Medium system and method for ex vivo expansion of nk cells

Abstract

This invention relates to a medium system and a method for ex vivo expansion of natural killer (NK) cells. This invention directly cultures Ficoll-separated PBMC by using immobilized anti-CD137 and RetroNectin, and uses OK-432 as a biological effector under the co-existence of GM-CSF, IL-4, IL-2, IL-15, and IL-21 for ex vivo activation and proliferation of NK cells, creating an efficient method for ex vivo expansion of NK cells. The expression rate of NK cells CD3-CD16+/CD56+ prepared by the method is as high as 92.5% or more. After 14 days of culture, NK cells can be expanded 1000 to 2000 times and have strong in vitro cytotoxic activity.

1 - 17 . (canceled) 
     
     
         18 . A culture medium system for ex vivo expansion of natural killer (NK) cells, comprising an induction medium and a proliferation medium, wherein
 (1) the induction medium comprises a basic culture medium and a group of induction factors comprising OK-432, IL-2, IL-15, and IL-21; and   (2) the proliferation medium comprises a basic culture medium and a group of proliferation factors comprising rhGM-CSF and rhIL-4.   
     
     
         19 . The culture medium system of  claim 18 , wherein the induction medium contains OK-432 at a concentration of 1˜1.5 μg/mL, IL-2 at a concentration of 500˜750 IU/mL, IL-15 at a concentration of 15˜30 ng/mL, and IL-21 at a concentration of 45˜60 ng/mL. 
     
     
         20 . The culture medium system of  claim 18 , wherein the proliferation medium contains rhGM-CSF at a concentration of 900˜1200 U/mL, and rhIL-4 at a concentration of 400˜500 U/mL. 
     
     
         21 . The culture medium system of  claim 18 , wherein the induction medium contains OK-432 at concentration of 1 μg/mL, IL-2 at a concentration of 500 IU/mL, IL-15 at a concentration of 20 ng/mL, and IL-21 at a concentration of 50 ng/mL. 
     
     
         22 . The culture medium system of  claim 18 , wherein the proliferation medium contains rhGM-CSF at a concentration of 1000 U/ml and rhIL-4 at a concentration of 500 U/ml. 
     
     
         23 . The culture medium system of  claim 18 , wherein the induction medium contains OK-432 at concentration of 1.5 μg/mL, IL-2 at a concentration of 750 IU/mL, IL-15 at a concentration of 30 ng/mL, and IL-21 at a concentration of 60 ng/mL. 
     
     
         24 . The culture medium system of  claim 18 , wherein the proliferation medium contains rhGM-CSF at a concentration of 1200 U/ml and rhIL-4 at a concentration of 500 U/ml. 
     
     
         25 . The culture medium system of  claim 18 , wherein the induction medium contains OK-432 at concentration of 1 μg/mL, IL-2 at a concentration of 500 IU/mL, IL-15 at a concentration of 15 ng/mL, and IL-21 at a concentration of 45 ng/mL. 
     
     
         26 . The culture medium system of  claim 18 , wherein the proliferation medium contains rhGM-CSF at a concentration of 900 U/ml and rhIL-4 at a concentration of 400 U/ml. 
     
     
         27 . The culture medium system of  claim 18 , wherein the induction medium, the proliferation medium or both further comprise 5% (v/v) autologous serum, 1% (v/v) glutamine and 1% (v/v) non-essential amino acids. 
     
     
         28 . The culture medium system of  claim 18 , wherein at least 90% of the expanded NK cells are CD3-CD16+/CD56+. 
     
     
         29 . The culture medium system of  claim 18 , wherein the number of NK cells after ex vivo expansion is at least 2500×10 6 . 
     
     
         30 . The culture medium system of  claim 18 , wherein the number of NK cells after ex vivo expansion is 1000 to 2000 times higher than the number of NK cells prior to such ex vivo expansion. 
     
     
         31 . The culture medium system of  claim 18 , wherein a population of peripheral blood mononuclear cells (PBMC) is cultured in said induction medium and subsequently cultured in said proliferation medium, thereby obtaining a population of NK cells. 
     
     
         32 . The culture medium system of  claim 18 , wherein:
 a) a population of peripheral blood mononuclear cells (PBMC) is cultured using said induction medium in a first cell culture flask pre-coated with anti-CD137;   b) suspension cells from the first cell culture flask are transferred to a second cell culture flask pre-coated with RetroNectin;   c) adherent dendritic cells in the first cell culture flask are cultured using said proliferation medium; and   d) suspended non-adherent dendritic cells in the first cell culture flask are transferred to the second cell culture flask and cells in said second culture flask are cultured using said proliferation medium for a period of time, thereby obtaining a population of NK cells.   
     
     
         33 . The culture medium system of  claim 18 , wherein:
 a) a population of peripheral blood mononuclear cells (PBMC) in a first cell culture flask pre-coated with anti-CD137 is cultured using said induction medium;   b) suspension cells from said first cell culture flask are transferred to a second cell culture flask pre-coated with RetroNectin;   c) adherent dendritic cells in said first cell culture flask are cultured using said proliferation medium;   d) non-adherent dendritic cells collected from the first cell culture flask after step c) are transferred to the second cell culture flask;   e) cells in said second cell culture flask are cultured using the proliferation medium for a first period of time; and   f) IL-2, IL-15 and IL-21 are added to said second cell culture flask and the cells are cultured for a second period of time, thereby obtaining a population of NK cells.   
     
     
         34 . The culture medium system of  claim 33 , wherein the first cell culture flask is pre-coated with anti-CD137 at a concentration of 5 μg/mL and the second cell culture flask is pre-coated with RetroNectin at a concentration of 5 μg/mL. 
     
     
         35 . The culture medium system of  claim 33 , wherein the first period of time is four to six days, and the second period of time is three days or more. 
     
     
         36 . The culture medium system of  claim 33 , wherein the sum of the first period of time and the second period of time is at least 7 days. 
     
     
         37 . The culture medium system of  claim 33 , wherein the sum of the first period of time and the second period of time is at least 14 days.