Pluripotent stem cell expansion and passage using a rocking platform bioreactor

Patent metadata

Field	Value
Publication number	US-9944894-B2
Application number	US-201514956408-A
Country	US
Kind code	B2
Filing date	Dec 2, 2015
Priority date	Jan 16, 2015
Publication date	Apr 17, 2018
Grant date	Apr 17, 2018

Abstract

Provided herein are novel methods for expansion and passaging of cell aggregates comprising stem cells and/or differentiated cells and comprising the use of closed systems on rocking platform bioreactors. The methods of the invention permit closed system serial passage expansion of pluripotent stem cells and/or progeny thereof with associated pluripotency markers and differentiation potential.

First claim

The invention claimed is:

1. A method for culturing cells in a closed system, the method comprising:
providing the cells to a culture vessel on a rocking platform bioreactor, wherein the cells are capable of forming cell aggregates in suspension;
expanding the cells in a culture medium to generate the cell aggregates in suspension;
performing an automated perfusion of the culture medium, wherein the automated perfusion is performed in absence of a filtration system while retaining the cell aggregates within the culture vessel;
flowing the cell aggregates across an in-line slicer grid to generate sliced cell aggregates; and
passaging the sliced cell aggregates in the closed system,
wherein the cell aggregates are passed through a mixing device for mixing the cell aggregates prior to flowing the cell aggregates across the in-line slicer grid, and
wherein the mixing device is placed between the culture vessel and the in-line slicer grid in the closed system.

2. The method of claim 1 , wherein the cells are of plant, animal, insect, or microbial origin.

3. The method of claim 1 , wherein the cells comprise pluripotent stem cells or differentiated human cells.

4. The method of claim 1 , wherein the cell aggregates are retained within the culture vessel by allowing the cell aggregates to settle by gravity.

5. The method of claim 1 , wherein the automated perfusion is performed without human intervention.

6. The method of claim 1 , wherein the in-line slicer grid comprises blades that are separated by a distance from about 20 microns to about 500 microns.

7. The method of claim 1 , wherein the in-line slicer grid comprises blades that are separated by a distance of about 100 microns.

8. The method of claim 1 , wherein the in-line slicer grid is coated with or comprises a hydrophobic material.

9. The method of claim 1 , wherein an average diameter of the cell aggregates is no more than 800 microns.

10. A method for culturing cells in a closed system, the method comprising:
providing the cells in a culture vessel on a rocking platform bioreactor, wherein the cells are capable of forming cell aggregates in suspension;
expanding the cells in a culture medium to generate the cell aggregates in suspension;
performing an automated perfusion of the cell culture medium while retaining the cell aggregates within the closed system, wherein the cell aggregates are retained within the closed system by allowing gravity settling of the cell aggregates;
flowing the cell aggregates across an in-line slicer grid to generate sliced cell aggregates; and
passaging the sliced cell aggregates in the closed system, wherein the cell aggregates are passed through a mixing device for mixing the cell aggregates prior to flowing the cell aggregates across the in-line slicer grid, and
wherein the mixing device is placed between the culture vessel and the in-line slicer grid in the closed system.

11. The method of claim 10 , wherein the culture vessel further comprises a tubing assembly comprising a dip tube and a gravity settling chamber.

12. The method of claim 11 , wherein the cell aggregates are retained within the gravity settling chamber.

13. The method of claim 10 , wherein the automated perfusion is performed in the absence of a filtration system.

14. The method of claim 10 , wherein flowing the cell aggregates across the in-line slicer grid is performed at flow rates from about 15 mL/min to about 150 mL/min.

15. The method of claim 10 , wherein the in-line slicer grid comprises blades that are separated by a distance from about 20 microns to about 500 microns.

16. The method of claim 15 , wherein the in-line slicer grid comprises blades that are separated by a distance of about 100 microns.

17. The method of claim 10 , wherein the step of passaging the sliced cell aggregates comprises culturing the sliced cell aggregates in absence of a ROCK inhibitor.

18. The method of claim 17 , wherein the sliced cell aggregates comprise pluripotent stem cells.

19. The method of claim 10 , wherein an average diameter of the cell aggregates is no more than 800 microns.

20. The method of claim 1 , wherein the in-line slicer grid is a polygonal slicer grid.

Assignees

• Gen Electric

Inventors

• Davis Brian Michael
• Conway Kenneth Roger
• Loghin Evelina Roxana
• Zhang Xiaohua

Classifications

• C12M41/48

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12M47/02

  Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02) · CPC title

• C12N5/0607

  Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696) · CPC title

• C12M47/00

  Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass (filters in general B01D24/00 - B01D41/00) · CPC title