Human Follicle Dermal Papilla Cells (HFDPC) Culture Protocol

Storage

Preparation for Culturing

Culturing HFDPC

Subculturing HFDPC

Materials

I. Storage

A. Cryopreserved Vials (602-05a)

Store the cryovials in a liquid nitrogen storage tank immediately upon arrival.

*Be sure to wear face protection mask and gloves when retrieving cryovials from the liquid nitrogen storage tank. The dramatic temperature change from the tank to the room could cause any trapped liquid nitrogen in the cryovials to burst and cause injury.

II. Preparation for Culturing

1. Ensure the Class II Biological Safety Cabinet, with HEPA filtered laminar airflow, is in proper working condition.
2. Sterilize the Biological Safety Cabinet with 70% alcohol.
3. Turn the Biological Safety Cabinet blower on for 10 minutes before beginning cell culture work.
4. Make sure all serological pipettes, pipette tips and reagent solutions are sterile.
5. Follow the standard sterilization technique and safety rules:
   a.    Do not pipette by mouth.
   b.    Always wear gloves and safety glasses when working with human cells even though all the strains have been
          tested negative for HIV, Hepatitis B and Hepatitis C.
   c.    Handle all cell culture work in a sterile hood.

III. Culturing HFDPC

A. Preparing Cell Culture Flasks for Culturing HFDPC

1. Take the Collagen Coated T-75 Flask (125-75) and Hair Follicle Dermal Papilla Cell Growth Medium (611-500) from the refrigerator and allow them to equilibrate to room temperature.
2. Decontaminate the bottle and flask with 70% alcohol in a sterile hood.
3. Pipette 15 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500)* to the Collagen Coated T-75 Flask (125-75).
   *Keep the medium to surface area ratio at 1-1.5mL per 5 cm².
   -  5 mL for a T-25 flask (SIAL0639) or a 60 mm tissue culture dish (SIAL0166).
   -  15 mL for a T-75 flask (SIAL0641) or a 100 mm tissue culture dish (SIAL0167).

B. Thawing and Plating HFDPC

1. Prepare a 15 mL sterile conical tube for washing HFDPC by adding 12 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500).
2. Remove the cryopreserved vial of HFDPC from the liquid nitrogen storage tank using proper protection for your eyes and hands.
3. Thaw the cells quickly by placing the lower half of the vial in a 37 °C water bath and watch the vial closely during the thawing process.
4. Take the vial out of the water bath when only small amount of ice left in the vial. Do not let cells thaw completely.
5. Take the vial out of the water bath and wipe dry.
6. Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet.
7. Remove the vial cap carefully. Do not touch the rim of the cap or the vial.
8. Disperse the cells in the vial by gently pipetting the cells 5 times with a 2 mL pipette. Be careful not to pipette too vigorously as to cause foaming.
9. Pipette the cell suspension (1 mL) from the vial into the prepared 15mL conical tube gently.
10. Centrifuge the cells at 200 x g for 5 minutes at room temperature to pellet the cells.
11. Aspirate the supernatant from the tube without disturbing the cell pellet.
12. Resuspend the cells in 2 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500) by gently pipetting the cells to break up the clumps.
13. Transfer the 2mL cell suspension to the Collagen Coated T-75 Flask (125-75) containing 15 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500).
14. Replace the cap tightly and swirl the flask gently to distribute the cells evenly in the flask.
15. Place the T-75 flask in a 37 ^oC, 5% CO₂ humidified incubator. Loosen the cap to allow gas exchange.
16. Change Hair Follicle Dermal Papilla Cell Growth Medium (611-500) every other day until the cells reach 60% confluent.
17. Double the Hair Follicle Dermal Papilla Cell Growth Medium (611-500) volume when the culture is >60% confluent or for weekend feedings.
18. Subculture the cells when the HFDPC reach 80% confluent.

IV. Subculturing HFDPC

A. Preparing Subculture Reagents

1. Remove the Trypsin-EDTA solution (T3924) and Trypsin Inhibitor (T6414) from the -20 °C freezer and thaw overnight in a refrigerator.
2. Make sure all the subculture reagents are thawed. Swirl each bottle gently several times to form homogeneous solutions.
3. Store all the subculture reagents at 4 °C for future use.
4. Aliquot Trypsin/EDTA solution (T3924) and store the unused portion at -20 °C if only a portion of the Trypsin/EDTA (T3924) is needed.

B. Preparing Culture Flask

1. Swirl the Collagen Coating Solution (125-50) bottle a few times to form a homogenous solution.
2. Take the Hair Follicle Dermal Papilla Cell Growth Medium (611-500) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
3. Add 10 mL of Collagen Coating Solution (125-50) to a T-225 flask (CLS431082) and rock the flask gently to distribute the solution evenly over the whole culture surface.
4. Coat the culture ware for 1-2 hours at room temperature.
5. Remove the Collagen Coating Solution (125-50) by aspiration in a sterile hood.
6. Wash the coated flask three times with Dulbecco’s Phosphate Buffered Saline (D8537) and aspirate completely from the flask in the last wash. The coated flask can be used immediately or stored in a sealed bag at 4 °C for up to 2 weeks.
7. Prepare the coated flask for subculturing by pipetting 45 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500) into this coated T-225 flask (CLS431082) and wait to be seeded.

B. Subculturing HFDPC

Trypsinize Cells at Room Temperature. Do Not Warm Any Reagents to 37 °C.

1. Remove the medium from culture flasks by aspiration.
2. Wash the monolayer of cells with HBSS (H6648) and remove the solution by aspiration.
3. Pipette 5 mL of Trypsin/EDTA Solution (T3924) into the T-75 flask. Rock the flask gently to ensure the solution covers all the cells.
4. Remove 4.5 mL of the solution immediately.
5. Re-cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope. It usually takes about 1 to 3 minutes for the cells to become rounded.
6. Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached.
7. Pipette 5 mL of Trypsin Inhibitor Solution (T6414) to the flask to inhibit further tryptic activity.
8. Transfer the cell suspension from the flask to a 50 mL sterile conical tube.
9. Rinse the flask with an additional 5 mL of Trypsin Inhibitor Solution (T6414) and transfer the solution into the same conical tube.
10. Examine the T-75 flask under a microscope. If there are >20% cells left in the flask, repeat Steps 2-9.
11. Centrifuge the conical tube at 220 x g for 5 minutes to pellet the cells.
12. Aspirate the supernatant from the tube without disturbing the cell pellet.
13. Flick the tip of the conical tube with your finger to loosen the cell pellet.
14. Resuspend the cells in 2 mL of Hair Follicle Dermal Papilla Cell Growth Medium (611-500) by gently pipetting the cells to break up the clumps.
15. Count the cells with a hemocytometer or cell counter. Inoculate at 10,000 cells per cm² for rapid growth, or at 6,000 cells per cm² for regular subculturing.