Pluripotent stem cell expansion and passage using a stirred tank bioreactor

• US 10,100,277 B2
• Filed: 03/21/2016
• Issued: 10/16/2018
• Est. Priority Date: 01/16/2015
• Status: Active Grant

• Current Assignee: Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Davis, Brian Michael, Conway, Kenneth Roger, Zhang, Xiaohua
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US15/075,211
• Publication Number:
  US 20160215257A1
• Time in Patent Office: 939 Days
• CPC Class Codes: C12M 27/02 Stirrer or mobile mixing elements
• CPC Class Codes: C12M 27/16 Vibrating; Shaking; Tilting
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 33/14 with filters, sieves or membranes
• CPC Class Codes: C12M 33/22 Settling tanks; Sedimentation by gravity settling tanks per se B01D21/02
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12M 45/02 by mechanical forces; Stirring; Trituration; Comminuting crushing, pulverizing, disintegrating in general B02C
• CPC Class Codes: C12M 47/00 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 Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

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 in stirred tank 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.

Claims

1. A method for culturing cells in a closed system, where the closed system comprises a cell culture vessel, a tubing assembly, a device for mixing cell aggregates, and a slicer grid, the method comprising:

providing the cells to the cell culture vessel;

expanding the cells in the cell culture vessel to generate the cell aggregates in the cell culture vessel;

conducting an automated perfusion of the cell aggregates in the cell culture vessel;

retaining the cell aggregates in the cell culture vessel by gravity settling of the cell aggregates during the perfusion;

harvesting the cell aggregates, and

conducting a serial passaging of the harvested cell aggregates in the closed system,

wherein the step of serial passaging comprises dissociating the harvested cell aggregates with the slicer grid mounted in line with a tubing of the tubing assembly and the device for mixing the cell aggregates.

2. The method of claim 1, wherein the serial passaging in the closed system is carried out in presence of a Rho-associated protein kinase (ROCK) inhibitor.

3. The method of claim 1, wherein the serial passaging in the closed system is carried out substantially in absence of an agent which maintains viability of passaged, monodispersed or disaggregated pluripotent cells.

4. The method of claim 3, wherein the agent which maintains the viability of passaged, monodispersed or disaggregated pluripotent cells is a Rho-associated protein kinase (ROCK) inhibitor.

5. The method of claim 1, wherein the cell aggregates which are expanded comprise human pluripotent stem cells.

6. The method of claim 1, wherein the cell culture vessel is a stirred tank bioreactor.

7. The method of claim 1, wherein said automated perfusion is carried out in the closed system without human intervention.

8. The method of claim 1, wherein the gravity settling chamber is at least 1 cm in length and positioned in an orientation that drives gravity settling of cell aggregates.

9. The method of claim 1, wherein the automated perfusion is conducted at a rate that allows gravity settling of cell aggregates of about 100 micron to about 800 micron diameter.

10. The method of claim 1, wherein an average diameter of each expanded cell aggregate is no more than about 800 micron in size.

11. The method of claim 1, wherein an average diameter of each expanded cell aggregate is no more than about 500 micron in size.

12. The method of claim 1, wherein a volume of the culture vessel is from about 50 mL to about 100 L.

13. A method for culturing cells in a closed system, where the closed system comprises a stirred tank bioreactor vessel, a tubing assembly, a device for mixing of cell aggregates, and a slicer grid, the method comprising:

providing the cells to the stirred tank bioreactor vessel;

expanding the cells in the stirred tank bioreactor vessel to generate cell aggregates;

conducting an automated perfusion of the cell aggregates in the stirred tank bioreactor vessel;

retaining the cell aggregates in the stirred tank bioreactor vessel by gravity settling of the cell aggregates during the perfusion;

harvesting the cell aggregates; and

conducting a serial passaging of the harvested cell aggregates in the closed system,

wherein the step of serial passaging comprises dissociating the harvested cell aggregates with the slicer grid mounted in line with the tubing of the tubing assembly and the device for mixing the cell aggregates.

14. The method of claim 13, wherein the serial passaging in the closed system is carried out substantially in absence of an agent which maintains viability of passaged, monodispersed or disaggregated pluripotent cells.