Isolation, Primary Culture, and Cryopreservation of Human Keratinocytes

Objective

A recommended procedure to isolate and establish a primary culture of human keratinocytes from foreskin tissue is described below. Subculture and cryopreservation procedures are also included.

Introduction

Primary human fibroblasts from skin (dermis) are useful for a number of scientific endeavors including the study of growth factor action, wound healing, toxicity/irritancy studies, and use as target cells for derivation of induced pluripotent stem cells. We offer a complete range of products for the isolation, growth, and cryopreservation of these cells in defined animal origin free or defined animal-containing conditions. The following protocol describes the isolation of cells from neonatal tissue (approximately 2 cm²). If isolation of keratinocytes from adult skin is desired, scale the protocol appropriately for the amount of tissue to be processed. The yield of epidermal cells from neonatal or adult tissue is similar (1–3 × 10⁶ cells/cm²).

• EpiLife (Cat. no. M-EPI-500-CA)
• S7 Supplement Defined, Animal Origin Free (AOF) (Cat. no. S-017-5)
• EpiLife Defined Growth Supplement (EDGS), defined, animal-containing supplement (Cat. no. S-012-5)
• Dispase Solution (Dispase in PBS pH 7.4 without Ca/Mg, 25 U/mL final) (Cat. no. 17105-041 and 10010-023)
• Antibiotic-Antimycotic 100X, Liquid (AA) (Cat. no. 15240-062)
• Trypan blue solution (Cat. no. 15260-061)
• Recombinant Trypsin/EDTA (rTE) (Cat. no. R-009-50)
• Defined trypsin inhibitor (DTI) (Cat. no. R-007-100)
• Coating Matrix Kit (Cat. no. R-011-K)
• Synth-a-Freeze (SAF) (Cat. no. R-005-50)
• Sterile forceps, scissors, and scalpel
• Absorbent underpads
• 15 mL and 50 mL conical centrifuge tubes, sterile
• 100 mm and T-75 sterile plastic culture dishes and culture flasks
• Sterile, individually-wrapped pipettes
• Sterile Pasteur pipettes

Preparing Tissue

1. To prepare animal origin-free, defined supplemented medium, add the following to one 500 mL bottle of EpiLife medium:

• S7 Supplement - 5 mL
• Antibiotic-Antimycotic 100X, Liquid 100X (AA) - 5 mL

To prepare defined (animal product-containing) supplemented medium, add the following to one 500 mL bottle of EpiLife medium 

• EpiLife Defined Growth Supplement (EDGS) - 5 mL
• Antibiotic-Antimycotic 100X, Liquid 100X (AA) - 5 mL

2. Place an absorbent underpad in the hood.
3. To a sterile 100 mm culture dish, add ~10 mL of the supplemented medium prepared in Step 1.
4. Obtain tissue and place the container with the tissue in the laminar flow hood. Dispose of any accompanying medium from the collection vessel by aspiration.
5. Remove the lid from the 100 mm dish and place it upside down in the hood for use in Step 8, below.
6. Using sterile forceps transfer the tissue to the culture dish prepared in Step 3.
7. Wash the tissue by agitating with forceps in the medium contained in the 100 mm dish.
8. Using sterile forceps, place and flatten the tissue onto the overturned lid of the 100 mm culture dish, epidermal side down. If the tissue is in a tubular configuration, use small sterile scissors to open the tissue and flatten onto the lid.
9. Trim away any fat and loose fascia using scissors and forceps. To keep the tissue from drying, rinse every few minutes in the medium in the 100 mm dish.
10. After the trimming is complete, cut the tissue into strips approximately 0.5 cm × 1.5 cm using a sterile scalpel.

Dispase Digestion

1. Add 5 mL Dispase Solution to a sterile 15 mL conical centrifuge tube.
2. Transfer the cut tissue to the tube containing 5 mL of Dispase Solution. Ensure the tissue pieces are submerged in solution. Cap the tube securely.
3. Remove outer gloves and wipe the outside of the tube with tuberculocidal solution. Label the tube appropriately.
4. Transfer the tube to a 4°C refrigerator.
5. Incubate for 16–21 hours at 4°C.

Isolating and Plating Epidermal Cells

1. After Dispase digestion, retrieve the tube containing the tissue and place the tube in the hood.
2. Obtain a sterile 100 mm culture dish, and remove and place the lid upside down in the hood.
3. Transfer the digested tissue and accompanying Dispase Solution into the bottom of the 100 mm culture dish, avoiding splashing. If any pieces of tissue remain in the bottle, use a sterile 1 mL pipette or sterile forceps and transfer the tissue pieces in the bottom of the100 mm culture dish.
4. Separate the epidermis from the dermis.

• Handling a few pieces at a time, move the tissue pieces to the overturned lid of the dish. Orient the strips so the epidermis is facing up.
• Hold the dermis of the tissue strip with one pair of sterile forceps and the edge of the epidermis with another pair of sterile forceps. Pull/peel the dermis and epidermis apart keeping pieces separated on the same lid. Working quickly, repeat the process for each tissue piece.

5. Add 5 mL rTE solution into a sterile 15 mL conical tube.
6. When all the pieces are separated, place the epidermal pieces into the tube containing 5 mL rTE solution. Cap the tube securely and wipe the outside with tuberculocidal solution. Label the tube appropriately. To isolate and culture dermal fibroblasts, refer to Isolation, Primary Culture, and Cryopreservation of Human Neonatal Fibroblasts protocol.
7. Incubate the epidermal pieces in the rTE solution for 30 minutes at 37°C in a water bath.
8. Gently agitate the tube every 5 minutes during the incubation.
9. Remove the tube from the water bath at the end of the 30 minute incubation, dry the outside of the tube, and agitate vigorously. Transfer the tube back to the culture hood.
10. Neutralize the rTE by adding 5 mL of DTI to the cell suspension using a 10 mL pipette and pipette the suspension up and down at least 6 times using the same pipette to release the epidermal cells.
11. Remove the cap from a 50 mL conical tube (keep the cap sterile) and insert a 70 μm cell strainer (Cat. no. 352350 from BD Biosciences) into the top of the tube. Pass the cell suspension through the cell strainer into the 50 mL conical tube. 
12. Rinse the tube with an additional 5 mL of DTI to collect any remaining cells and epidermal strips. Pass through the cell strainer.
13. Remove and discard the strainer. The volume of the cell suspension in the 50 mL conical tube is now ~15 mL.
14. Pellet the cells by centrifugation at 180 × g for 7–10 minutes.
15. Carefully aspirate the supernatant without dislodging the cell pellet.
16. Resuspend the cell pellet in 10 mL supplemented EpiLife prepared in Step 1. Pipette up and down 7-10 times with a 10 mL pipette to ensure a uniform cell suspension.
17. Determine the concentration of viable cells/mL and calculate the culture surface area required for treatment with Coating Matrix. Place the remaining cell suspension at 4ºC until needed.
    
    
    • Add a 20 µL aliquot of the cell suspension from Step 16 to a sterile tube containing 20 µL of Trypan Blue solution and determine the total number of viable cells in the preparation using a hemocytometer.
    • For epidermal cells isolated from adult tissue, plate 2 × 10⁴ viable epidermal cells/cm²; or from neonatal tissue, plate 4 × 10³ viable epidermal cells/cm².
    
    
18. Determine the number of flasks needed for primary culture and prepare the culture surface with Coating Matrix as described below.
    
    
    • Obtain and label the required number of flasks.
    • You need one Coating Matrix kit per every 750 cm² of surface to be coated.
    • Add 67 µL of Dilution Medium for each cm² to be coated. For example, add 5 mL to a T-75 flask.
    • Add 0.67 µL Coating Matrix for each cm² to be coated directly to the Dilution Medium in each flask. For example, add 50 µL to each T-75 flasks containing 5 mL of Dilution Medium.
    • Swirl flasks thoroughly to mix and coat the surface of each flask. Incubate at room temperature for 30 minutes.
    • Aspirate the Coating Matrix solution from flasks using a Pasteur pipette under vacuum.
      
      
19. Retrieve the epidermal cell suspension from 4°C.
20. Dilute the cell suspension in supplemented EpiLife medium to the appropriate volume and density of viable cells for seeding primary cultures and seed coated flasks prepared in Step 18.
21. Rock each flask back and forth to ensure that the surface of the flask is uniformly covered, and incubate in a 37°C, 95% air/5% CO₂ saturated humidity chamber. Allow the primary culture to incubate undisturbed for at least 48 hours.

Primary Culture

Change the culture medium after 48–72 hours of initial incubation and then at least once every 48 hours thereafter. Use the appropriate medium prepared above for medium changes.

Once the cultures are ~50% confluent, feed at least 15 mL medium per T-75 flask every day. Once the cultures are ~80% confluent, subculture and/or cryopreserve the cells using Synth-a-Freeze® (SAF) cryopreservation medium as described below.


Subculture of Primary Keratinocytes

Use the following protocol to harvest keratinocytes from primary cultures as primary cultures of human keratinocytes are particularly difficult to release from the culture surface. The protocol described below is for a 75 cm² flask. If differently sized vessels are used, modify the protocol proportionately.

1. Remove the medium from the flask and add 3 mL rTE to the cell layer, incubate at room temperature for 1 minute.
   
   Note:   It is not necessary to rinse the cell layer with PBS first because the supplemented keratinocyte media do not contain serum and have very low concentrations of Ca++.
   
   
2. After 1 minute, remove the rTE solution and replace with 3 mL fresh rTE solution. Incubate at room temperature for an additional 2 minutes.
3. Remove the rTE added in Step 2 and replace with 3 mL fresh rTE.
4. Continue incubation at room temperature for a total of 10 minutes.
5. Observe the cells under a microscope. When the cells have become completely rounded (between 12–15 minutes), release the cells from the surface of the flask by holding the flask in one hand and gently tapping the side of the flask with the knuckle of the other hand. If the cells do not release from the surface, continue the incubation for an additional two minutes and try again. Do not try to release the cells by forceful rapping against your hand or other object. Using unnecessary force may cause cell lysis.
6. Once the cells have released from the flask, add 5 mL DTI solution to the flask and pipette the solution over the surface of the flask to suspend the cells and break up clumps of cells.
7. Transfer the cell suspension to a 15 mL conical centrifuge tube. Repeat Steps 6–7.
8. Pellet the cells by centrifugation at 180 × g for 7–10 minutes.
9. Carefully aspirate the supernatant without dislodging the cell pellet.
10. For subculture, resuspend the cell pellet in 10 mL supplemented EpiLife medium and determine the concentration of the cells using a hemocytometer. Seed new flasks at 5 × 10³ cells/cm².

Cryopreservation of Primary Keratinocytes

1. Follow Steps 1–9 in Subculture of Primary Keratinocytes.
2. Resuspend the cell pellet in 3–5 mL Synth-a-Freeze cryopreservation solution.
3. Determine the concentration of the cells using a hemocytometer.
4. Cryopreserve cells at a density of 0.5–2 × 10⁶ cells/mL using a controlled-rate freezer or other appropriate device, then transfer to liquid nitrogen storage (vapor phase).