Method for dissociating cell aggregates

Patent metadata

Field	Value
Publication number	US-11242503-B2
Application number	US-201716072379-A
Country	US
Kind code	B2
Filing date	Jan 25, 2017
Priority date	Jan 26, 2016
Publication date	Feb 8, 2022
Grant date	Feb 8, 2022

Abstract

A method for dissociating cell aggregates in an agitated reactor. The method comprises providing a cell culture comprising cell aggregates in the agitated reactor, contacting the cell aggregates with a dissociation reagent, generating a dissociation force in the agitated reactor and exposing the contacted cell aggregates to the generated dissociation force under conditions sufficient to dissociate the contacted cell aggregates. The method may be used in a process for passaging cells and/or generating dissociated differentiated cells from stem and/or progenitor cells.

First claim

We claim:

1. A method for dissociating cell aggregates in an agitated reactor, the method comprising:
providing a cell culture having a volume of at least about 500 mL comprising cell aggregates in the agitated reactor;
contacting the cell aggregates with a dissociation reagent; and
applying a dissociation force generated by the agitated reactor to the cell aggregates under conditions sufficient to dissociate the cell aggregates, comprising exposing the cell aggregates to the applied dissociation force for 4 hours or less.

2. The method of claim 1 , wherein the contacting occurs:
before or at substantially the same time as the applying of the dissociation force in the agitated reactor.

3. The method of claim 1 , wherein the dissociation force is generated by movement of a stirrer, impeller, paddle, or wheel, by rocking, or by forced fluid flow entering the agitated reactor.

4. The method of claim 1 , wherein the providing step comprises exposing the cell culture to a culture force in the agitated reactor and wherein the dissociation force is about 50% to 500% of the culture force.

5. The method of claim 1 , further comprising:
washing the cell culture prior to contacting the cell aggregates with the dissociation reagent;
neutralizing and/or diluting the dissociation reagent; and/or
washing the dissociated cells.

6. The method of claim 1 , wherein the method is carried out in a closed system.

7. The method of claim 1 , wherein the agitated reactor is a stirred tank reactor, wave-mixed/rocking reactor, up and down agitation reactor, spinner flask, shake flask, shaken bioreactor, paddle mixer, or vertical wheel bioreactor.

8. The method of claim 1 , wherein the cell culture comprises a volume of about 500 mL-2,000 L.

9. The method of claim 1 , wherein the cell culture comprises about 1×10 6 cells/mL to 1×10 15 cells/mL.

10. The method of claim 1 , wherein the dissociated aggregates have a cell viability between about 50% and 100%.

11. The method of claim 1 , wherein the dissociated aggregates comprise:
aggregates that are at least 50% smaller than the provided cell aggregates; or
substantially single cells.

12. The method of claim 1 , wherein the provided cell aggregates have a diameter of about 150 microns to 800 microns.

13. The method of claim 1 , wherein the cell aggregates substantially comprise:
pluripotent stem cells;
multipotent stem and/or progenitor cells; and/or
somatic cells.

14. The method of claim 1 , wherein the dissociation force generated comprises a Kolmogorov eddy size less than the size of the largest cell aggregate and greater than the diameter of a cell in the cell aggregates at Reynolds number >1000.

15. The method of claim 1 , wherein the contacting occurs after the applying of the dissociation force in the agitated reactor.

16. The method of claim 1 , wherein the providing step comprises exposing the cell culture to a culture force in the agitated reactor and wherein the dissociation force is about 100% of the culture force.

17. The method of claim 1 , wherein the cell culture comprises a volume of between about 1 L to 1,000 L.

18. The method of claim 1 , wherein the dissociated aggregates have a cell viability greater than 80%.

19. The method of claim 1 , wherein the provided cell aggregates have a diameter of about 200 microns to 400 microns.

20. The method of claim 1 , wherein the agitated reactor is a stirred tank reactor.

21. The method of claim 1 , wherein the cell aggregates are exposed to the applied dissociation force for about 10 minutes.

22. A method for passaging cells, the method comprising:
providing a cell culture having a volume of at least about 500 mL comprising cell aggregates in a first agitated reactor;
contacting the cell aggregates with a dissociation reagent;
applying a dissociation force generated by the agitated reactor to the cell aggregates under conditions sufficient to dissociate the cell aggregates, comprising exposing the cell aggregates to the applied dissociation force for 4 hours or less; and
culturing at least a portion of the dissociated cell aggregates, thereby passaging the cells.

23. The method of claim 22 , wherein the portion of the dissociated aggregates is cultured in the first agitated reactor.

24. The method of claim 22 , wherein the portion of the dissociated aggregates is cultured in a second agitated reactor.

25. The method of claim 24 , wherein the second agitated reactor is a different type of agitated reactor and/or is a different size than the first agitated reactor.

26. The method of claim 24 , wherein the second agitated reactor is larger than the first agitated reactor.

27. A method for generating dissociated differentiated cells from a stem or progenitor cell population within an agitated reactor, the method comprising:
providing a cell culture having a volume of at least about 500 mL comprising a population of stem and/or progenitor cells in the agitated reactor;
differentiating the stem and/or progenitor cells into differentiated cell aggregates within the agitated reactor under conditions suitable for differentiation;
contacting the differentiated cell aggregates with a dissociation reagent; and
applying a dissociation force generated by the agitated reactor to the cell aggregates under conditions sufficient to dissociate the differentiated cell aggregates, comprising exposing the cell aggregates to the applied dissociation force for 4 hours or less.

Assignees

• Centre For Commercialization Of Regenerative Medicine
• Governing Council Univ Toronto

Inventors

• Timmins Nicholas
• Chan Lesley

Classifications

• C12M45/02 Primary

  by mechanical forces; Stirring; Trituration; Comminuting (crushing, pulverizing, disintegrating in general B02C) · CPC title

• C12N2521/00

  Culture process characterised by the use of hydrostatic pressure, flow or shear forces · CPC title

• C12N2513/00

  3D culture · CPC title

• C12M27/02

  Stirrer or mobile mixing elements · CPC title

• C12M45/09

  by enzymatic treatment · CPC title