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1. (WO2018161063) METHODS AND SYSTEMS FOR FUNCTIONAL MATURATION OF iPSC AND ESC DERIVED CARDIOMYOCYTES
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CLAIMS

What is claimed is:

1. A method of maturing functionally immature cardiomyocytes, the method comprising:

a) providing a system configured to culture, electrically pace, and monitor beating of beating cells;

b) culturing immature cardiomyocytes in the system;

c) monitoring the immature cardiomyocytes to characterize cardiomyocyte beating as synchronized or not synchronized; and

d) if synchronized, electrically pacing the immature cardiomyocytes according to a pulse profile that induces maturation until the immature

cardiomyocytes mature into functionally adult cardiomyocytes.

2. The method of claim 1 , wherein the system comprises a cell-substrate impedance monitoring electrode array positioned on a substrate and operably connected to an impedance analyzer to monitor cell-substrate impedance of a cell population cultured in the system.

3. The method of claim 1 , wherein the system comprises an extracellular recording electrode array operably connected to an extracellular recording amplifier to conduct extracellular recording of a cell population cultured in the system.

4. The method of claim 1 , wherein the system comprises a cell-substrate impedance monitoring electrode array on a substrate, an extracellular recording electrode array on the substrate, an impedance analyzer, and an extracellular recording amplifier, wherein the system is configured to monitor cell-substrate impedance and conduct extracellular recording of a cell population cultured in the system.

5. The method of claim 1 , wherein the immature cardiomyocytes are derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

6. The method of claim 1 , wherein the step of monitoring the immature cardiomyocytes comprises monitoring cell-substrate impedance of the immature

cardiomyocytes.

7. The method of claim 1 , wherein the step of monitoring the immature cardiomyocytes comprises performing extracellular recording of the immature

cardiomyocytes.

8. The method of claim 1, wherein characterizing whether cardiomyocyte beating is synchronized is by way of determining a beating rate of the immature cardiomyocytes and comparing the beating rate over time.

9. The method of claim 1, wherein if the cardiomyocyte beating is not synchronized, the method further comprises electrically pacing the immature cardiomyocytes until cardiomyocyte beating is synchronized.

10. The method of claim 9, wherein the electric pacing to synchronize beating is at a constant frequency.

11. The method of claim 1 , wherein the pulse profile comprises a rectangular pulse shape.

12. The method of claim 1, wherein the pulse profile comprises a pulse intensity of 0.7 V to 1 V.

13. The method of claim 1, wherein the pulse profile comprises a pulse duration from 0.1 milliseconds to 0.2 milliseconds.

14. The method of claim 1, wherein the pulse profile comprises a pulse frequency between 1 Hz and 2 Hz.

15. The method of claim 1, wherein the pulse profile comprises an increasing pulse frequency over time.

16. The method of claim 15, wherein the increasing pulse frequency increases over days to weeks.

17. The method of claim 15, wherein the increasing pulse frequency doubles in frequency over time.

18. The method of claim 15, wherein the increasing pulse frequency increases from 0.75 Hz to 2 Hz.

19. The method of claim 1 , wherein the functionally adult or mature

cardiomyocytes comprise a positive beating force-frequency relationship and immature cardiomyocytes comprise a negative beating force-frequency relationship.

20. The method of claim 1 , wherein the immature cardiomyocytes are paced until displaying a positive beating force-frequency relationship.

21. A method of characterizing an effect of a compound on cardiomyocyte beating, the method comprising:

a) providing a system configured to culture, electrically pace, and monitor beating of beating cells;

b) culturing immature cardiomyocytes in the system;

c) electrically pacing the immature cardiomyocytes according to a pulse profile until the cardiomyocytes are functionally mature;

d) adding a compound suspected of having an effect on cardiomyocyte beating force or cardiomyocyte beating rate to the functionally mature

cardiomyocytes;

e) electrically monitoring the cultured cardiomyocytes before and after compound addition;

f) determining before and after the compound addition, at least one parameter that characterizes a beating force or a beating rate;

g) comparing the determined at least one parameter before and after the compound addition thereby identify a difference in response to the compound addition; and

h) characterizing the compound as:

a positive inotropic compound if the beating force increases after compound addition or a negative inotropic compound if the beating force decrease after compound addition, and/or

a positive chronotropic compound if the beating rate increases after the compound addition or a negative chronotropic compound if the beating rate decreases after the compound addition.

22. The method of claim 21, wherein the immature cardiomyocytes are derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

23. The method of claim 21, wherein the immature cardiomyocytes are cultured until beating is synchronized.

24. The method of claim 23, wherein synchronized beating is determined by way of electrical measurement.

25. The method of claim 24, wherein the electrical measurement is selected from the group consisting of cell-substrate impedance monitoring, extracellular recording, and both cell-substrate impedance monitoring and extracellular recording.

26. The method of claim 23, wherein if the beating is not synchronized, the method further comprises electrically pacing the cardiomyocytes until cardiomyocyte beating is synchronized.

27. The method of claim 21, wherein the cardiomyocytes are paced until displaying a positive beating force-frequency relationship.

28. The method of claim 21, wherein the pulse profile comprises a rectangular pulse shape.

29. The method of claim 21, wherein the pulse profile comprise an intensity of 0.7 V to 1 V.

30. The method of claim 21, wherein the pulse profile comprises a pulse duration from 0.1 millisecond to 0.2 milliseconds.

31. The method of claim 21, wherein the pulse profile comprises a pulse frequency between 1 Hz and 2 Hz.

32. The method of claim 21, wherein the pulse profile comprises an increasing pulse frequency over time.

33. The method of claim 32, wherein the increasing pulse frequency increases over days to weeks.

34. The method of claim 33, wherein the increasing pulse frequency doubles in frequency over time.

35. The method of claim 33, wherein the increasing pulse frequency increases from 0.75 Hz to 2 Hz.

36. The method of claim 21, wherein the compound is a suspected vasodilator.

37. The method of claim 21, wherein the step of monitoring the cultured cardiomyocytes is selected from the group consisting of monitoring cell-substrate impedance of the cardiomyocytes, performing extracellular recording of the cardiomyocytes, and both monitoring cell-substrate impedance of the cardiomyocytes and performing extracellular recording of the cardiomyocytes.

38. A method of characterizing an effect of a compound on cardiomyocyte beating, the method comprising:

a) providing a system configured to culture, electrically pace, and monitor beating of beating cells;

b) culturing two populations of immature cardiomyocytes in the system; c) adding a compound suspected of having an effect on cardiomyocyte maturation to one of the populations of immature cardiomyocytes;

d) electrically pacing the two populations of immature cardiomyocytes according to a pulse profile that functionally matures immature cardiomyocytes until at least one of the two populations of cardiomyocytes is functionally mature; and e) characterizing the compound as further driving maturation if the population with compound addition functionally matures before the other cardiomyocyte population.

39. The method of claim 38, wherein the immature cardiomyocytes are derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

40. The method of claim 38, wherein the immature cardiomyocytes are cultured until beating is synchronized.

41. The method of claim 40, wherein synchronized beating is determined by way of electrical measurement.

42. The method of claim 40, wherein if the beating is not synchronized, the method further comprises electrically pacing the cardiomyocytes until cardiomyocyte beating is synchronized.

43. The method of claim 38, wherein the pulse profile comprises a rectangular pulse shape.

44. The method of claim 38, wherein the pulse profile comprise an intensity of 0.7 V to 1 V.

45. The method of claim 38, wherein the pulse profile comprises a pulse duration from 0.1 millisecond to 0.2 milliseconds.

46. The method of claim 38, wherein the pulse profile comprises a pulse frequency between 1 Hz and 2 Hz.

47. The method of claim 38, wherein the pulse profile comprises an increasing pulse frequency over time.

48. The method of claim 38, wherein the increasing pulse frequency increases over days to weeks.

49. The method of claim 38, wherein the increasing pulse frequency doubles in frequency over time.

50. The method of claim 38, wherein the increasing pulse frequency increases from 0.75 Hz to 2 Hz.

51. The method of claim 38, wherein maturity is determined by a force-frequency relationship, further wherein a mature cardiomyocyte population is characterized by a positive force-frequency relationship and an immature cardiomyocyte population is characterized by a negative force-frequency relationship, wherein the method further comprises electrically monitoring the two populations of cardiomyocytes to determine beating amplitude.

52. The method of claim 51, wherein electric monitoring is selected from the group consisting of cell-substrate impedance monitoring, extracellular recording, and both cell-substrate impedance monitoring and extracellular recording.

53. A system for the functional maturation of immature cardiomyocytes, the system comprising:

a) an electronic pulse generator configured to deliver electronic pulses according to a pulse profile that induces maturation of immature cardiomyocytes; b) a device station configured to engage a cell culture device and deliver the electronic pulses from the pulse generator to the engaged cell culture device; c) a cell culture device comprising a substrate configured to culture cells, each substrate comprising an electrode array addressable by the device station when engaged; and

d) a module for electrically monitoring cultured cells.

54. The system of claim 53, wherein the pulse generator delivers electronic pulses comprising a pulse shape as a rectangular shape.

55. The system of claim 53, wherein the pulse generator delivers an intensity of 0.7 V to 1 V.

56. The system of claim 53, wherein the pulse generator delivers a pulse duration from 0.1 millisecond to 0.2 milliseconds.

57. The system of claim 53, wherein the device station comprises an interface comprising a plurality of electrical contacts and a switch capable of independently delivering the electronic pulses to each of the electrical contacts.

58. The system of claim 53, wherein the device station is configured to a multi-well plate, optionally a plurality of multi-well plates.

59. The system of claim 53, wherein the electronic pulse generator and the device station are configured to deliver a pulse simultaneously to more than one well of the multi-well plate.

60. The system of claim 53, wherein the electronic pulse generator and the device station are configured to deliver a pulse simultaneously to all wells of the multi-well plate.

61. The system of claim 53, wherein the electronic pulse generator and the device station are configured to deliver a pulse simultaneously to more than one multi-well plate.

62. The system of claim 53, wherein the electronic pulse generator and the device station are configured to deliver different pulse profiles to different wells of the multi-well plate.

63. The system of claim 53, wherein the device station is configured to engage one or more cell culture flasks.

64. The system of claim 53, wherein the cell culture device is a flask.

65. The system of claim 53, wherein the culture device is a multi-well plate, optionally selected from the group consisting of a 6 well plate, a 48 well plate, and a 96 well plate.

66. The system of claim 53, wherein the cell culture device comprises immature cardiomyocytes on the substrate.

67. The system of claim 66, wherein the immature cardiomyoctes are derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

68. The system of claim 53, wherein the module for electrically monitoring cultured cells comprises an impedance analyzer operably connected for monitoring cell-substrate impedance of a cell population cultured in the cell culture device.

69. The system of claim 53, the module for electrically monitoring cultured cells comprises an extracellular recording amplifier operably connected for extracellular recording of a cell population cultured in the cell culture device.

70. The system of claim 53, wherein the system measures beating amplitude of the cardiomyocytes, determines a beating force-frequency relationship of the cardiomyocytes and reports a status of mature or immature, wherein the mature status is reported if the force-frequency relationship is positive and the immature status is reported if the force-frequency relationship is negative.