These cells generate rAAV preparations with high titers of infectious particles which are essentially free of adenoviruses for biological, preclinical, clinical or pharmaceutical applications.

HelaRC32 is a stable packaging cell line expressing the rep and cap genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly which constitutes an attractive alternative to transient transfection protocols.

This is a clone of HeLa cells that harbors one to two rep-cap gene copies per cell. Upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the inverted terminal repeats (ITRs) deleted.

1. Check all containers for leakage or breakage.

2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below --130°C, preferably in liquid nitrogen vapor, until ready for use.

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).

2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 10 minutes.

4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm2 or a 75 cm2 culture flask.  It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product.