Tissue: Blood
Donor History: Normal tissue
Growth Properties: Suspension, round
Storage Condition: Vapor phase of liquid nitrogen, or below -130°C.
Shipping Conditions: Ship with dry ice.
Product Format: Frozen
Intended Use: This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
BioSafety: II
Growth Conditions: PriCoat™ T25 Flasks (G299) are recommended for optimal cell culture. PriGrow X Series Medium (TM4121) + 1% Penicillin/Streptomycin Solution (G255), +37.0°C, 5% CO₂.
Unpacking and Storage Instructions:
1. Visually examine the packaging containers for signs of leakage or breakage.
2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use.
To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability.
Thawing Protocol:
1. Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.
2. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.
3. Transfer the cell suspension into a 15ml sterile conical tube containing 10ml of pre-warmed, complete growth media. Centrifuge cells at 300xg for 10 minutes.
4. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and perform a cell count.
6. Refer to the "Subculturing Protocol" Section for information regarding T Cell Expansion.
Subculture Protocol
To expand T cells:
1. Seed 5.0x105 cells in a 24-well plate with 1ml of TM4121 supplemented with 100 IU/mL IL-2.
2. Activate T cells with antibodies capable of binding CD3 and CD28 as per manufacturer protocols.
3. Incubate the cells at the recommended conditions for up to 3 days.
4. On day 2 or 3, transfer cells into larger culture vessels, as per cell density. Centrifugation is not required; simply transfer the culfutre suspension and add additional media as required. Supplement with additional IL-2 to a final concentration of 100 IU/mL.
5. Perform a viabile cell count on day 5 and subculture cells as per steps 1 to 3, above.
6. Continue to expand T-cells by repeating the subculture procedure every 3 days. Do not wait longer than 3 days between subcutlures.
7. Cells are expected to expand for 9-12 days upon activation before entering a resting phase.
Cryopreservation We recommend using serum-free CryoGuard™ Freezing Media (TM078).