Human Mononuclear Cells – Periphery Blood (HMNC-PB) Culture Protocol

I. Storage

A. Cryopreserved Vials (690PB-100a)

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 HMNC-PB

A. Preparing Cell Culture Flasks for Culturing HMNC-PB

1. Take the Blood Cell Growth Medium (615-250) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
2. Pipette 9 mL of Blood Cell Growth Medium (615-250) to a 15 mL conical tube.
3. Pipette 15 mL of Blood Cell Growth Medium (615-250)* to a T-75 flask (SIAL0641).
   * Keep the medium to surface area ratio at 1mL per 5 cm².
   For example:
   -  7.5 mL for a T-25 flask (SIAL0639) or a 60 mm tissue culture dish (SIAL0166).
   -  22.5 mL for a T-75 flask (SIAL0641) or a 100 mm tissue culture dish (SIAL0167).

B. Thawing and Plating HMNC-PB

1. Remove the cryopreserved vial of HMNC-PB from the liquid nitrogen storage tank using proper protection for your eyes and hands.
2. Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
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. Remove the vial from the water bath when only a small amount of ice is left in the vial. Do not let cells thaw completely.
5. Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet.
6. Remove the vial cap carefully. Do not touch the rim of the cap or the vial with your hands to avoid contamination.
7. Resuspend 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.
8. Transfer the cell suspension to the 15 mL conical tube prepared in Section IIIA Step 2.
9. Centrifuge at 400 x g for 5 minutes to pellet the cells.
10. Aspirate the supernatant from the tube without disturbing the cell pellet.
11. Flick the tip of the conical tube with your finger to loosen the cell pellet.
12. Resuspend the HMNC in 5 mL of Blood Cell Growth Medium (615-250) by gently pipetting the cells.
13. Transfer 5 mL of HMNC to the T-75 prepared in Section IIIA Step 3. HMNC should float in the Blood Cell Growth Medium (615-250).
14. Incubate HMNC in a 37 ^oC, 5% CO₂ humidified incubator.
15. HMNC can be cultured for 3-4 days.