NOTE: Not for human or diagnostic use.

Procedure for Use of Human T-Cell Growth Factor (TCGF)

1. Prepare isolated human T-lymphocytes from blood using a cell separation medium such as Histopaque or lymphocytic separation medium (LSM). Wash T-cells 3 times in RPMI-1640 fetal bovine serum 10%.
2. Expose T-cells at a concentration of 1 x 10⁶ cells per mL to a mitogen such as phytohemagglutinin (PHA) to activate cells for TCGF-dependent growth in vitro. Incubate activated cells 5 days at 37°C in 5% CO².
3. Wash cells 3 times in RPMI-1640 fetal bovine serum 10%. Resuspend cells to a concentration of 5 x 10⁵ cells/mL in RPMI-1640 supplemented with 10 to 20% fetal bovine serum and 10% volume of TCGF.
4. To split cultures, pellet cells and resuspend back to 4 to 6 x 10⁵ cells/mL every 3 to 5 days using fresh RPMI-1640 fetal bovine serum 10 to 20% plus a 10% volume of TCGF.
5. To establish long-term cultures of T-lymphocytes, repeat step 4 as necessary.

Production of TCGF

Human T-cell growth factor (TCGF) is prepared from pooled human blood leukocytes, separated on Ficoll-Hypaque and stimulated with purified phytohemagglutinin (PHA). Following induction with PHA, the leukocytes are incubated for 48-72 hours in RPMI-1640 and the crude TCGF fluids harvested.

Purification

Crude TCGF fluids are clarified by centrifugation, purified by chromatographic methods and the purified TCGF sterile filtered into vials.

Quality Control

Each lot of TCGF is screened and found to be free of bacteria, yeasts, molds and mycoplasma by accepted laboratory methods. The product is lectin-free and capable of stimulating long-term growth of adult human T-lymphocytes when assayed by long-term proliferation assays in vitro or by tritiated thymidine uptake.

1. Measure DNA synthesis by tritiated thymidine incorporation assay: 640 half-maximal units/ml.
2. In vitro proliferation assay in lectin activated T-cells: Use of a 10%(V/V) concentration of IL-2 in cell culture growth medium supports a T-cell dependent cell population doubling in 72 hours.