The tumor is washed using HBSS/G.
The tumor is then trimmed into small pieces and incubated in a medium containing (DNase) and collagenase at 36 - 38°C for 35-45 minutes.
The cells are centrifuged and the cell pellet is re-suspended in HBSS/G.
Additional dissociation medium is added to the remaining tumor fragments and the dissociation procedure is repeated for a total of three dissociations.
A minimum of nine vials is prepared for controlled rate freezing from +4°C to -90°C with a rate of decrease of 1°C/minute until a temperature of -40°C is reached.
The frozen tumor cells are then stored in quarantine under controlled conditions in the vapor phase of liquid nitrogen at a temperature ≤ -110°C.
Vials are irradiated (200,000 RADS) using a gamma radioactive source.
One vial is used for quality control testing.
Eight remaining vials are maintained in a separate quarantine area of the liquid nitrogen freezer.
Release tests are performed (appearance, tumor cell number and viability, purity, identity, potency, sterility and endotoxins).
The eight vials of patient doses, BCG for the first two vaccinations and paperwork are shipped to the designated pharmacy.