B-cell cultivation method

Abstract

Herein is reported a method for co-cultivating one or more B-cells comprising the step of incubating the one or more B-cells with EL4-B5 cells, whereby the EL4-B5 cells have been obtained/are from a cultivation of EL4-B5 cells that has a cell density of from 600,000 cells/ml up to 1,500,000 cells/ml.

1 . A method for co-cultivating one or more B-cells comprising the steps of
 cultivating EL4-B5 cells to a cell density of from 600,000 cells/ml to 1,500,000 cells/ml, and   incubating the one or more B-cells with an aliquot of the EL4-B5 cells obtained in the previous step.   
     
     
         2 . The method according to  claim 1 , wherein the cultivation of the EL4-B5 cells is to a cell density of 650,000 cells/ml to 1,450,000 cells/ml. 
     
     
         3 . The method according to  claim 1 , wherein the cultivation of the EL4-B5 cells is to a cell density of more than 1,000,000 cells/ml to 1,500,000 cells/ml. 
     
     
         4 . The method according to  claim 1 , wherein the cultivation of the EL4-B5 is to a cell density of 650,000 cells/ml to 825,000 cells/ml. 
     
     
         5 . The method according to  claim 1 , wherein the cultivation of the EL4-B5 cells is to a cell density of 1,400,000 cells/ml to 1,500,000 cells/ml. 
     
     
         6 . The method according to  claim 1 , wherein the incubating is additionally in the presence of a feeder mix. 
     
     
         7 . The method according to  claim 6 , wherein the feeder mix comprises one or more of:
 i) interleukin-1 beta and tumor necrosis factor alpha,   ii) interleukin-2 (IL-2) and/or interleukin-10 (IL-10),   iii)  Staphylococcus aureus  strain Cowan's cells (SAC),   iv) interleukin-21 (IL-21) and optionally interleukin-2 (IL-2),   v) B-cell activation factor of the tumor necrosis factor family (BAFF),   vi) interleukin-6 (IL-6),   vii) interleukin-4 (IL-4), and   viii) thymocyte cultivation supernatant.   
     
     
         8 . The method according to  claim 6 , wherein the feeder mix comprises IL-1β, TNF-α, IL-10 and one or more of the members selected from the group consisting of: IL-21, SAC, BAFF, IL-2, IL-4, and IL-6. 
     
     
         9 . The method according to  claim 1 , wherein the EL4-B5 cells are, after the cultivating and prior to the incubating, irradiated with γ-radiation. 
     
     
         10 . The method according to  claim 1 , wherein the method is for the co-cultivation of one B-cell. 
     
     
         11 . The method according to  claim 10 , wherein the one B-cell is a single deposited B-cell. 
     
     
         12 . The method according to  claim 1 , wherein the incubating is for 5 to 14 days. 
     
     
         13 . A method for producing an antibody, the method comprising performing the method according to  claim 1 , thereby producing the antibody. 
     
     
         14 . The method according to  claim 13 , wherein the antibody is produced by the one or more B cells. 
     
     
         15 . The method according to  claim 14 , wherein the antibody is produced by the one or more B cells and secreted into cell culture supernatant. 
     
     
         16 . The method according to  claim 14 , further comprising recovering the antibody from the one or more B cells. 
     
     
         17 . The method according to  claim 15 , further comprising recovering the antibody from the cell culture supernatant.