A Novel Process Technology for Automated NK Cell Culture and Enrichment

Natural killer (NK) cells are a promising tool for cancer therapy due to their ability to detect and eliminate cancer cells. Using NK cells for therapeutic cell transfer in the clinic requires high numbers of these cells. Therefore a method for automated expansion of human NK cells is developed.

Automation of NK cell expansion was done by use of the CliniMACS^® Prodigy instrument, a novel technology for cell processing in a clinical environment. Automated Ficoll density gradient centrifugation was performed to remove erythrocytes and granulocytes from buffy coat samples of healthy donors and to obtain peripheral blood mononuclear cells (PBMC). This automated method yielded a suitable number of PBMC and a better depletion of granulocytes compared to the manual PBMC preparation. PBMC were cultivated with IL-2 and OKT-3 leading to an expansion of NK cells, but also of NK like T cells and T cells. NK cell numbers up to 5,1x10⁸were reached with expansion factors of 54-208-fold after three weeks. NK cell purities of 5-52% were obtained.

Pure NK preparations are preferred with regard to analysis of clinical outcome and to reduce the risk of graft versus host disease possibly caused by contaminating T cells.

Removing co-cultured T and NK like T cells by CD3-depletion during the expansion phase was possible and resulted in a higher purity of NK cells (56 - 96%). The cells retained their proliferative capacity after depletion and expansion factors of 30-156-fold were obtained.

Flow cytometry analysis revealed comparable expression of CD16, NKp30, NKp46 and NKG2D for manually or automatically expanded NK cells either from CD3-depleted or undepleted cultures.

Results show that large scale automation of NK cell expansion is possible by use of the CliniMACS^® Prodigy. CD3 depletion of unwanted T and NK like T cells during culture is feasible.