Cell culture medium and bioprocess optimization

Patent metadata

Field	Value
Publication number	US-2016251614-A1
Application number	US-201415033531-A
Country	US
Kind code	A1
Filing date	Nov 3, 2014
Priority date	Nov 1, 2013
Publication date	Sep 1, 2016
Grant date	—

Abstract

The invention provides a chemically defined cell culture media and methods of using the media. The invention also provides an inverse screen to identify small molecules and synergies stimulating proliferation in a chemically defined medium. In this chemical-genetics approach, a compound-protein interaction data-base is used to systematically score genetic targets on a screen-wide scale to extract further information about cell growth. Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). Polyamines were identified as important components that enables the CDM to support the long-term maintenance of C1.8 cells and Kc cells (such as Kc167 cells). Our cumulative target scoring approach improves on traditional chemical-genetics methods and is extensible to biological processes in other species.

First claim

1 . An aqueous chemically defined media for supporting long-term growth of Drosophila cell lines, the media comprising ZO media supplemented with insulin, a disaccharide, ascorbic acid, and at least one of glutamine and glutamate, wherein the pH of the media is about 6 to about 8.

2 . The media of claim 1 comprising about 95% to about 99% water.

3 . The media of claim 1 wherein the insulin is present at a concentration of at least about 1 ng μg/mL.

4 . The media of claim 1 wherein the disaccharide is present at a concentration of at least about 10 mM.

5 . The media of claim 1 wherein the ascorbic acid is present at a concentration of at least about 40 ng/mL.

6 . The media of claim 1 wherein the ascorbic acid is in the form of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

7 . The media of claim 1 wherein the glutamine is in the form of a dipeptide.

8 . The media of claim 1 wherein at least one of glutamine and glutamate is present at a concentration of at least about 5 μg/mL.

9 . The media of claim 1 wherein the pH is about 6.5 to about 7.3.

10 . The media of claim 1 further comprising a polyamine compound.

11 . The media of claim 10 wherein the polyamine compound is present at a concentration of at least about 0.25 μM.

12 . The media of claim 11 wherein the polyamine compound is present at a concentration of about 0.5 μM to about 20 μM.

13 . The media of claim 10 wherein the polyamine compound is spermidine, spermine, or putrescine.

14 . The media of claim 1 further comprising a BTK inhibitor.

15 . The media of claim 10 further comprising a BTK inhibitor.

16 . The media of claim 14 wherein the BTK inhibitor is terreic acid or LFM-A13 (α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide).

17 . The media of claim 1 wherein the media comprises insulin at a concentration of about 3-10 μg/mL, trehalose at a concentration of about 20-30 mM, L-alanyl-L-glutamine at a concentration of about 10-20 μM, and L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate at a concentration of about 50-150 nM.

18 . The media of claim 17 wherein the pH is about 6.7 to about 6.8, the media comprises a polyamine at a concentration of about 0.5 μM to about 20 μM, and the polyamine is spermidine.

19 . (canceled)

20 . (canceled)

21 . An aqueous serum-free chemically defined media for supporting long-term growth of Drosophila cell lines, the media consisting essentially of ZO media supplemented with insulin at a concentration of about 1 ng/mL-10 μg/mL, trehalose at a concentration of about 20-30 mM, L-alanyl-L-glutamine at a concentration of about 1-20 μM, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate at a concentration of about 40-150 nM, and at least one of glutamine and glutamate at a concentration of about 1 μM to about 20 μM, wherein the pH of the media is about 6 to about 8.

22 . A method for screening small molecule compounds comprising:
a) combining Drosophila cells, one or more test compounds, and a Drosophila cell media, wherein the Drosophila cell media is an aqueous chemically defined media comprising ZO media supplemented with insulin, a disaccharide, L-ascorbic acid 2-phosphate, and at least one of glutamine and glutamate, wherein the pH of the media is about 6 to about 8; b) analyzing the cells after a period of time to score the screen for increases or decreases in the number of cells that are in the presence of the one or more test compounds to obtain compound scores (z i ); c) identifying test compounds that increase the number of Drosophila cells in the media and test compounds that modify the number of Drosophila cells in the media, change the morphology of the cells, or affect the amount of cell signaling as measured by reporter genes or chemical dyes, thereby identifying compounds of interest; d) querying the Search Tool for Interactions of Chemicals (STITCH) database for all test compounds to determine test compound-protein interaction scores (s i ); and e) optionally identifying species homologs and percent identity matches (q i ) for orthologous Drosophila proteins to identified mouse or human protein-drug interactions; thereby translating screen wide compound proliferation data into biologically relevant protein data by identifying significant protein targets (p-values) from the protein score, Σ(s i ·q i ·z i 2 ), which follows a chi-squared distribution with degrees of freedom n.

23 .- 28 . (canceled)

Assignees

• Univ Notre Dame Du Lac

Inventors

• Zartman Jeremiah
• Burnette Miranda
• Li Jun
• Brito-Robinson Teresa

Classifications

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• G06F19/18

  Physics · mapped topic

• C12N2500/38

  Vitamins · CPC title

• C12N2500/32

  Amino acids · CPC title

• G06F19/28

  Physics · mapped topic