System and method for microfluidic cell culture

• US 10,421,936 B2
• Filed: 12/13/2017
• Issued: 09/24/2019
• Est. Priority Date: 07/07/2010
• Status: Active Grant

• Current Assignee: University of British Columbia
• Original Assignee: University of British Columbia
• Inventors: Hansen, Carl L. G., Lecault, Veronique, Piret, James M., Singhal, Anupam
• Primary Examiner(s): Beisner, William H.
• Assistant Examiner(s): Henkel, Danielle B
• Application Number:
  US15/841,194
• Publication Number:
  US 20180163166A1
• Time in Patent Office: 650 Days
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 29/10 Perfusion

Abstract

Microfluidic devices and methods for perfusing a cell with perfusion fluid are provided herein, wherein the gravitational forces acting on the cell to keep the cell at or near a retainer or a retaining position exceed the hydrodynamic forces acting on the cell to move it toward an outlet. Also provided, are methods for assaying cell products within the microfluidic device.

Claims

1. A method for selecting a cell, or a clone thereof, from a population of cells, comprising:

introducing the population of cells into 1,600 to 20,000 microfluidic chambers of a microfluidic device via a single introduction port, wherein each microfluidic chamber comprises an inlet, the single introduction port is in fluid communication with a flow channel that is in fluid communication with the inlets of the microfluidic chambers, and wherein individual cells of the population are retained in unique microfluidic chambers of the microfluidic device, and the cells are transported via the introduction port and flow channel into the unique microfluidic chambers;

providing a cell culture medium to the plurality of microfluidic chambers via the flow channel and the inlets of the chambers;

exchanging the cell culture medium in the individual microfluidic chambers via the flow channel and the inlets of the chambers, to create a plurality of individual clonal cell populations, wherein the individual clonal cell populations are retained in the same microfluidic chamber as their respective parental cells;

measuring a cell product secreted by the individual clonal cell populations, or subset thereof, within one or more of the microfluidic chambers; and

selecting one or more individual clonal cell populations from the plurality to provide one or more selected individual clonal cell populations.

2. The method of claim 1, wherein selecting one or more individual clonal cell populations from the plurality is based on a characteristic selected from quantity of secreted product, quality of secreted product, proliferation, morphology, gene expression, fluorescent reporter, surface proteins, genealogical pedigree, viability, apoptosis, autophagy, metabolism, clone homogeneity, or clone heterogeneity.

3. The method of claim 1, wherein exchanging the cell culture medium is via diffusion or convection.

4. The method of claim 1, wherein exchanging the cell culture medium is via diffusion.

5. The method of claim 1, wherein exchanging the cell culture medium is continuous.

6. The method of claim 1, wherein the individual cells of the population are retained in the unique microfluidic chambers via a cell trap.

7. The method of claim 6, wherein the cell trap is a mechanical trap, a hydrodynamic trap, a hydrodynamic balancing trap, a dielectrophoretic trap, a magnetic trap, an acoustic trap or an optical trap.

8. The method of claim 6, wherein the cell trap is a dielectrophoretic trap.

9. The method of claim 1, wherein the individual cells of the population are retained in the unique microfluidic chambers at a concentration of greater than or equal to 50,000 cells per mL.

10. The method of claim 1, wherein the individual cells of the population are retained in the unique microfluidic chambers at a concentration of greater than or equal to 250,000 cells per mL.

11. The method of claim 1, wherein the population of cells is derived from an adherent cell line.

12. The method of claim 1, wherein the population of cells grows while suspended in cell culture medium.

13. The method of claim 1, wherein exchanging the cell culture medium is via diffusion, and the individual cells of the population are retained in the unique microfluidic chambers via dieletrophoretic cell traps.

14. The method of claim 13, wherein the individual cells of the population are retained in the unique microfluidic chambers at a concentration of greater than or equal to 250,000 cells per mL.

15. The method of claim 13, wherein the individual cells of the population are retained in the unique microfluidic chambers at a concentration of greater than or equal to 250,000 cells per mL, but less than or equal to 100,000,000 cells per mL.

16. The method of claim 1, further comprising measuring the average cell growth rate in individual chambers.

17. The method of claim 13, further comprising measuring the average cell growth rate in individual chambers.

18. The method of claim 1, wherein measuring the cell product comprises flowing a fluid comprising an immunostaining agent, an enzymatic reagent, a dye or a functionalized bead into the individual chambers.

19. The method of claim 13, wherein measuring the cell product comprises flowing a fluid comprising an immunostaining agent, an enzymatic reagent, a dye or a functionalized bead into the individual chambers.

20. The method of claim 18, wherein measuring the cell product comprises flowing a fluid comprising an immunostaining agent into the individual chambers.

21. The method of claim 18, wherein measuring the cell product comprises flowing a fluid comprising a functionalized bead into the individual chambers.

22. The method of claim 19, wherein measuring the cell product comprises flowing a fluid comprising an immunostaining agent into the individual chambers.

23. The method of claim 19, wherein measuring the cell product comprises flowing a fluid comprising a functionalized bead into the individual chambers.

24. The method of claim 1, further comprising recovering the selected individual clonal cell population, or a subset thereof.

25. The method of claim 1, wherein the cell product is an antibody.

26. The method of claim 4, wherein the cell product is an antibody.

27. The method of claim 13, wherein the cell product is an antibody.

28. The method of claim 2, wherein selection is based on the quantity of the secreted product.

29. The method of claim 28, wherein the secreted product is IgG1 antibody.