Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

All vertebrate animal use protocols have been approved by the University of Minnesota Institutional Animal Care and Use Committee in accordance with recommended NIH and government guidelines.

1. Feeder Layer Initialization and Subculturing

1. Thaw one cell vial containing frozen Swiss mouse 3T3 from the liquid nitrogen tank by placing the vial in a 37 °C water bath for 1-2 min. The number of cells in the vial is approximately 1 x 10⁶.
2. Remove the 3T3 cell vial when the final sliver of ice has melted. Wipe the tube with a 70% alcohol swab. Then, transfer the vial to a biosafety cabinet.
3. Transfer the cells slowly into a T-150 flask, and rinse the 3T3 vial with 1 mL of CGM medium. Slowly add 30 mL of pre-warmed 3T3 Complete Growth Medium (CGM) to the cells. Gently rock the flask to evenly spread the 3T3 fibroblast cells. Label the flask with cell line details, date, and passage number appropriately.
4. Incubate the cells at 37 °C with 5% CO₂ and 95% humidity.
   NOTE: According to the ATCC, the doubling time of Swiss mouse 3T3 is 18 h; when confluent (contact inhibited), the density is about 40,000 cells per cm². We use a particular line of 3T3 within 10-15 passages after initialization. More rapid growth or the presence of spindle-shaped cells can occur at higher passages and is usually a sign that the 3T3 will not perform as well as feeder cells for adult mouse keratinocytes. If rapid growth occurs, it is better to initiate a new line from lower passage cells.
5. Change media after 24 h to remove dead cells and remaining DMSO (cryopreservation agent), and twice weekly thereafter. Allow cells to proliferate around 80% confluence and use T-150 flasks for culture initiation. Use T-225 flasks for subculture after initialization.
6. To subculture 3T3 fibroblast cells, remove the flask from the cell culture incubator and wash twice with cold sterile PBS (1x) with gentamycin. Pipet 10 mL of pre-warmed (37 °C water bath) trypsin solution (0.25%) into each T-225 flask.
   1. Place the flask on a warming plate for 3-8 min. Remove the flask and gently use palms to tap the walls of the flask to detach cells and confirm cell detachment under inverted microscope. Trypsin solution may appear cloudy with detached 3T3 cells.
7. Pipet the trypsin solution 3–4x to wash the cell growth surface of the flask and transfer the trypsinized cell to 30 mL of CGM in a 50 mL conical tube. Rewash the flask 3–5x with 10 mL of the CGM-trypsin-cell mixture from the 50 mL conical tube.
   NOTE: The contents of two flask contents can be added to a 50 mL tube; if only one flask is used, then place 10 mL of trypsin/cells into 20 mL of CGM in a 50 mL tube.
8. Centrifuge the 50 mL conical tube at 160 x g for 7 min at 4 °C.
9. Now aspirate the supernatant and resuspend the cell pellet with 5 mL of CGM, pipetting gently ~10x to completely disrupt the cell pellet. Add 10 mL of CGM to bring the total volume of the 50 mL conical tube to 15 mL.
   1. Mix again ~10x and place 5 mL of the cell suspension into 3 separate T-150 flasks. The subculture ratio is 1:3 in T-225 flasks or 1:4 in T-150 flasks. Add 30 mL of pre-warmed 3T3 CGM to each of the flasks.
10. Label the flask with the cell line name, the date of cell seeding, and the passage number.
11. To freeze down 3T3 cells following centrifugation in the subculture step, remove the 50 mL tube from the centrifuge and place on an ice bucket. Aspirate the supernatant and resuspend the cell pellet with 1 mL of 5% DMSO-DMEM mixture (see Table 1) for each flask harvested.
12. Place 1 mL of DMSO-DMEM mixture with cell suspension into each cryovial (one cryovial per 3T3 flask harvested). Label the vial with the passage number, the cell line name (3T3), and the date that the cells were frozen.
13. Place these 3T3 cell vials into cell freezer jar (see Table of Materials) and transfer to -70 °C freezer for >5 h. Careful remove and transfer the vials from the cell freezer jar and place in a liquid nitrogen storage tank for long-term storage until next use. Enter information about the cell line name, the location in tank and the date in the laboratory liquid nitrogen inventory sheet.

2. Preparation of 3T3 Feeder Layer for X-ray Irradiation and Seeding

1. Prepare 3T3 cells one week before their irradiation and allow them to grow ~100% confluence. The 3T3 cells are typically within the passages of 120 to 130.
2. For successful clonogenic assays of primary keratinocytes, before seeding 3T3 cells, surface coat the 60 mm Petri dishes with a collagen.
   1. Pipet around 2–3 mL of the collagen-coating solution into 60 mm Petri dishes to coat the bottom surface and remove the excess collagen-coating liquid (see Table 1). Transfer the Petri dishes to a 37 °C incubator with 5% CO₂ for a minimum of 1 h. Do not let the dishes dry in incubator.
3. X-ray irradiation step: Follow steps 1.4–1.6. Use 50 mL of fresh CGM to re-suspend the cell pellet, close the conical tube and seal with paraffin sealing film. To avoid any contamination, use plastic bags to double bag the paraffin-sealed cell containing tube and place on ice. A mitomycin based alternative feeder layer can be used for keratinocyte culture⁷.
4. Irradiate fibroblast 3T3 cells in the 50 mL conical tube with a 5,000 rads (50 Cgy) dose with a biological x-ray machine. Irradiate these cells along with the CGM media.
   1. Following X-ray irradiation, centrifuge cells at 160 x g for 7 min at 4 °C. Aspirate the supernatant media and gently resuspend cell pellet with 5 mL of CGM triturating gently ~10x. Now bring the final volume to 30 mL with additional 25 mL of medium and triturate 10x. These triturating steps are crucial for uniform 3T3 fibroblast layer for clonogenicity assay.
5. Viable Cell counting: Count the keratinocytes using a regular glass hemocytometer. Take approximately 0.5 mL of the cells and place into a 1.5 mL tube. Remove 200 µL of cell mixture and add an equal volume of 0.4% Trypan blue solution and mixture 3–4x. Transfer cells with a hemocytometer and count all nucleated cells (small gold and pink cells) as viable cells. Calculate the final concentration of viable gold cells.
6. Pipette 1 x 10⁶ 3T3 cells (a minimum of 7 x 10⁵) cells may be used) into 60 mm collagen-coated Petri dishes (step 2.2) and gently add 4 mL of modified high-calcium Williams E media (see Table 1). Allow freshly seeded 3T3 cells to attach for 24 h. On the next day, harvest the fresh primary keratinocyte and seed them on top of attached 3T3 fibroblast layer for clonogenic assay as indicated below.

3. Primary Keratinocyte Harvesting and Seeding

1. Euthanize four to five adult female mice via CO₂ inhalation according to approved animal facility/IACUC standards. However, this protocol may also be used for single female/male mice.
   1. Clip approximately 9 cm² of the dorsal fur with an electric animal clipper and place all clipped mice into a 500 mL jar with enough povidone iodine antiseptic solution (not scrub) to cover them for 1–2 min. Shake the jar well to evenly coat the antiseptic solution over the mice.
   2. Pour off the solution and rinse with clean water until the liquid runs clear. Repeat the same antiseptic wash followed by a water rinse. After the antiseptic wash with iodine solution, soak all the mice in 70% ethanol for 5–10 min.
      NOTE: Mice with light fur (e.g., BALB/c) will retain a slight yellowish color from the antiseptic iodine washes, while darker mice (e.g., C57BL/6) will not. Notably, cell viability or culture growth is not affected due to the yellow color.
2. Carefully remove the clipped dorsal skin using thumb forceps and scissors in a laminar flow hood. Place the excised dorsal skin in conical tube filled with PBS/2x gentamycin. Avoid using ventral side skin to prevent mammary cell contamination in culture.
3. Using autoclaved forceps and a scalpel, place one dorsal skin at a time hairy side down onto a thin Petri dish. Start scraping all subcutaneous tissue including fat from the ventral side of the skin tissue until it is semi-translucent. Try to remove maximum traces of subcutaneous tissue without tearing the skin (hair follicle part). Also, do not scrape for a long time and avoid skin drying.
   1. Keep scraped skin in PBS solution until all other remaining skins are processed. Using a scalpel, slice skins into 0.5 cm x 1–1.5 cm strips and place the hairy side up onto a sterile Petri dish.
4. Carefully pipette 20 mL of PBS/2x Gentamicin solution mixed with trypsin (0.25%) into a 100 mm x 20 mm plastic Petri dish. Using sterile forceps, transfer the skins and float them hairy side up on the surface of trypsin solution for 2 h in a 32 °C incubator.
   NOTE: Trypsinization time and temperature are crucial for good yield of highly culturable primary keratinocytes. Although other methods may provide good yields of viable cells, the culturability of the keratinocytes has been less satisfactory.
   1. During this 2 h incubation time, coat dishes with collagen coating and place them at 37 °C for 1 h (see step 3.1.2). For clonogenic assays, the 60 mm Petri dishes have already been coated 24 h prior to keratinocyte harvesting to allow the irradiated 3T3 feeder layer to attach to bottom and spread evenly.
      NOTE: Coating of culture dishes for clonogenic culture is very important for proper attachment, colony formation, and ultimate growth/proliferation of mice epidermal keratinocytes.
5. Epidermal scraping step: Arrange a sterile plastic square Petri dish and create a surface at a 30° incline for proper epidermal scraping with 15 mL of harvesting medium (see Table 1). Carefully remove a floating skin strip from the trypsin solution with curved forceps. With a new scalpel blade, scrape off the epidermis and hairs into the medium.
   1. Do not use excessive force, but use sufficient force to scrape the epidermis, or it will affect the cell viability.
   2. During epidermis scraping, keep the blade perpendicular to the skin piece. If the blade is angled toward the motion of the blade, there are more chances of skin tearing. If the blade is angled away from the blade movement, there are more chances of insufficient epidermis removal.
6. Carefully pour the harvesting medium containing scraped epidermal cells into a sterile 60 mL jar (autoclavable and reusable) with a 1.5 inch magnetic stir bar. Rinse the Petri dish with additional harvesting medium to collect remaining epidermal cells and bring the final volume to 30 mL. Use a magnetic stirrer and stir at 100 rpm for 20 min at room temperature. This process will remove the trapped epidermal cells from hairs.
7. Place a sterile 70 µm cell strainer on the top of a 50 mL conical tube. The cell strainer fits on top of a 50 mL conical tube.
   1. Take the jar inside a biosafety hood. Remove the stir bar and pour the contents into strainer filter attached to a 50 mL conical tube. Strain out undesired hair and sheets of stratum corneum.
   2. Press the hairs along with stratum corneum materials within the strainer and manipulate them to release the trapped hair cells. Use 5 mL of harvesting medium to allow remaining trapped cells to release and flow into the tube. Bring the total volume up to 50 mL in conical tube.
8. Cap the 50 mL conical tube with cell filtrate and centrifuge at 160 x g for 7 min at 4 °C. Next aspirate the supernatant and add 5 mL of harvesting medium. Resuspend the cell pellet by triturating gently ~20x with a 5 mL pipet.
   1. At this step, keep the cells in an icebox to avoid any aggregation. Add 25 mL of harvesting medium and triturate again (20–25x).
   2. To ensure an accurate keratinocyte count at this step, take 1 mL of cell suspension and add 19 mL harvesting medium. Now the cell dilution is 1:20 in 50 mL conical tube. If keratinocytes are harvested from a single mouse, adjust the volume of the cell suspension. Instead of 30 mL, use 15 mL and make a 1:10 dilution for counting the cells.
9. Pipet 0.5 mL of diluted cells from the 50 mL conical tube (1:20 dilution) and transfer to a 1.5 mL autoclaved microcentrifuge tube. Remove 0.2 mL (200 µL) of cell mix to another 1.5 mL microcentrifuge tube and add an equal volume (200 µL) of 0.4% Trypan blue solution. Gently mix this solution 3x and transfer cells to a hemocytometer for counting nucleated keratinocytes.
   1. Score all dark blue cells positive for Trypan blue as non-viable dead cells, and score small gold and pinkish cells as viable cells. The final keratinocyte yield per mouse should be around 30 million viable cells.
10. Centrifuge the original 50 mL conical tube (from step 3.8) for 7 min at 160 x g at 4 °C. At this step, resuspend the cells in desired medium and make the required dilution for seeding cells.
    1. For mass culture, seed 2 to 4 million viable gold cells in each 35 mm Petri dish in cell culture medium (KGM-type medium + supplements for monolayer cultures) (see Table 1). For a clonogenic (colony formation) assay, seed 1,000 cells per 60 mm Petri dish on X-ray irradiated Swiss mouse 3T3 feeder layers with collagen-coating and modified William’s E medium with supplements and serum.
    2. Use a total of 2 to 4 mL medium for 35 mm and 60 mm Petri dishes, respectively. Also, formulate DMEM and Williams E medium procured from ATCC with reduced levels of sodium bicarbonate for use at 5% CO₂.
11. Grow the culture cells (mass or clonogenic) at 32 °C, 95% humidity with 5% CO₂. Change the media one day after initial seeding for mass culture, and then 3x weekly thereafter. However, for clonal assays, the first medium change is 2 days after cell seeding and 3x weekly thereafter.
12. For clonal culture, cultivate cells at two- and four-week intervals. After completion of clonal cultures (2 or 4 weeks), aspirate the medium. Fix the colonies in 10% buffered formalin overnight at room temperature and stain with 0.5% rhodamine B in autoclave water for 1 h. Then, remove the rhodamine B stain with a pipette from the edge of the dishes.
    1. Rinse the dishes in cold autoclaved water until the dishes run clear. Put the dishes inclined on the lid of the dishes and let them dry for final colony counting. Notably, when performing clonogenic culture assay, never pipet <1 mL of cells. If cells are concentrated, serially dilute the cell concentration.