CHOLESTEROL-BASED MEDIA SUPPLEMENTS FOR CELL CULTURE
The disclosure provides compositions and methods for high density cell culture and banking of cholesterol auxotrophic cells.
1 . A method, comprising:
culturing a population of cholesterol auxotrophic cells in a bioreactor that contains culture medium supplemented with a composition, the composition comprising
cholesterol associated with a carrier, and
free cholesterol,
wherein the ratio of free cholesterol to carrier-associated cholesterol is at least 1:5.
2 - 3 . (canceled)
4 . A method, comprising:
providing a population of cholesterol auxotrophic cells; and
culturing the cells in a bioreactor comprising culture medium supplemented with a composition, the composition comprising (a) cholesterol, cyclodextrin, lipids and ethanol, or (b) cholesterol complexed with cyclodextrin, free cholesterol and ethanol, wherein the ratio of complexed cholesterol:free cholesterol is about 1:12 to about 1:2.
5 . The method of claim 4 , wherein the ratio of complexed cholesterol:free cholesterol is about 1:8.
6 . The method of claim 4 , wherein the concentration of cholesterol complexed with cyclodextrin is about 2.5 to about 5 mg/L.
7 . (canceled)
8 . The method of claim 4 , wherein the concentration of free cholesterol is about 10 to about 20 mg/L.
9 . (canceled)
10 . The method of claim 4 , wherein the free cholesterol is synthetic cholesterol.
11 . The method of claim 4 , wherein the cholesterol in (i) is synthetic cholesterol.
12 . The method of claim 4 , wherein the cyclodextrin is methyl-β-cyclodextrin (mβCD).
13 . The method of claim 4 , wherein the cholesterol auxotrophic cells are NS0 cells.
14 . The method of claim 4 , wherein the bioreactor is a disposable bag.
15 . The method of claim 4 , wherein the bioreactor is a polymer-based bioreactor.
16 . The method of claim 15 , wherein the polymer-based bioreactor comprises linear low-density polyethylene (LLDPE).
17 . The method of claim 4 , further comprising one or more lipid(s).
18 . The method of claim 17 , wherein the one or more lipid(s) is/are selected from oleic acid and linoleic acid.
19 . The method of claim 4 , wherein the cells are cultured to a density of about 2×10 5 viable cells/ml to about 3×10 7 viable cells/ml.
20 . The method of 19 , wherein the cell viability is about 70% to about 100%.
21 . (canceled)
22 . The method of claim 4 , wherein the population of cells comprises recombinant cells expressing one or more gene(s) encoding one or more protein(s).
23 . The method of claim 22 , wherein the one or more protein(s) is a monoclonal antibody.
24 . The method of claim 23 , wherein the monoclonal antibody is natalizumab.
25 - 26 . (canceled)
27 . Cell culture medium or supernatant produced by the method of claim 1 .
28 - 29 . (canceled)
30 . A composition comprising cholesterol, cyclodextrin, lipids and ethanol.
31 - 40 . (canceled)
41 . A method, comprising:
dissolving about 10 mg/ml synthetic cholesterol, about 1 mg/ml oleic acid, and about 1 mg/ml linoleic acid in absolute ethanol to form a solution; and
adding to the solution cholesterol complexed with methyl-β-cyclodextrin (mβCD) at a concentration of about 2.2 to about 2.5 mg/ml.