Patent US10087408

System and method for microfluidic cell culture
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US10087408

1. A method of culturing a cell, the method comprising: (21) (5)

retaining the cell at a retaining position within an individual chamber of a microfabricated device;

perfusing the cell with a perfusion fluid by flowing the perfusion fluid into the individual chamber through an inlet and out of the chamber through an outlet,

wherein the outlet is positioned such that gravitational forces acting on the cell to keep it at or near the retaining position exceed hydrodynamic forces acting on the cell to move it toward the outlet;

culturing the cell within the chamber and monitoring a response in the chamber; and

selectively recovering the cell or a clonal population thereof from the individual chamber based on the response in the monitoring step.

2. The method of claim 1, further comprising regulating osmolarity of the perfusion fluid within the chamber. (3) (0)

3. The method of claim 2, wherein regulating osmolarity of the perfusion fluid within the chamber comprises placing the chamber in gaseous communication with a bathing fluid, wherein the bathing fluid has a volume greater than the chamber volume. (1) (0)

21. The method of claim 2, wherein the bathing fluid is located in a reservoir, wherein the reservoir is separated from the chamber by a gas-permeable polydimethylsiloxane (PDMS) layer. (0)

22. The method of claim 2, wherein regulating osmolarity within the chamber comprises placing the chamber in gaseous communication with an osmolarity regulator. (1) (0)

5. The method of claim 1, wherein a speed of the perfusion fluid is decreased to less than 50 μm/s as the perfusion fluid approaches the retaining position. (1) (0)

7. The method of claim 1, wherein the chamber has a top and a bottom, and the retaining position is at the bottom. (1) (0)

10. The method of claim 1, wherein the cell is a suspension cell. (0)

11. The method of claim 1, wherein the perfusion fluid comprises any one or more of a cell culture medium, an immunostaining agent, an enzymatic reagent, a dye, a buffer, an oil, and a bead-containing solution. (0)

12. The method of claim 1, wherein x is less than or equal to y and x is the length of the shortest distance between the inlet and the outlet and y is the length of the shortest distance between the retaining position and a region of the chamber that is interposed directly between the inlet and outlet positions. (1) (0)

14. The method of claim 1, further comprising a step of flowing the cell into the chamber prior to retaining the cell at the retaining position. (0)

15. The method of claim 1, further comprising isolating a clone of the cell. (0)

16. The method of claim 1, further comprising tracking the progeny of the cell. (0)

17. The method of claim 1, wherein the flow of the perfusing fluid is intermittent. (0)

18. The method of claim 1, wherein the flowing of the perfusing fluid is continuous. (0)

19. The method of claim 1, further comprising characterizing the cells by lineage staining, antibody staining, enzymatic assaying, RT-PCR analysis, sequencing, functional assaying, or bead capturing to characterize the cells. (1) (0)

24. The method of claim 1, wherein the chamber volume is less than about 10 nL. (1) (0)

26. The method of claim 1, further comprising imaging the chamber to obtain an image of the chamber comprising the cell. (1) (0)

28. The method of claim 1, wherein the cellular response is growth rate kinetics. (0)

29. The method of claim 1, wherein the cellular response is cell death. (0)

30. The method of claim 1, wherein the cellular response is determined using a fluorescent reporter. (0)

31. The method of claim 1, wherein recovering the cell comprises piercing the chamber and aspirating the chamber's contents or a portion thereof to obtain the recovered cell. (0)

32. The method of claim 1, wherein the inlet is operable as the outlet. (0)

33. The method of claim 1, wherein the outlet is operable as the inlet. (0)

34. The method of claim 1, further comprising exchanging the perfusion fluid. (0)

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Patent US10087408

System and method for microfluidic cell culture

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