American Fluoroseal Corporation

There is considerable interest in autologous cytotoxic cells for adoptive cellular immunotherapy of cancer. However, isolation and expansion of primary cytotoxic cells is technically difficult. Alternatively, some groups have proposed using cytotoxic cell lines such as NK-92, for immunotherapy. 

NK-92 is a highly cytotoxic natural killer cell with significantly higher cytolytic activity against a broader target range than lymphokine activated killer cells and which can be easily cultured in vitro.

A clinical trial in cancer patients is currently underway in Europe and clinical trials for in vivo therapy and ex vivo purging are under consideration at the FDA. In this work, we describe large-scale, clinical expansion of this cell line under GMP conditions. Optimal expansion of NK-92 (maximum density: 0.8-1.0 x 106 cells/ml) was achieved using X-Vivo 10 (BioWhittaker) media supplemented with 500 U/ml of rhIL-2 (Proleukin, Chiron).

Addition of 1-2.5% human serum and amino acids (L-asparagine, L-glutamine and L-serine) resulted in a 20-75% increase in maximum density.

Cells could be effectively cultured in gas-permeable bag culture system, allowing for expansion in a completely "closed" system.

Optimal expansion was seen with VueLife (Afc Corp) and X-Fold (Nexell Corp) culture systems (maximum density: 1.0-1.2 x 106/ml). Under culture/feeding conditions described, >109 cells/liter can be obtained and NK-92 can be expanded to > 1010 cells per incubator without any specialized equipment.

Expanded NK-92 exhibit high viability (80-95%) and maintain their high cytolytic activity. It is feasible, using the conditions defined here, to achieve large-scale expansion of NK-92 under GMP conditions for clinical immunotherapy