer to trap hairs or any debris and obtain a single-cell suspension of epidermal cells. Transfer the desired volume of this cell suspension into a collagen-coated plate containing an adherent layer of growth-arrested feeder cells. Supplement with a suitable media that favors keratinocyte survival.

During culture, the keratinocytes attach to the feeder layer. Additionally, these feeder cells secrete growth factors and synthesize extracellular matrix proteins that support the growth of keratinocytes. Depending on the proliferative capability of individual keratinocytes, they start forming colonies. Remove the media and fix the colonies. Stain the cells with a suitable dye to detect the colonies for counting. In the following protocol, we will perform the clonogenic assay of primary keratinocytes harvested from the adult mice following treatment with test compounds.

- After harvesting dorsal skin samples from euthanized adult mice, use autoclaved forceps and a scalpel to place one dorsal skin at a time, hairy-side down, into a thin Petri dish and scrape off the subcutaneous tissue, including the fat from the ventral side of the skin tissue, until the tissue is semi-translucent.

Place this scraped skin in PBS until all other remaining skins are processed. Then, use a scalpel to slice the skin samples into 0.5 x 1 to 1.5 centimeter strips. Place the strips hairy-side up in a sterile Petri dish before floating the samples hairy-side up on the surface of a 20 milliliter PBS plus 2x Gentamicin solution supplemented with 0.25% Trypsin in a plastic 100 x 20 millimeter Petri dish for 2 hours at 32 degrees Celsius.

At the end of the incubation, place a sterile plastic square Petri dish containing 15 milliliters of harvesting medium at a 30-degree incline, and use curved forceps to carefully transfer a floating skin strip into the dish. Holding a new scalpel blade at a perpendicular angle to the skin and using sufficient but not excessive force, scrape off the epidermis and the hairs from the sample into the medium.

When all the strips have been scraped, carefully decant the epidermal cell-containing supernatant into a sterile 60-milliliter jar containing a 1.5 inch magnetic stir bar, and rinse the Petri dish with additional harvesting medium to collect any remaining epidermal cells.

Bring the final volume in the jar to 30 milliliters with fresh medium, and stir the epidermal cell solution at 100 rotations per minute for 20 minutes at room temperature. At the end of the stirring incubation, in a biosafety cabinet, remove the stir bar and filter the cell solution through a 70-micrometer strainer into a 50 milliliter conical tube.

Use forceps and then a pipette to press the hairs and the stratum corneum materials through the strainer to manipulate the tissue to release the trapped hair cells, and use an additional 5 milliliters of harvesting medium to release the remained trapped hair cells into the tube.

- It is critical that the epidermal cells and hairs are manipulated so as to release the cells from the hair follicles.

- Bring the total volume in the tube up to 50 milliliters with fresh medium and collect the cell filtrate by centrifugation. Resuspend the pellet in 5 milliliters of fresh harvesting medium and about 20 triturations with a 5-milliliter pipette. Take 0.5 milliliters of the 1:20 dilution and transfer it to a 2-milliliter microfuge tube.

After mixing the sample carefully, take 200 microliters from the microfuge tube and mix gently with an equal volume of 0.4% Trypan Blue solution. Gently mix this solution three times and transfer the cells to a hemacytometer for counting nucleated keratinocytes.

Score all dark blue cells as non-viable cells, and score small golden pinkish cells as viable cells. The viability averages about 80%, and the final keratinocyte yield per mouse should be about 30 million viable cells. After counting, collect the cells with another centrifugation, and for mass culture, resuspend 2 to 4 times 10 to the sixth viable keratinocytes per 35 millimeter Petri dish in 2 milliliters of cell culture medium.

For a clonogenic colony formation assay, resuspend the cells at a 1 times 10 to the third keratinocyte per 4 milliliters of modified William's E Medium with supplements and serum concentration per collagen-coated 60 millimeter Petri dish on X-ray irradiated Swiss mouse 3T3 feeder layers.

For mass culture, grow the cultures at 32 degrees Celsius in 5% carbon dioxide for the appropriate cell culture period, changing the medium 24 hours after the initial seeding for mass culture and 3 times a week thereafter.

At the end of the clonal culture, aspirate the medium and fix the colonies in 10% buffered formalin overnight at room temperature. The next morning, stain the colonies with 0.5% Rhodamine B in autoclaved water for one hour before rinsing the dishes in cold autoclaved water until the water runs clear. Then, incline the dishes on their lids to dry before counting the colonies.