Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

Castiglia S, Adamini A, Rustichelli D, Castello L, Mareschi K, Pinnetta G, Leone M, Mandese A, Ferrero I, Mesiano G, Fagioli F. 
Source: J Transl Med
Publication Date: (2018)
Issue: 16: 237
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:

Comparison of different media to expand t cells over a time period of 20 days. X-vivo 15 , TexMacs and cell genix were compared and checked for viability,  identity and fold increase. No FBS, HS or HSA has been added

CIK (CD3+CD56+)  generation: 30×10exp6 of cells were seeded in 15 ml of culture medium. We cultured the CIKs in T75 fasks (Corning Incorporated, NY, USA).  

At Day 0, 1000  U/ml IFN-? was added (Boehringer Ingelheim, Vienna, Austria).
At Day 1, 50  ng/ml CD3 Pure, which is IgG anti-CD3 (OKT-3) (170-076-124, Miltenyi Biotec, Bergisch Gladbach, Germany), and 300 IU/ml of Proleukin which is IL-2 cytokines (Novartis, Origgio, VA, Italy) were added [4].
Fresh medium and Proleukin (300 U/ml) were added weekly (every 3  days) during culture, and the cell concentration was maintained at 1-1.5×106 cells [17]. The fold increase of CIKs was calculated as follow: (%CD3+×total cells counted at Day 20–21)/(%CD3+×seeded cells at Day 0). After 20–21  days, CIK cells culture was stopped and the quality controls were performed (cell count and viability, immunophenotype and sterility).


Cytokine-induced killer (CIK) cells are a very promising cell population raising growing interest in the field of cellular antitumor therapy. The aim of our study was to validate the most advantageous expansion method for this advanced therapy medicinal product (ATMP) and to translate it from preclinical field to good manufacturing practices (GMP). GMP ensures that ATMP are consistently produced and controlled to the quality standards required to their intended use. For this reason, the use of the xenogenic sera tended to be minimized by GMP for their high variability and the associated risk of transmitting infectious agents.