阅读说明：本技术 用于iPSC和ESC衍生的心肌细胞的功能成熟的方法和系统 (Methods and systems for functional maturation of iPSC and ESC derived cardiomyocytes ) 是由 X·张 Y·A·阿巴西 李楠 王小波 于 2018-03-03 设计创作，主要内容包括：本发明为诱导未成熟心肌细胞功能成熟的方法,所述未成熟心肌细胞衍生自诱导的多能干细胞(iPSC)和胚胎干细胞(ESC),所述方法为通过根据诱导成熟的脉冲谱使未成熟心肌细胞电起搏,直到未成熟心肌细胞成熟为功能上的成体心肌细胞时为止。(The present invention is a method of inducing functional maturation of immature cardiomyocytes derived from induced pluripotent stem cells (ipscs) and Embryonic Stem Cells (ESCs) by electrically pacing the immature cardiomyocytes according to a pulse profile that induces maturation until the immature cardiomyocytes mature into functional adult cardiomyocytes.)

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

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

b) Culturing immature cardiomyocytes in the system;

c) Monitoring immature cardiomyocytes to characterize cardiomyocyte beating as synchronous or asynchronous; and

d) If synchronized, the immature cardiomyocytes are electrically paced 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 an array of cell-substrate impedance monitoring electrodes on a substrate and in operable connection with an impedance analyzer for monitoring the cell-substrate impedance of a population of cells cultured in the system.

3. The method of claim 1, wherein the system comprises an extracellular recording electrode array, said array being operably connected to an extracellular recording amplifier for extracellular recording of a population of cells 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 a substrate, an impedance analyzer, and an extracellular recording amplifier, wherein the system is configured to monitor cell-substrate impedance and perform extracellular recording of a population of cells 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 the cell-substrate impedance of the immature cardiomyocytes.

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

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

9. The method of claim 1, wherein if the cardiomyocyte beats asynchronously, the method further comprises electrically pacing the immature cardiomyocyte until the cardiomyocyte beats synchronously.

10. The method of claim 9, wherein the electrical pacing to synchronize beats is at a constant rate.

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

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

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

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

15. The method of claim 1, wherein the pulse spectrum includes a pulse frequency that increases with time.

16. The method of claim 15, wherein the increased pulse frequency is increased over a period of days to weeks.

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

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

19. The method of claim 1, wherein the functionally adult or mature cardiomyocytes comprise a positive beating power-frequency relationship and the immature cardiomyocytes comprise a negative beating power-frequency relationship.

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

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

a) Providing a system configured to culture a beating cell, electrically pace the beating cell, and monitor the beating of the beating cell;

b) Culturing immature cardiomyocytes in the system;

c) Electrically pacing the immature cardiomyocytes according to a pulse profile until the cardiomyocyte function is mature;

d) Adding to functionally mature cardiomyocytes a compound suspected of having an effect on cardiomyocyte beating power or cardiomyocyte beating rate;

e) Electrical monitoring of cultured cardiomyocytes before and after compound addition;

f) Determining at least one parameter indicative of the stroke power or rate before and after the addition of the compound;

g) Comparing at least one parameter determined before and after the addition of the compound, thereby identifying a difference in response to the addition of the compound; and is

h) Characterizing the compound as:

A positive inotropic compound if the pulsatile force increases after addition of the compound, or a negative inotropic compound if the pulsatile force decreases after addition of the compound, and/or

A positive chronotropic compound if the pulse rate increases after addition of the compound, or a negative chronotropic compound if the pulse rate decreases after addition of the compound.

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 said immature cardiomyocytes are cultured until pulse synchronization.

24. The method of claim 23, wherein the synchronized beats are determined by 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, cell-substrate impedance monitoring, and extracellular recording.

26. The method of claim 23, wherein if the beats are not synchronized, the method further comprises electrically pacing the cardiomyocyte until the cardiomyocyte beats are synchronized.

27. The method of claim 21 wherein the cardiomyocytes are paced until a positive beating power-frequency relationship is exhibited.

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

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

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

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

32. The method of claim 21, wherein the pulse spectrum includes a pulse frequency that increases with time.

33. The method of claim 32, wherein the increased pulse frequency is increased over a period of days to weeks.

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

35. The method of claim 33, wherein the increasing pulse frequency is increased from 0.75Hz 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 the cell-substrate impedance of the cardiomyocytes, making extracellular recordings of the cardiomyocytes, and monitoring the cell-substrate impedance of the cardiomyocytes and making extracellular recordings of the cardiomyocytes.

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

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

b) Culturing two populations of immature cardiomyocytes in this system;

c) Adding a compound suspected of having an effect on cardiomyocyte maturation to one of the population of immature cardiomyocytes;

d) Electrically pacing the two immature cardiomyocyte populations according to a pulse profile that functionally matures the immature cardiomyocytes until at least one of the two cardiomyocyte populations is functionally matured; and is

e) A compound is characterized as further driving maturation if the cardiomyocyte population to which it is added functionally matures before another 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 said immature cardiomyocytes are cultured until pulse synchronization.

41. The method of claim 40, wherein the synchronized beats are determined by electrical measurement.

42. The method of claim 40, wherein if the beats are not synchronized, the method further comprises electrically pacing the cardiomyocyte until the cardiomyocyte beats are synchronized.

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

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

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

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

47. The method of claim 38, wherein the pulse spectrum includes a pulse frequency that increases with time.

48. The method of claim 38, wherein the increased pulse frequency is increased over a period of days to weeks.

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

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

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

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

53. A system for 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) An apparatus station configured to engage the cell culture apparatus and to deliver the electronic pulse from the pulse generator to the engaged cell culture apparatus;

c) a cell culture device comprising substrates configured to culture cells, each substrate comprising an array of electrodes addressable by a device station when engaged; and

d) A module for electrical monitoring of cultured cells.

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

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

56. The system of claim 53, wherein the pulse generator delivers a pulse duration of from 0.1 milliseconds 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 an electrical pulse to each electrical contact.

58. The system of claim 53, wherein the device station is configured as 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 pulses to more than one well of the multi-well plate simultaneously.

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

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

62. The system of claim 53, wherein the electronic pulse generator and device station are configured to deliver different pulse spectra to different wells of a 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 apparatus 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 assembly comprises immature cardiomyocytes on a substrate.

67. The system of claim 66, wherein the immature cardiomyocytes are derived from Induced Pluripotent Stem Cells (iPSCs) or Embryonic Stem Cells (ESCs).

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

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

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

Technical Field

The present invention relates to methods of electrically inducing iPSC and ESC derived cardiomyocytes into cells with an adult-like phenotype and electrically monitoring the maturation process.

Background

induced pluripotent stem cells (ipscs) are a type of pluripotent stem cells that can be generated directly from adult cells. This technology was initiated ten years ago by introducing four specific genes encoding the transcription factors Oct3/4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under Embryonic Stem (ES) cell culture conditions. Takahashi et al (2006). Takahashi et al later demonstrated that factors also act in humans. Takahashi et al (2007).

The use of the iPSC has wide prospects in regenerative medicine application and drug discovery and development. In particular, ipscs can differentiate into many different types of cells for potential therapy, such as neurons, cardiomyocytes, and hepatocytes. These cells can also be used to understand underlying disease mechanisms and to screen for therapeutic agents that can be used to alleviate disease conditions. One of the first cell types derived from iPSC technology was cardiomyocytes. These iPSC-derived cardiomyocytes appear to express the appropriate pool of ion channels as well as structural and other proteins found in normal cardiomyocytes. However, it is challenging to fully differentiate ipscs into cardiomyocytes with adult phenotypes. In contrast, its development is usually arrested at an immature stage, immature cardiomyocytes tend to have rounded morphology, sarcomeric disorders, lack of T-tubes, different gene expression profiles, and different action potential distributions compared to the mature or adult phenotype. Immature cardiomyocytes have limited potential as a therapeutic approach. Yang et al (2014).

A number of different approaches have been taken to improve the maturation state of iPSC-derived cardiomyocytes, including long-term cardiomyocyte culture (Lundy et al, 2013), culturing cells on substrates with stiffness approximating native myocardium (Jacot et al, 2008), seeding cells onto patterned substrates that provide topological cues (McDevitt et al, 2002), applying mechanical loads to cells (Zimmermann et al, 2002), and directed electrical stimulation (Sathaye et al, 2006). All of these approaches can result in cardiomyocytes that are structurally and functionally similar to the adult-like phenotype. However, each system has its weaknesses, such as difficulty in achieving high throughput screening, and requires a high level of technical knowledge.

Accordingly, there remains a need for improved systems and methods for further maturation of cardiomyocyte precursor cells, including iPSC-derived and Embryonic Stem Cell (ESC) -derived cardiomyocytes, into cardiomyocytes having a mature or adult-like phenotype.

Brief description of the invention

The present invention addresses the above-described deficiencies and provides related benefits. In particular, the present invention provides methods and systems for electrically inducing further maturation of iPSC or ESC derived cardiomyocytes and electrically monitoring their maturation to a more image-like phenotype. This is achieved, at least in part, by extending the electrical pacing and monitoring features of the xCELLigence CardioECR system developed by ACEA Biosciences, inc (San Diego, CA), which systematically paces ipscs and other immature cardiomyocytes (pace) using specially developed pulse spectra that induce maturation, while also monitoring the pulse spectra of cultured cells to monitor the maturation process.

More specifically, in one aspect of the present invention, there is provided a method of maturing functionally immature cardiomyocytes, the method comprising: providing a system configured to culture a beating cell, electrically pace the beating cell, and monitor the beating cell; culturing immature cardiomyocytes in said system; monitoring immature cardiomyocytes to characterize the cardiomyocyte beating as synchronized or unsynchronized; and, if synchronized, electrically pacing the immature cardiomyocytes according to the pulse profile until the immature cardiomyocytes mature into functionally adult (functional adult) cardiomyocytes.

In some embodiments, the system includes an array of impedance monitoring electrodes on the substrate and in operable connection with the impedance analyzer for monitoring the cell-substrate impedance of the cell population cultured in the system. In other embodiments, the system includes an extracellular recording electrode array operably connected to an extracellular recording amplifier for extracellular recording of a population of cells cultured in the system. In other embodiments, by providing both, such as an impedance monitoring electrode array on a substrate, an extracellular recording electrode array on a substrate, an impedance analyzer, and an extracellular recording amplifier, the system is configured to monitor cell-substrate impedance and perform extracellular recording of a population of cells cultured in the system. In some embodiments, the electrodes are shared between the cell-substrate impedance monitoring electrode array and the extracellular recording electrode array, but in other embodiments each array has its own unique pair of electrodes.

The improvements provided include the ability to initiate differentiation of cardiomyocytes from different levels or degrees of immature differentiation, regardless of the beating rate in culture. In other words, the methods herein can be used to induce maturation of any population of cardiomyocytes characterized by a negative force-frequency relationship that is considered immature. For this reason, cardiomyocytes characterized as immature (also referred to herein as having an embryonic phenotype) can be used as long as they undergo excitation-contraction coupling. Thus, immature cardiomyocytes can be derived from induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs).

In addition, immature cardiomyocytes can be electrically monitored to characterize their stage as immature or adult, and to assess whether they are suitable to undergo further differentiation. Among the useful means of monitoring cardiomyocyte maturation include monitoring the cell-substrate impedance of the cardiomyocytes. In other embodiments, immature cardiomyocytes are monitored electrically by extracellular recording of cardiomyocytes. In one hybrid approach, immature cardiomyocytes are monitored electrically by cell-substrate impedance monitoring of the cardiomyocytes and extracellular recording of the cardiomyocytes. Electrical measurement of cardiomyocyte beating makes it possible to determine a force-frequency relationship, wherein a positive force-frequency relationship is indicative of a mature cardiomyocyte population and a negative force-frequency relationship is indicative of an immature cardiomyocyte population.

In addition, electrical monitoring of immature cardiomyocytes can be used to determine whether the cardiomyocyte beats synchronously, e.g., by determining the beat rate of the immature cardiomyocytes and comparing the beat rate over time. If the cardiomyocytes beat asynchronously, the immature cardiomyocytes may require additional culture. Electrical pacing of immature cardiomyocytes can also be used to induce synchronized beating of immature cardiomyocytes. Electrically induced synchronized beats may be achieved by applying electrical pulses at a constant frequency. In particular, the electrical pulses are typically provided at or about the same rate as the pulsatile rate of the immature cells. Generally, a pulse frequency of about 0.5Hz to 1Hz can help synchronize the beats.

Once the beats are synchronized, the immature cardiomyocytes are electrically paced according to a pulse profile to induce further maturation into a functionally mature or adult phenotype. These pulse profiles were established to induce maturation of iPSC-derived cardiomyocytes, ESC-derived cardiomyocytes, and functionally immature cardiomyocytes. An exemplary pulse spectrum has a rectangular pulse shape. An exemplary pulse intensity is 0.7V to 1V, and intensities or voltages up to a few volts (e.g., 2V or 3V) are acceptable. The pulse intensity should be high enough to pace the immature cardiomyocytes (i.e., each applied pulse results in a beating cycle of the cardiomyocytes in the device).

An exemplary pulse spectrum has a pulse duration or width of 0.1 to 0.2 milliseconds, with durations of up to 10 milliseconds being acceptable. With appropriate application of pulse strength, the pulse duration should be long enough to pace the cardiomyocytes (i.e., each applied pulse contributes to the pulse cycle of the cardiomyocytes in the device). In general, as short a pulse duration as possible should be applied as long as it promotes effective pacing of the cardiomyocytes (i.e., each applied pulse promotes a pulse cycle of the cardiomyocytes in the well).

Differentiating immature cardiomyocytes using the system is primarily accomplished by pacing immature cardiomyocytes over time by applying a pulse profile with varying pulse frequency. The skilled person will appreciate that the pulse profile may vary depending on the pulse rate of the immature cardiomyocytes and may also vary depending on the method used to differentiate the immature cardiomyocytes into mature cardiomyocytes. As an example, the pulse profile will typically start at a frequency that is consistent or nearly consistent with the pulse rate of immature cardiomyocytes, which can vary. In most cases, the pulse spectrum will start with a slower pulse frequency, and the frequency increases over time. This initial lower pulse rate should be appropriate so that the cardiomyocytes can follow the pacing pulses (i.e., each pacing pulse causes one beat cycle of the cardiomyocytes in the device). As an example, a pulse spectrum with a pulse frequency varying between 0.5Hz and 2Hz over time is established. As another example, the pulse spectrum includes a pulse frequency that varies between 1Hz and 2Hz over time. As another example, the pulse spectrum includes a pulse frequency that increases from 0.75Hz to 2Hz over time. As another example, cardiomyocytes of certain genotypes or phenotypes (such as diseased genotypes/phenotypes) may require a lower initial pulse frequency, e.g., 0.5Hz or 0.3Hz or even lower, in order for the cardiomyocytes to be effectively paced. On the other hand, the pulse rate may increase over time from an initially low pulse rate to a higher pulse rate, such as 3Hz or even higher, depending on the requirements of the assay that will use the cardiomyocytes being paced. The skilled person will appreciate that the time interval between increasing the pulse frequency may also vary, but in some embodiments the time interval for increasing the pulse frequency is 2-6 days. In other embodiments, the time course of increasing the pulse frequency is 1 to 5 weeks. As a non-limiting example, further differentiation of immature cardiomyocytes can occur when pulsed at 0.75Hz for 1 week, 1.5Hz for another week, and 2Hz for another week. Preferably, the increasing pulse frequency doubles in frequency over time. Cardiomyocyte maturation can be followed by periodically testing the force-frequency relationship of the beating culture to determine whether the force-frequency relationship is increasing (indicating maturation) or decreasing (indicating non-maturation). In particular, the test may include gradually increasing the pacing rate of the cardiomyocytes while recording the beat amplitude-a surrogate indicator of force. If the beat amplitude/electrical pacing rate relationship is negative, the cardiomyocyte is considered immature; conversely, if the amplitude/electrical pacing rate relationship is positive, the cardiomyocytes are considered "mature".

Functionally mature cardiomyocytes, or adult cardiomyocytes, have a positive force-frequency relationship; whereas immature cardiomyocytes have a negative force-frequency relationship. Thus, by electrically monitoring some parameter corresponding to the stroke force (such as a measure of the pulse amplitude), the force-frequency relationship can be evaluated in order to monitor the maturation process. As such, the method includes electrically pacing immature cardiomyocytes until the cardiomyocytes are characterized as having a positive force-frequency relationship indicative of an adult phenotype.

In a related aspect of the invention, methods of characterizing the effect of a compound on the beating of a cardiomyocyte are provided. The method comprises the following steps: providing a system configured to culture a contractile cell, electrically pace it, and monitor its pulsation; culturing immature cardiomyocytes in said system; electrically pacing the immature cardiomyocytes according to a pulse profile until the cardiomyocyte function is mature; adding to functionally mature cardiomyocytes a compound suspected of having an effect on cardiomyocyte beating power or cardiomyocyte beating rate; electrical monitoring of cultured cardiomyocytes before and after compound addition; determining at least one parameter indicative of beat amplitude or beat rate before and after addition of the compound; comparing the at least one parameter determined before and after the addition of the compound, thereby identifying a difference in response to the addition of the compound; and characterizing the compound as: a positive inotropic compound if the beating amplitude increases after addition of the compound, or a negative inotropic compound if the beating amplitude decreases after addition of the compound; and/or a positive going compound if the pulse rate increases after the compound is added, or a negative going compound if the pulse rate decreases after the compound is added.

In view of the technical improvements herein, the methods may be used with immature cardiomyocytes derived from induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs). Preferably, immature cardiomyocytes are cultured until the pulse synchronization. The synchronous beats are preferably determined by electrical measurements, but may also be determined optically. As non-limiting examples, the electrical measurement may be cell-substrate impedance monitoring, extracellular recording, or both cell-substrate impedance monitoring and extracellular recording. If the beats are not synchronized, the cardiomyocytes can be electrically paced until the beats are synchronized. Such electrical pulses may have a constant frequency.

Pacing myocardial cells to test the effect of one or more compounds to obtain a more mature or adult phenotype, the pacing comprising applying a pulse profile capable of inducing further maturation. Such a pulse spectrum can be characterized as having a rectangular pulse shape, an intensity of about 0.1V to 0.2V (up to 2 volts or 3 volts), and a pulse duration from 0.1 milliseconds to 0.2 milliseconds (up to 10 milliseconds).

Further differentiation of the immature cardiomyocyte population into a cardiomyocyte population that can be characterized as having an adult phenotype can be achieved by applying a pulse profile with varying pulse frequencies. Specifically, the pulse spectrum starts at a slower pulse frequency, which increases over time. As a non-limiting example, a pulse profile for cardiomyocyte differentiation with a pulse frequency varying between 0.3Hz and 3Hz over time was established. More preferably, the pulse spectrum comprises a pulse frequency varying between 0.5Hz and 2Hz over time. More preferably, the pulse spectrum comprises a pulse frequency varying between 1Hz and 2Hz over time. In a particularly preferred method, the pulse spectrum comprises a pulse frequency that increases from 0.75Hz to 2Hz over time. The time interval between increasing the pulse frequency may vary, but in some embodiments the time interval for increasing the pulse frequency is 2-6 days. In other embodiments, the time course of increasing the pulse frequency is 1 to 5 weeks. As a further guide, immature cardiomyocytes can be pulsed at 0.75Hz for 1 week, 1.5Hz for another week, and 2Hz for another week to induce and maintain the mature phenotype. Preferably, increasing the pulse frequency doubles the frequency over time.

Combining synchronized pulsatile cultures with high resolution electrical monitoring of the cultures also makes it possible to identify changes in pulsatile amplitude and pulsatile rate in response to compound administration. To this end, the method allows to characterize the effect of potential drugs on cardiomyocytes and can therefore be used to develop new therapies or new therapeutic uses of known compounds. By way of non-limiting example, the method can electrically detect a change in the population of cardiomyocytes in response to administration of a vasodilator. These changes can be assessed by electrical monitoring of cultured cardiomyocytes before and after compound addition, by monitoring of cell-substrate impedance, extracellular recording of immature cardiomyocytes, or both cell-substrate impedance monitoring of cardiomyocytes and extracellular recording of immature cardiomyocytes.

In a further related aspect, there is provided a method of characterizing the effect of a compound on cardiomyocyte maturation, the method comprising: providing a system configured to culture a contractile cell, electrically pace the contractile cell, and monitor contractile cell beating; culturing immature cardiomyocytes in the system; electrically pacing immature cardiomyocytes according to a pulse profile that matures cardiomyocyte function; adding a compound suspected to have an effect on cardiomyocyte maturation; electrical monitoring of cultured cardiomyocytes before and after compound addition; determining at least one parameter characterizing the pulsatility force-frequency relationship of the monitored cardiomyocyte before and after the compound addition; comparing at least one parameter determined before and after the addition of the compound, thereby identifying a difference in response to the addition of the compound.

In yet another related aspect, there is provided a method of characterizing the effect of a compound on the beating of a cardiomyocyte, the method comprising: providing a system configured to culture a contractile cell, electrically pace the contractile cell, and monitor contractile cell beating; culturing two populations of immature cardiomyocytes in the system; adding a compound suspected of having an effect on cardiomyocyte maturation to a population of immature cardiomyocytes; electrically pacing the two populations of immature cardiomyocytes according to a pulse profile that functionally matures the immature cardiomyocytes until at least one of the two populations of cardiomyocytes is functionally mature; and, characterizing the compound as further driving maturation if the functional maturation of the population of added compounds precedes that of another population of cardiomyocytes.

In yet another related aspect, a system for functional maturation of immature cardiomyocytes is provided. The system comprises: an electronic pulse generator configured to deliver electronic pulses according to a pulse profile that induces maturation of immature cardiomyocytes; an apparatus station configured to engage the cell culture apparatus and to deliver the electronic pulse from the pulse generator to the engaged cell culture apparatus; a cell culture device having substrates configured to culture cells, each substrate having an array of electrodes addressable by the device station when engaged; and a module for electrical monitoring of the cultured cells.

In view of the methods for cardiomyocyte differentiation described herein, the pulse generator can deliver an electronic pulse having a pulse shape with a rectangular shape. In some embodiments, the pulse generator delivers an intensity of 0.1V to 0.2V to 2 volts. In other embodiments, the pulse generator delivers an intensity of up to 3 volts. In some embodiments, the pulse generator delivers a pulse duration of 0.1 milliseconds to 0.2 milliseconds to 10 milliseconds.

In some embodiments, the device station has an interface with a plurality of electrical contacts and a switch capable of independently delivering an electronic pulse to each electrical contact. In some embodiments, the device station is configured to accept a multi-well plate, optionally a plurality of multi-well plates. In such embodiments, the electronic pulse generator and device station may be configured to deliver pulses to more than one well of a multi-well plate simultaneously. Further, the electronic pulse generator and the device station may be configured to deliver pulses to all wells of the multi-well plate simultaneously. Further, in some embodiments, the electronic pulse generator and the device station are configured to deliver pulses to more than one multi-well plate simultaneously. In some embodiments, the electronic pulse generator and the device station are configured to deliver different pulse spectra to different wells of the multi-well plate. In other embodiments, the device station is configured to engage one or more cell culture flasks.

In some embodiments, the cell culture apparatus is a flask. In other embodiments, the culture device is a multi-well plate, optionally selected from a 6-well plate, a 48-well plate, and a 96-well plate.

In some embodiments, the system comprises immature cardiomyocytes cultured on a substrate of a cell culture device. Exemplary cardiomyocytes include immature cardiomyocytes derived from induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs).

The means for electrical monitoring of cultured cells may include an impedance analyzer operably connected to monitor a cell-substrate impedance of a population of cells cultured in the cell culture apparatus. In other embodiments, the module for electrical monitoring of cultured cells comprises an extracellular recording amplifier operably connected to perform extracellular recording of a population of cells cultured in the cell culture apparatus. In a further embodiment, a module for electrical monitoring of cultured cells includes an impedance analyzer operatively connected to monitor a cell-substrate impedance of a population of cells cultured in a cell culture device and an extracellular recording amplifier operatively connected to perform extracellular recording of the population of cells cultured in the cell culture device.

Brief Description of Drawings

fig. 1 depicts an exemplary system 100 for inducing and monitoring functional maturation of iPSC and ESC derived cardiomyocytes, and for screening for inotropic and chronotropic compounds, as well as compounds that affect maturation of immature cardiomyocytes, depicting a base unit 110 housing an electronic pulse generator module 110A, an integrated impedance analyzer module 110B, and an extracellular recording amplifier module 110C; an apparatus station 120 implemented in the form of an electronic multi-well plate station and a cell culture apparatus 130 implemented in the form of an electronic plate.

FIG. 2A depicts a schematic of an electrode configuration in which two recording electrodes 12A each have a diameter of 100 μm; the distance between the two recording electrodes 12A was 2.98 mm; the diameter of the circle in the "circle-on-line" electrode elements 19A is 90 μm, and the center-to-center distance between two adjacent circle-on-line electrode elements 19A is 110 μm; the gap between the two shared impedance/electrical stimulation electrodes 16A, 18A, and covering the recording electrode 12A, is about 290 μm. Each of the electrodes 12A, 14A, 16A, 18A is connected to a connection pad 24A via an electrical trace 26A.

fig. 2B depicts a schematic of an electrode configuration in which each electrode array 10B includes two annular, extracellular recording electrodes 12B and a single unitary reference electrode 14B. The electrode structures 16B, 18B are switched by electronic switches for both conducting electrical pulses and monitoring cell impedance (albeit at different points in time). In this exemplary embodiment, the diameter of each recording electrode 12B is 60 μm; the distance between the two recording electrodes 12B was 2 mm; the diameter of the circle in the on-line circular electrode elements 19B is 90 μm, and the center distance between two adjacent on-line circular electrode elements 19B is 110 μm; the gap between the two resistive electrode structures 16B, 18B and covering the recording electrode 12B is about 290 μm.

Fig. 2C depicts a schematic of an electrode configuration in which each electrode array 10C is configured on a non-conductive substrate associated with a single well. The electrode array 10C includes a circular extracellular recording electrode 12C and a unitary integral (unit-piece) reference electrode 14C. The electrode structures 16C, 18C perform impedance measurements and electrical pulses of the cells at different points in time by electronic switching. In this example, a plurality of "circle-on-circle line" electrode elements 19C form an interdigitated electrode structure 16C, 18C. In this exemplary embodiment, the diameter of the recording electrode 12C is 80 μm; the distance between the two recording electrodes 12C was 1.44 mm; the diameter of a circle in the on-line circular electrode elements 19C is 90 μm, and the center-to-center distance between two adjacent on-line circular electrode elements 19C is 110 μm.

Fig. 2D depicts a schematic diagram in which an electrode array 10D includes two circular ring-shaped extracellular recording electrodes 12D and a single unitary reference electrode 14D. The electrode structures 16D, 18D are used for cell impedance monitoring. The electrode structures 20D, 22D are used for electrical pulse and cell impedance monitoring. In this exemplary embodiment, the diameter of each recording electrode 12D is 60 μm; the distance between the two recording electrodes 12D is 2 mm; the diameter of the circle in the on-line circular electrode elements 19D is 90 μm, and the center distance between two adjacent on-line circular electrode elements 19D is 110 μm; the gap between the two impedance electrode structures 16D, 22D and covering the recording electrode 12D is about 290 μm.

FIG. 3 is a graph depicting the beat cycle of cardiomyocytes with a mature or adult-like phenotype.

Fig. 4 is a table showing corresponding screen shots of pulse settings and beat activity recorded for 6 seconds continuously before pacing, after pacing at 1Hz, after pacing at 1.5Hz, and after pacing at 2 Hz.

Fig. 5 is a graph comparing beat amplitude to beat frequency for electrically paced cardiomyocytes to spontaneously beating cardiomyocytes, showing that the beat amplitude (beat power) increases with increasing electrical pacing frequency. Here, the force-frequency relationship is enhanced in paced cells and attenuated in spontaneously beating cells.

Fig. 6 is a series of graphs showing that the pulse rate of iPSC matured cardiomyocytes improved in response to administration of isoproterenol as compared to spontaneously pulsating cardiomyocytes.

Fig. 7 is a series of graphs showing that the pulse rate of iPSC matured cardiomyocytes improved in response to administration of milrinone compared to spontaneously pulsating cardiomyocytes.

Fig. 8 is a summary table showing the effect of positive inotropic compounds on contractile activity of functionally mature iCell cardiomyocytes quantified after pacing at representative concentrations. Data are presented as mean ± SD. (N ═ 5).

Detailed description of the preferred embodiments

The present invention relates to systems and methods for culturing immature cardiomyocytes, electrically pacing them, and monitoring their maturation into cells with an adult-like phenotype. In particular, the systems and methods described above are capable of inducing iPSC-derived cardiomyocytes and Embryonic Stem Cell (ESC) -derived cardiomyocytes and electrically monitoring their functional maturation. In addition, the systems and methods provide novel methods for testing inotropic, chronotropic, and test compounds that affect the maturation process of cardiomyocytes.

A. System for cellular electrical pacing and measurement

One of the core advantages of the systems described herein is their ability to electrically induce further maturation of embryonic stem cell-derived cardiomyocytes and iPSC-derived cardiomyocytes. A preferred system pairs an electrode-based monitoring system with an electrical stimulation system to provide immediate feedback on the functional phenotype of the mature cell population in response to the electrical stimulation. In a preferred method, the system includes electrical stimulation for inducing differentiation while providing high resolution parallel impedance-based monitoring and/or parallel extracellular recording-based monitoring of mature cardiomyocytes, thereby filling a significant technical gap in vitro monitoring of cardiomyocyte maturation.

Referring to fig. 1-2D, a system 100 for functionally maturing immature cardiomyocytes comprises: an electronic pulse generator 110A configured to deliver electronic pulses according to a pulse profile that induces maturation of immature cardiomyocytes; an apparatus station 120 configured to engage the cell culture apparatus 130 and deliver the electronic pulse from the pulse generator 110A to the engaged cell culture apparatus 130; a cell culture apparatus 130 having substrates configured for culturing cells, each substrate having an electrode array 10A-D addressable by it when engaged by apparatus station 120; and modules 110B, 110C for electrical monitoring of cultured cells.

Pulse generator 110A generates and delivers electronic pulses to device station 120 where they are routed to a population of cells cultured in cell culture device 130 to electrically pace immature cardiomyocytes. The pulses generated and delivered may themselves have any desired waveform that induces maturation, but in a preferred embodiment, the pulse generator 110A generates and delivers the pulses in the form of a rectangular waveform. The pulse generator 110A can also generate other waveforms, such as a sinusoidal waveform, a triangular waveform, and/or a sawtooth waveform. The pulse duration or pulse width may also vary as desired, but is typically between about 0.1 milliseconds to about 10 milliseconds, but more typically 0.1 milliseconds to 0.2 milliseconds. Non-limiting examples of voltages that are particularly useful in iPSC maturation can be up to about 3 volts, 2 volts, but more typically 0.7V to 1V.

Pulse generator 110A is programmable so that it can selectively deliver electrical stimulation pulses to each electrode array 10A-D in each well independently of the other electrode arrays 10A-D in the other wells, and so that it can simultaneously deliver pulses to each electrode array 10A-D as needed. User programming may be performed by inserting raw pulse parameters or by preprogrammed selectable options, wherein the user defines one or more pulse spectra and assigns the one or more pulse spectra to one or more apertures of the device 130. As used herein, "pulse spectrum" refers to a current that is pulsed at a pulse intensity (or voltage) and a pulse frequency over a pulse duration (or width). The pulse spectrum may be programmed by a user or may be pre-programmed. To this end, in some embodiments, multiple pulse spectra or pulse sub-spectra are programmed or preprogrammed for series operation, wherein a series of pulse sub-spectra maintain the same waveform and voltage, but with increasing pulse frequency. As a non-limiting example, the pulse spectrum may be a series of: a first pulse sub-spectrum at about 1Hz, followed by a second pulse sub-spectrum having a frequency of about 1.5Hz, followed by a third pulse sub-spectrum having a frequency of about 2 Hz. Further, the series may be programmed or preprogrammed to perform the spectra over a predetermined period of time (e.g., without limitation, one week per pulse of the sub-spectra).

The user programmable feature is particularly useful because, as demonstrated herein, a typical experiment in which pacing an iPSC, inducing its maturation into cardiomyocytes with a more adult-like phenotype, may take more than about three weeks, which limits access to the device station. By providing a user programmable feature, multiple experiments can be performed for multiple users across multiple cell culture devices 130 with the same electronic pulse generator 110A and device station 120. Alternatively, it may be desirable to vary the pulse profile between wells of the same multi-well device 130 to study the effect of the pulses themselves, for example, to further modulate maturation in the presence of test compounds (such as compounds believed to drive maturation).

In a related embodiment, the pulse generator 110A is preprogrammed with instructions to assess the maturation of immature cardiomyocytes or ipscs into cardiomyocytes, and pulse programming is performed according to the assessment. That is, the system 100 may be equipped with a feedback loop that adjusts the pacing of the cardiomyocytes according to the maturation of the electrical measurements. In such embodiments, pulse generator 110A is loaded with one or more pulsatile parameters indicative of maturation (e.g., surrogate markers of force-frequency relationship) for comparison to parameters calculated from real-time measurements taken during maturation, and the pulse spectrum is adjusted accordingly. That is, by forming a database of control pulse parameters indicative of maturation and corresponding pulse profiles for further maturation, the system 100 can automatically assess the stage of maturation and adjust pacing of the cardiomyocytes according to the database to further mature the cell population or to signal the completion of the maturation process.

Monitoring maturation of immature cardiomyocytes or ipscs into mature cardiomyocytes was performed by monitoring excitation-contraction coupling of cells undergoing further maturation. The term excitation-contraction coupling (ECC) is used to describe the physiological process of converting electrical stimulation into a mechanical response. The process is the basis of muscle physiology, where electrical stimulation can be an action potential and the mechanical response is in the form of contraction. Excitation-contraction coupling in cardiomyocytes is commonly referred to as beating. That is, the beating of the cardiomyocytes itself corresponds to excitation-contraction coupling of the cells.

The beating spectrum of a population of cardiomyocytes (in particular its beating force-frequency relationship) was found to be an accurate predictor of its phenotype, since early immature cardiomyocytes and ipscs tend to beat at irregular frequencies, and as the synchronous beating frequency increases due to electrical stimulation, the beating amplitude or force tends to decrease. To this end, the pulse generator comprises hardware and software, or is coupled to a unit with suitable hardware and software, to evaluate the excitation-contraction coupling or beating of the cell population in culture, and the force-frequency relationship can be determined.

Coupled excitation-contraction monitoring of cells can be performed using cell-substrate impedance, extracellular recording of cells, by a combination of cell-substrate impedance and extracellular recording, or by optical systems such as resonant waveguide or resonant waveguide grating sensors. Thus, the central base 110 housing the pulse generator 110A may also include one or more of impedance analyzer functionality, extracellular recording functionality, or resonant waveguide functionality. Such functionality may be facilitated by integrating the required hardware and software into the pulse generator, or by communicatively coupling the pulse generator 110A with a separate impedance analyzer 110B, extracellular recording amplifier 110C, optical or resonant waveguide system. Most preferably, the pulse generator 110A is integrated into a single base unit 110, which also comprises at least an impedance analyzer 110B for analyzing the cell-substrate impedance.

Cell substrate impedance monitoring is a non-invasive method for monitoring living cells. In brief overview, microelectrodes 16A-D, 18A-D of appropriate geometry are fabricated onto the bottom surface of a substrate, such as a multi-well plate or similar device 130, with the microelectrodes 16A-D, 18A-D exposed to the inner bore of the plate/device 130. Cells are introduced into the wells, brought into contact with and attached to the electrode surface. The presence, absence or change of cell attachment affects the electron and ion channels on the electrode sensor surface. When the biological state of the cell changes (e.g., changes in morphology), an analog electronic readout signal is automatically measured in real time and converted to a digital signal for processing and analysis. Furthermore, monitoring of cell-substrate impedance at different frequencies can reveal differences in cell behavior. In general, fluctuations in cell-basal impedance at lower frequencies tend to reflect changes in the spacing under or between cells, while fluctuations in cell-basal impedance at higher frequencies tend to reflect changes in the overall cell coverage of the electrode.

In the xCELLigence RTCA system, the impedance measurements are converted to a single parameter, called the "cellular index". A cellular index is automatically derived and provided based on the measured electrode impedance values. The cellular index obtained for a given well reflects: 1) how many cells attached to the electrode surface in the well, and 2) how well the cells attached to the electrode surface in the well. Thus, the more cells of the same type that are attached to the surface of an electrode under similar physiological conditions, the greater the cellular index. Similarly, the greater the cell attachment to the electrode surface (e.g., a greater contact area, by more cells spreading to have a greater contact area, or cells more tightly attached to the electrode surface), the greater the cell index.

Still further, it has been found that converting the millisecond resolution of cardiomyocyte beat to impedance-based parameters, such as Cell Index (CI) and/or Cell Change Index (CCI), allows a high degree of resolution of the force-frequency relationship that is closely related to the state of maturation of the cardiomyocytes. Cell Indices (CI) and Cell Change Indices (CCI) have been discussed in detail in US 7,470,533, US 7,560,269, US 8,026,080, US 8,263,375, US 9,612,234, and elsewhere. Thus, in a preferred embodiment, the pulse generator 110A is loaded with or in communication with a computer configured to calculate, by suitable programming, a Cellular Index (CI) for one or more time points of one or more wells of the multi-well device. In some preferred embodiments, the software can also calculate a Cell Change Index (CCI) from impedance measurements of one or more wells of the multi-well device. The software preferably can generate a plot of impedance-based parameters over time, such as, but not limited to, impedance-based curves selected from impedance measurements, CI or CCI, which depict stroke rate and beat frequency. The software can also perform other analyses such as calculating cell numbers from the CI, generating dose response curves based on the impedance data, calculating IC values based on the impedance values, and calculating kinetic parameters of the cell excitation cycle from the impedance-based parameters and the impedance-based curves. Non-limiting examples of methods for calculating and comparing the beat parameters can be found in US 9,612,234 and examples I and II.

Excitation-contraction coupled monitoring can also be measured, at least in part, using extracellular recordings of cells. In extracellular recording, an extracellular recording amplifier 110C is communicatively coupled to the pair of extracellular recording electrodes 12A-D, 14A-D on the substrate to allow amplification and recording of the voltage signal between the recording electrodes 12A-D and the reference electrodes 14A-D. That is, the extracellular voltage signal is recorded as the potential difference between the recording electrode 12A-D and the reference electrode 14A-D. Such voltages are induced on the electrodes 12A-D, 14A-D as a result of opening and/or closing of different ion channels across the cell membrane during the duration of the action potential, resulting in ionic currents or movement through the cell culture medium or solution. Extracellular recordings of cardiomyocyte populations are discussed in detail in US 9,612,234 and US 2011/0039294.

System 100 also includes device station 120, device station 120 configured to engage one or more cell culture devices 130 so that electrical pulses can be delivered from pulse generator 110A to cell culture devices 130 to pace the cardiomyocytes. Device station 120 is also preferably configured to be placed in an incubator or have temperature and/or humidity controls that promote cell growth and/or differentiation. In some embodiments, the device station 120 is configured to engage a cell culture device 130 implemented in a flask. In a preferred embodiment, the device station 120 includes one or more platforms or one or more slots for positioning one or more multi-well devices 130. One or more platforms or one or more sockets may include sockets, pins, or other electrically conductive structures for electrically connecting device 130 with device station 120 (e.g., through interaction of connection pads 24A). System 100 may be configured to be sufficiently portable so that porous device 130 may be positioned in a tissue culture chamber during electrical stimulation, extracellular recording, or impedance monitoring.

Device station 120 may also include electronic circuitry for connecting to an electronic pulse generator, and may include electronic switches that can make and break connections to each electrode array 10A-D within multi-well device 130 used in system 100. The switching of the device station 120 may be controlled by a software program within the pulse generator 110A.

Cell culture apparatus 130 is configured to culture cells and to transmit and receive electrical signals. This is achieved by providing a chamber-like housing with suitable electrode sensors 10A-D. The sensors 10A-D are configured to deliver electrical pulses to the cultured cells and to detect changes in beating frequency and amplitude through cell-substrate impedance measurements and/or extracellular recordings of the cells. Examples of such configurations are illustrated in fig. 2A-D.

preferably, the device 130 comprises two or more holes; however, again, the device 130 may have any number of wells required for a particular experiment. For example, the device can have 1 well, 2 wells, 3 wells, 6 wells, 8 wells, 12 wells, 24 wells, 36 wells, 96 wells, 384 wells, and the like. Preferably, the diameter of the hole is from about 1 mm to about 20 mm at the bottom of the hole (the end disposed on the substrate), more preferably from about 2mm to about 8 mm. The holes may have a uniform diameter or may taper towards the bottom such that the diameter of the holes at the end in contact with the base substrate is less than the diameter of the opposite end.

The surface of the substrate is suitable for cell attachment and cell growth. Preferably, the non-conductive substrate is planar and flat or approximately flat. The substrate may be composed of a variety of non-conductive materials known in the art, including, but not limited to, silicon dioxide on silicon, silicon-on-insulator (SOI) wafers, glass (e.g., quartz glass, lead glass, or borosilicate glass), sapphire, ceramics, polymers, fiberglass, plastics, such as polyimides (e.g., polyimide film available from Kapton, DuPont), polystyrene, polycarbonate, polyvinyl chloride, polyester, polypropylene, and urea resins. Preferably, the substrate is biocompatible with excitable cells such as ipscs and cardiomyocytes; however, non-biocompatible materials can be made biocompatible by applying a biocompatible or biomolecular coating with a suitable material, such as a biocompatible polymer or other material.

The substrate may be rigid, but in other embodiments the substrate has an elastic modulus of 1kPa to 50kPa, 5kPa to 15kPa, 8kPa to 12kPa, or 10kPa, approximating the stiffness of a native myocardium. Such a substrate can be formed as taught by Jacot et al (2008), for example, by polymerizing a 3% -7% acrylamide solution on a glass cover slip, and then coating the gel with 0.5mg/mL collagen bound by the heterobifunctional crosslinker N-sulfosuccinimidyl-6- [4 '-azido-2' -nitrophenylamino ] hexanoate (sulfo-SANPAH, Pierce Biotechnology, Rockville, IL). The tensile modulus of elasticity can be measured by calculating the slope of a stress-strain curve obtained by hanging a weight on a 15mm thick cylinder of gel. Jacot et al (2008).

In some embodiments, the substrate comprises at least one patterned island (patterned island) that improves cardiomyocyte arrangement. As non-limiting examples, the substrate may be patterned as follows: a polymer film (such as polyacrylamide doped with acryloyl-NHS) was added and fibronectin was imprinted onto the gel surface by poly (dimethylsiloxane) (PDMS) to form covalently bound fibronectin islets of about 50 μm x 50 μm. In such embodiments, it may be preferred to isolate the patterned islands with cell-repellent acrylamide. Further guidance regarding patterned islands can be found in Werley et al (2017).

To improve production efficiency, the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D of the present invention may be applied to a substrate, and then the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D applied to the substrate may be joined to a plate having no bottom holes (bottomless wells), or the electrodes may be applied to holes already having the substrate as a bottom. The electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D may be formed from a larger sheet of conductive metal, such as via laser ablation of a metal film, and may be applied directly to the substrate. Alternatively, the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D may be printed on the substrate using a printing technique (e.g., a printing technique similar to ink jet printing) in which a conductive fluid having an Ultraviolet (UV) curable monomer, polymer or compound is printed on the substrate and then a light source is applied to cure the applied conductive fluid to form the electrodes. The skilled person will appreciate that the conductive material may be applied directly to the planar substrate, or may be inserted into a groove laser ablated or formed in the surface of the substrate. Glue (e.g. UV curable glue) may be applied between the substrate and the electrodes or on the electrodes to increase the security. Furthermore, when applying the conductive fluid, it may be preferable to apply a mask prior to applying the fluid to further define the electrode area.

Electrode sensors 10A-D are an electrode array configured to deliver electrical pulses to cells on a substrate and to monitor the beating of the cells. Monitoring pulsation is performed using cell basal impedance monitoring and/or extracellular recording of the cells. Examples of suitable configurations of the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D are shown in FIGS. 2A-2D.

In some embodiments, the device comprises a separate electrode for each function. For example, in some embodiments, the device includes an electrode for electrically stimulating the cells, a different electrode for monitoring the cell-substrate impedance of cells attached to the substrate, and also a different electrode for extracellular recording of the cells. In other embodiments, the electrodes are shared between different pairs of functions. For example, one electrode of a pair of electrostimulation electrodes is shared with the other electrode to function as a cell-substrate impedance monitoring electrode pair, or shared with the other electrode to function as an extracellular recording electrode pair.

in other embodiments, the same pair of electrodes performs two different functions. For example, when impedance is not measured, a pair of cell-substrate impedance monitoring electrodes may be used for electrical stimulation.

Preferably, the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D within each well of the device are individually addressed, meaning that the electrical traces 26A and connection pads 24A of the arrays are configured such that each array can be independently connected with the device station 120 and the electronic pulse generator 110A such that each electrode pair can be operated independently of the other electrode pairs in the other electrode wells. This allows each aperture within the device 130 to be independently monitored and electrically stimulated as desired.

The electrical traces 26A of conductive material used to connect each of the electrodes 12A-D, 14A-D, 16A-D, 18A-D, 20D, 22D to the corresponding connection pads 24A may be made of any conductive material. The traces 26A may be located on a surface of the substrate and may optionally be covered by an insulating layer. Alternatively, the traces may be disposed in a second plane of the substrate.

Turning now to the cell-substrate impedance monitoring electrode pairs 16A-D, 18A-D, each pair comprises two or more electrode structures configured with dimensions and spacing such that when connected to an impedance analyzer they can function as a unit, generating an electric field in a spatial region around the impedance electrode structures. In a preferred embodiment, the impedance electrode pair 16A-D, 18A-D comprises two impedance measuring electrode structures, each impedance measuring electrode structure comprising a plurality of electrode elements 19A or substructures branching from the electrode structure. In a preferred embodiment, the electrode structures in each pair have substantially the same surface area.

With the preferred arrangement of the electrode elements 19A forming the impedance monitoring electrode pairs 16A-D, 18A-D and the gaps between the electrodes 16A-D, 18A-D in a given pair and the electrode bus, all cells (wherever they fall and attach to an impedance measuring electrode pair) can make a similar contribution to the measured total impedance change of that electrode pair. Thus, when a measurement voltage is applied to any given pair of impedance-measuring electrodes 16A-D, 18A-D, it is desirable to have similar electric field strengths at any two locations within the pair of impedance-measuring electrodes 16A-D, 18A-D. At any given position of the electrode pair, the field strength is related to the potential difference between the closest point on the first electrode structure of the electrode pair and the closest point on the second electrode structure of the electrode pair. Therefore, it is desirable to have similar potential drops across all electrode elements 19A and all electrode busses of a given electrode pair. Based on this requirement, it is desirable to have an approximately uniform distribution of electrode resistance across the electrode pair, wherein the electrode resistance at the location of interest is equal to the sum of the electrode resistance between the closest point on the first electrode structure (i.e., the point on the first electrode structure closest to the location of interest) and the first connection pad connected to the first electrode structure and the electrode resistance between the closest point on the second electrode structure (i.e., the point on the second electrode structure closest to the location of interest) and the second connection pad connected to the second electrode structure.

A substantially uniform distribution across the impedance measuring electrode pairs 16A-D, 18A-D may be achieved, for example, by: electrode structures and electrode buses having particular spacings and dimensions (length, width, thickness, and geometry) such that the resistance at any single location on a pair of electrodes is approximately equal to the resistance at any other single location on the pair of electrodes. In most embodiments, the electrode elements (or electrode structures) of a given electrode pair will have uniform spacing and have similar thickness and width, the electrode busses of a given electrode pair will have similar thickness and width, and the electrode traces 26A leading from a given electrode pair to the connection pads 24A will have very similar thickness and width. Thus, in these preferred embodiments, the electrode structure pair is designed such that the length and geometry of the electrode elements or structures, the length and geometry of the electrode traces, and the length and geometry of the bus lines provide for an approximately uniform electrode resistance distribution across the electrode pair.

When integrating a configuration with two or more apertures, the impedance-measuring electrodes 16A-D, 18A-D between the electrode arrays 10A-D of different apertures may be completely independent of each other by being connected to completely separate connection pads 24A, or may share a common connection pad 24A. For example, one of the impedance-measuring electrode structures of a first via may be connected to a connection pad 24A, which is also connected to the impedance-measuring electrode structure of another via.

Next, the extracellular recording electrodes 12A-D, 14A-D are discussed, and in general, extracellular recording is performed by amplifying and recording the voltage signal between the recording electrode(s) 12A-D and the reference electrode(s) 14A-D. In order to improve the uniformity and reproducibility of the recorded voltage signals, it is desirable to minimize any contribution of the electrical signals from the reference electrodes 14A-D to the recorded voltage signals and to ensure that most, if not all, of the recorded voltage signals originate from the recording electrodes 12A-D. Thus, in general, it is desirable and recognized that reference electrodes 14A-D have a small electrode impedance. A small electrode impedance can be achieved by using reference electrodes 14A-D with a large effective surface area: the ratio of the surface area of reference electrode 14A-D to the surface area of recording electrode 12A-D is increased to one hundred or even thousands of times.

The reference electrodes 14A-D may generally be unitary or unbranched electrodes, and may have simple geometric shapes such as circular, square, or other shapes. In other embodiments, the reference electrodes 14A-D have a branched configuration, which can make the reference electrode surface larger. In some embodiments, the ratio of the surface area of the reference electrode 14A-D to the surface area of the recording electrode 12A-D is greater than 2. In other embodiments, the ratio of the surface area of the reference electrode 14A-D to the surface area of the recording electrode 12A-D is 10 or greater than 10. In other embodiments, the ratio of the surface area of the reference electrode 14A-D to the surface area of the recording electrode 12A-D is 100 or greater than 100. In other embodiments, the ratio of the surface area of the reference electrode 14A-D to the surface area of the recording electrode 12A-D is 1000 or greater than 1000. In other embodiments, the ratio of the surface area of the reference electrodes 14A-D to the surface area of the recording electrodes 12A-D is 10,000 or greater than 10,000.

In some embodiments, the device 130 simultaneously measures impedance and performs extracellular recordings. However, some devices 130 do not perform impedance monitoring and extracellular recording simultaneously, but rather share one or both electrodes between the impedance measurement electrode pair and the extracellular recording electrode pair. When using an impedance electrode in the form of an interdigitated electrode structure with a plurality of electrode elements, the shared electrode is typically used as the reference electrode in the pair of extracellular recording electrodes. This is achieved when the surface area of the impedance monitoring electrode is large enough to act as a reference electrode than the surface area of the recording electrode. The skilled person will appreciate that by electrically switching the pair of interdigitated impedance electrodes from impedance monitoring to performing the single reference electrode function, the surface area ratio of the composite interdigitated electrode to the recording electrode will be greatly increased and may therefore be preferred in some circumstances. Further, when the reference electrode is sufficiently larger than the impedance measuring electrode, the impedance measuring electrode may also be used as the recording electrode, but this is not preferable. While not preferred, this approach is more likely to be used when using an impedance measuring electrode configuration with a small working electrode and a large counter electrode, as previously detailed in the art.

B. Functional maturation of immature cardiomyocytes derived from pluripotent stem cells (ipscs) using electrical stimulation

iPSC cardiomyocytes have been shown to often exhibit immature or embryonic phenotypes as demonstrated by gene expression analysis, structural studies, and functional studies. One of the hallmarks of the immature phenotype of iPSC cardiomyocytes is a negative force-frequency relationship, in which increased beating rate does not increase amplitude or beating force. Conversely, as shown in fig. 5, the amplitude or pulsatile force tends to decrease or weaken. To this end, the present invention provides methods of monitoring this force-frequency relationship and orchestrates it with methods developed to induce further maturation of iPSC-and ESC-derived cardiomyocytes into cardiomyocytes with a more mature or more adult-like phenotype. In particular, the systems and methods herein not only induce iPSC and ESC derived cardiomyocyte functional maturation, but also monitor maturation electronically by measuring the pulse amplitude and calculating the force-frequency relationship. The result is a population of functionally mature cardiomyocytes that respond appropriately in a positive force-frequency relationship, which can then be used for subsequent testing of inotropic and chronotropic compounds or for maturation process studies.

In view of the above, there is provided a method of maturing functional immature cardiomyocytes, the method comprising: providing a system, preferably having an electrode sensor array, configured to culture a beating cell, electrically pace the beating cell, and monitor the beating of the beating cell; culturing immature cardiomyocytes in said system; monitoring immature cardiomyocytes to characterize cardiomyocyte beating as synchronous or asynchronous; and, if synchronized, electrically paces the immature cardiomyocytes according to the pulse profile until the immature cardiomyocytes mature into functional adult cardiomyocytes.

As with the system 100 described above and with brief reference to fig. 1-2D, the impedance-monitoring electrode arrays 16A-D, 18A-D may be positioned on a substrate and may be operably connected with an impedance analyzer 110B to monitor the cell-substrate impedance of a population of cells cultured in the system 100. Alternatively, the extracellular recording electrode arrays 12A-D, 14A-D may be operably connected to an extracellular recording amplifier 110C for extracellular recording of a population of cells cultured in the system. Still further, the system 100 may include impedance monitoring electrode arrays 16A-D, 18A-D on a substrate, extracellular recording electrode arrays 12A-D, 14A-D on a substrate, an impedance analyzer 110B and an extracellular recording amplifier 110C, wherein the system 100 is configured to monitor cell-substrate impedance and perform extracellular recording of a population of cells cultured in the system 100.

As a non-limiting example, the method may be implemented using an xcelligene RTCA CardioECR system (ACEA Biosciences, inc. san Diego, CA) as depicted in fig. 1 by: seeding iPSC-derived cardiomyocytes into wells of CardioECR plates (E-Plate CardioECR 48) at appropriate densities; allowing cells to attach, grow and achieve spontaneous beating rhythm while continuously monitoring spontaneous beating activity using CardioECR system; iPSC-derived cardiomyocytes were then subjected to directed electrical pacing after spontaneous synchronized beating of the cardiomyocyte monolayer was observed during 4 weeks post-inoculation, while beating activity was continuously monitored.

The skilled person will appreciate that the method may be used to differentiate various cardiomyocyte precursor cells. In particular, the method can be used to further differentiate ipscs or ESCs that have partially differentiated into immature cardiomyocytes. Conceptually, the differentiation of ESCs into terminally differentiated cardiomyocytes was not different from iPSCs. Both are dependent on regulatory signaling pathways (activin/node, TGF β, GSK3, Wnt, BMP, etc.) that lead to embryonic development in vivo and follow the same general procedure. Moran et al (2010). After 7 days of differentiation, cardiomyocytes derived from PSC began to contract. Moran et al (2010). However, as already suggested before, such PSCs have an immature phenotype. Immature cardiomyocytes tend to differ in gene expression profiles and in action potential profiles compared to mature cardiomyocytes. Moran et al (2010). Thus, it can be determined whether the cardiomyocytes are immature by gene expression analysis or, more preferably, using the electrical monitoring methods described herein. In particular, beat frequency, contractility, beat amplitude, and action potential can be assessed using cell-substrate impedance monitoring and/or extracellular recordings. Furthermore, the force-frequency relationship can then be evaluated by comparing the beat amplitude at increasing beat frequencies (induced by pacing). An increase in beating amplitude with increasing frequency is indicative of mature cardiomyocytes; while a decrease in beat amplitude with increasing frequency is indicative of immature cardiomyocytes. Thus, cardiac myocytes can be electrically tested by measuring the amplitude of the beat while increasing the frequency of the beat to determine whether they have an adult or immature phenotype. This test may be performed at different times throughout the maturation process to assess the degree of maturation or as feedback to adjust the pulse frequency.

It should be added that the methods are not limited to any particular species, but that the most commonly used cells for these methods are from the same species that is ultimately diagnosed or treated with a therapeutic agent. For this reason, when making measurements that may lead to human therapy such as treatment of heart disease, the human iPSC is most commonly used. When performing veterinary studies, ipscs or ESCs from the same species to be diagnosed or treated are most commonly used. Still further, when the objective is to diagnose or treat a human patient, the ipscs are most preferably derived from cells collected from the same patient to be treated or diagnosed.

The method of the invention is further advantageous in that no special cell culture medium is required. Conventional cell culture media can be used. However, the system may also incorporate the use of specialized media that can further promote maturation in addition to electrical pacing.

Although the method is described as being ready for further differentiation of immature cardiomyocytes, the initial differentiation procedure can also be performed on a cell culture device and monitored electrically. In addition, a coating for differentiating ipscs into mature cardiomyocytes may also be applied to the cell culture device and a corresponding procedure performed.

Further, in some embodiments, maturation of the immature cardiomyocytes is performed by arranging the immature cardiomyocytes on a patterned substrate. By way of non-limiting example, culturing cardiomyocytes on fibronectin patterned islets separated by cell-repellent naked (bare) polyacrylamide showed improved spontaneous beating. Werley et al (2017). As such, the methods herein may include culturing immature cardiomyocytes on a patterned substrate, such as, but not limited to, a fibronectin pattern as taught or suggested by Werley et al, with the addition of electrodes for pulsing the cardiomyocytes and their electrical monitoring. That is, the iPSC maturation device can be patterned for further drive in maturation.

In some embodiments, the immature cardiomyocytes are cultured on a substrate having an elastic modulus approaching the natural myocardial stiffness of 1 to 50kPa, 5 to 15kPa, 8 to 12kPa, or 10 kPa. Such a substrate may be formed following the teachings of Jacot et al (2008), with the addition of electrodes for pulsing the cardiomyocytes and their electrical monitoring. That is, the iPSC maturation device may have an elastic modulus that mimics the myocardium.

In some embodiments, the immature cardiomyocytes are cultured in the presence of a vascular-like network. As a non-limiting example, immature cardiomyocytes were seeded on the vascular network of Endothelial Cells (EC). Vascular ECs produce a variety of autocrine and paracrine factors, including VEGF, angiogenin, and nitric oxide, which affect the metabolism, growth, contractility, and rhythmicity of cardiac myocytes. Brustaert (2003). In other embodiments, immature cardiomyocytes are seeded on a vascular network of fibroblasts. In other embodiments, immature cardiomyocytes are seeded on the vascular network of Human Umbilical Vein Endothelial Cells (HUVECs) and human adipose basal cells (hascs). Adipose basal cells (hascs) have been shown to produce a number of angiogenic factors and cytokines, including VEGF, hepatocyte growth factor, and angiogenin. In other embodiments, immature cardiomyocytes are seeded on the vascular network of HUVECs and human foreskin fibroblasts. Each of the above has proven beneficial for culturing cardiomyocytes. Vourenpaa et al. Each of which may be used with the methods and systems herein.

Looking back at the electrical monitoring of immature cardiomyocytes to determine whether the beating is synchronized, it is preferred that the beating of the cells is synchronized before the pulse profile of the immature cardiomyocytes further differentiate into mature cardiomyocytes is performed. Electrical monitoring may include monitoring the cell-substrate impedance of immature cardiomyocytes, extracellular recording of immature cardiomyocytes, or both. More preferably, the beating parameters (whether raw impedance values or cell indices) are obtained by cell-substrate impedance monitoring and then compared over time to determine if the beats are synchronized. Specifically, it is determined whether the beats are synchronized by determining the beat rate (also referred to as "beat frequency"), and comparing the beat rate over time. That is, a population with beat asynchrony will not generate a continuous and reproducible impedance-based curve. Pulsatile cultures also did not produce reproducible pulsatile peaks (fig. 3 depicts a series of pulsatile peaks). Instead, the curve may shift or lack refinement (refinement). The beat is monitored until synchronization, which typically lasts from about 4 days to several weeks.

it has been found that synchronized beats can be achieved more quickly by electrically pacing immature cardiomyocytes. That is, if the cardiomyocyte beats out of sync, the method can further include electrically pacing the immature cardiomyocyte until the cardiomyocyte beats in sync. Electrically pacing the cardiomyocytes to synchronize the beats generally maintains a slower pulse rate compared to the pulse profile used for differentiation. That is, immature cardiomyocytes can be paced slowly but stably until the beat is synchronized. Typically, pacing is at a constant rate. Most preferably, the pacing is at the same pace as the rate of beating of immature cardiomyocytes in culture. The frequency is typically about 0.5Hz to about 1Hz, but may also be slower, such as 0.5Hz, 0.1Hz, 0.2Hz, 0.3Hz, or 0.4 Hz. The diseased genotype/phenotype may have a slow beat rate (e.g., 0.5Hz-0.5 Hz).

once the beats are synchronized, the method includes electrotransport of the pulse profile into immature cardiomyocytes, thereby inducing further differentiation. That is, immature cardiomyocytes are paced or stimulated at a pulse rate to drive maturation using an appropriately selected pulse intensity or amplitude. The pulse intensity should be high enough to pace the cardiomyocytes (i.e., each applied pulse results in one beat cycle of the cardiomyocytes in the hole). On the other hand, the pulse intensity or amplitude should be within a certain range, since very high field strengths may have undesired effects on the cardiac muscle cells. For example, when the pulse intensity is too high, water electrolysis may occur on the electrodes, resulting in a local pH change in the cell culture medium and bubbling of O2 and/or H2, thereby producing an undesirable physiological effect on the cardiomyocytes. The exact range of pulse intensities that effectively paces cardiomyocytes under any given experimental condition may also depend on the electrode geometry used in the wells of the device. For example, an electrode array with a larger spacing between adjacent electrodes will require a greater pulse intensity to achieve the same pacing effect than an electrode array with a smaller spacing. For example, electrode geometries with large distances between adjacent electrodes may require large pulse intensities of up to a few volts (e.g., 3V) or even higher.

An exemplary pulse spectrum has a pulse duration or width of 0.1 to 0.2 milliseconds, with durations of up to 10 milliseconds being acceptable. The cardiomyocytes should be stimulated or paced with an appropriately selected pulse duration. With appropriate application of pulse intensity, the pulse duration should be long enough to pace the cardiomyocytes (i.e., each applied pulse causes one pulse cycle of the cardiomyocytes in the well). On the other hand, the pulse duration does not have to be too long, since too long a pulse duration may have undesired effects on the cardiac muscle cells. For example, when the pulse duration is too long, a large average effective direct current DC field bias may be applied to the electrodes (e.g., for a pulse duration of 700 milliseconds at 1Hz, a 1V pulse means a DC voltage of 0.7V (═ 1V x 700 milliseconds x 1Hz) applied to the electrodes), resulting in water electrolysis at the electrodes and causing undesirable effects such as local pH changes in the cell culture medium and bubbling of O2 and/or H2, and undesirable physiological effects on the cardiomyocytes. In general, the duration of the applied pulses should be as short as possible, so long as effective pacing of the cardiomyocytes is induced (i.e., each applied pulse causes one beat cycle of the cardiomyocytes in the well).

Most preferably, the pulse spectrum has a rectangular pulse shape and an intensity or voltage of about 0.7V to 1V (but may be up to about 2 volts). In other embodiments, the voltage is up to 3 volts. In some cases, the intensity increases as the pacing rate increases. The pulse duration or pulse width is typically about 0.1 to 0.2 milliseconds, but may be as long as 10 milliseconds, and may also increase as the pacing rate increases.

Although pulse intensity and duration may increase over time, the acceleration of differentiation of immature cardiomyocytes is believed to be primarily due to an increase in the beating rate of the cell culture, which is achieved by increasing electrical pacing. In experiments, it was found that increasing the beating rate of immature cardiomyocyte cultures over time increases the differentiation rate. To this end, it is demonstrated herein that it can be accomplished within weeks, which would take months to around a year. To this end, differentiation of immature cardiomyocytes is primarily accomplished by pacing immature cardiomyocytes over time by applying a pulse profile with varying pulse frequency.

The skilled person will appreciate that the pulse profile may vary with the pulse rate of the immature cardiomyocytes and also with the method used to differentiate the immature cardiomyocytes into mature cardiomyocytes. As an example, the frequency at which the pulse profile begins is typically coincident or nearly coincident with the beat rate of immature cardiomyocytes, which can vary. In most cases, the pulse spectrum will start with a slower pulse frequency, and the frequency increases over time. This initial lower pulse rate should be appropriate so that the cardiomyocytes can follow up with the pacing pulses (i.e., each pacing pulse can cause one beat cycle of the cardiomyocytes in the well). Experimentally, an adult-like or mature phenotype can be obtained more quickly when pacing is increased stepwise over time in increments of about 0.5Hz until a final pacing rate of 2Hz is reached. Beneficial results were surprisingly achieved when the pacing rate was maintained at the same rate for one week, and then paced at an increased rate of about 0.5Hz per week until a pacing rate of 2Hz was reached. However, it is expected that shorter times (e.g., days) will be acceptable as will longer durations (e.g., weeks).

As an example, a pulse spectrum with a pulse frequency varying between 0.5Hz and 2Hz over time was developed. As another example, the pulse spectrum includes a pulse frequency that varies between 1Hz and 2Hz over time. As another example, the pulse spectrum includes a pulse frequency that increases from 0.75Hz to 2Hz over time. As another example, cardiomyocytes of certain genotypes or phenotypes (such as diseased genotypes/phenotypes) may require a lower initial pulse frequency, e.g., 0.5Hz or 0.3Hz or even lower, in order for the cardiomyocytes to be effectively paced. On the other hand, the pulse rate may increase over time from an initially low pulse rate to a higher pulse rate, such as 3Hz or even higher, depending on the requirements of the assay in which these paced cardiomyocytes are to be used. The skilled person will appreciate that the time period between increasing the pulse frequency may also vary, but in some embodiments the time course of increasing the pulse frequency is 2-6 days. In other embodiments, the time course of increasing the pulse frequency is 1 to 5 weeks. As a non-limiting example, further differentiation of immature cardiomyocytes can occur when pulsed at 0.75Hz for 1 week, 1.5Hz for one more week, and 2Hz for one more week. Increasing the pulse frequency is preferably frequency doubling over time. Cardiomyocyte maturation can be followed by periodically testing the force-frequency relationship of the beating culture to determine whether the force-frequency relationship is increasing (indicating maturation) or decreasing (indicating maturation). In particular, the test may include gradually increasing the pacing rate of the cardiomyocytes while recording the beat amplitude as a surrogate indicator of force. If the beat amplitude/electrical pacing rate relationship is negative, then the cardiomyocyte is considered immature; conversely, if the amplitude/electrical pacing rate relationship is positive, the cardiomyocytes are considered "mature".

Thus, inducing differentiation of immature cardiomyocytes into cardiomyocytes with an adult phenotype may comprise electrically pacing the cardiomyocytes by applying a pulse profile having a pulse frequency between 1Hz and 2 Hz. Most preferably, the pulse spectrum increases the pulse frequency over time. In embodiments, where the immature cardiomyocytes beat at a frequency of less than 1Hz, the applied pulse frequency may double in frequency over time. Immature cardiomyocytes were pulsed until a positive force-frequency relationship was obtained. Specifically, functionally mature cardiomyocytes have a positive force frequency; whereas immature cardiomyocytes have negative force-frequency.

In some embodiments, a compound that drives cardiomyocyte maturation is added to immature cardiomyocytes to help drive maturation. In some embodiments, a growth factor or growth hormone is added to the immature cardiomyocytes. In some embodiments, the angiogenic factor is added to immature cardiomyocytes. In some embodiments, angiogenin is added to immature cardiomyocytes. Angiogenin is a family of growth factors, including glycoproteins angiogenin 1 and 2 and ortholog 4. In other embodiments, VEGF is added.

triiodo-L-thyronine (T3) is an essential growth hormone for optimal cardiac growth. Yang et al (2014). Yang found that treatment of immature cardiomyocytes with T3 increased cardiomyocyte size, increased sarcomere length, decreased proliferation, increased contractile force production, enhanced calcium handling properties, and increased maximal mitochondrial respiration capacity. Thus, when coupled with electrical pacing, the addition of growth hormone can further accelerate the maturation process.

As further demonstrated above, example III describes the use of the xcelligene RTCA CardioECR system to measure and monitor dynamic changes in pulsatile activity during chronic application of electrical pacing to iPSC cardiomyocytes. Cells were then tested in example IV to assess functional maturation by electrically pacing cells over a 40 minute interval to determine if the cells responded with a positive force-frequency relationship. The confirmation results are shown in FIGS. 4 to 5.

As a further demonstration, example V provides a test using inotropic compounds to determine if the treated cells are responding properly, wherein the meaning of "responding properly" is that positive inotropic compound treatment will result in an increase in pulsatile amplitude, while negative inotropic compound treatment will result in a decrease in pulsatile amplitude. The results are depicted in FIGS. 6A-B. Overall, an increase in beat rate (B) was detected in both spontaneously beating cardiomyocytes and chronically paced cardiomyocytes after exposure to isoproterenol, whereas a positive inotropic effect, reflected by an increase in beat amplitude (a), was observed only in paced cells. Data are presented as mean ± SD (N ═ 5)

C. Screening for inotropic and/or chronotropic compounds using iPSC-or EC-derived cardiomyocytes with a more adult-like phenotype

The force-changing agent, or the force-changing drug, is a drug that changes the force of heart contraction. The force-changing medicines have two types: positive inotropic drugs and negative inotropic drugs. Positive inotropic agents enhance the strength of the heartbeat, while negative inotropic agents attenuate the strength of the heartbeat. Both types of inotropic agents are used to treat a number of different cardiovascular disorders. Accordingly, the present invention provides methods for screening inotropic agents for potential therapeutic use.

The type of inotropic agent administered depends on the condition. Positive inotropic drugs enhance the contraction of the heart so that the heart can pump more blood with fewer heartbeats. Such drugs are commonly administered to patients with congestive heart failure or cardiomyopathy. These drugs may also be administered to patients who have had a recent heart attack. In some cases, inotropic agents are administered to patients with cardiac weakening after cardiac surgery (in the case of cardiogenic shock). Examples of positive inotropic agents include digoxin, amiodarone (amidarone), berberine, levosimendan, omecamivo (omeamikamitiv), dopamine, dobutamine, dopexamine, epinephrine, isoproterenol, angiotensin (antimention).

Conversely, negative inotropic drugs attenuate the contraction of the heart and slow down the heart rhythm. These drugs are used to treat hypertension (elevated blood pressure), chronic heart failure, abnormal heart rhythm (arrhythmia) and chest pain (angina pectoris). They are sometimes used in heart attack patients to relieve heart pressure and prevent future heart attacks. Examples of negative inotropic agents include diltiazem, verapamil, clevidipine, quinidine, procainamide, propylpiramide (dispyramide), and flecainide.

Therefore, the use of iPSC-or ESC-derived cardiomyocytes with a more adult-like phenotype to screen for inotropic compounds may be an important tool compared to immature cardiomyocytes and may provide more reproducible results. Accordingly, the invention also provides methods of screening for potential inotropic compounds using functionally mature cardiomyocytes. Once a compound is identified, the compound can be provided with a pharmaceutically acceptable carrier suitable for the intended route of administration and administered to a patient in need thereof, such as a patient suffering from congestive heart failure, cardiomyopathy, hypertension (elevated blood pressure), chronic heart failure, abnormal heart rhythm (arrhythmia), and/or chest pain (angina pectoris). Alternatively, the medication may be prescribed after cardiac surgery.

Compounds that affect the rate of pulsation are called chronotropic compounds or chronotropic drugs. Positive chronotropic drugs increase the rate of pulsatility and include most adrenergic agonists. These drugs include atropine, dopamine, epinephrine, isoproterenol, milrinone, and theophylline. Negative chronotropic drugs reduce the rate of pulsatility and include beta blockers such as metoprolol, acetylcholine, digoxin, diltiazem, and verapamil.

As demonstrated herein, the method is capable of resolving beat power or amplitude and beat frequency with high resolution. Experimentally, ipscs differentiated using the pacing method were also demonstrated herein to respond to cardiac drugs. To this end, there is provided a method of characterizing the effect of a compound on the beating of a cardiomyocyte, the method comprising: providing a system having an electrode sensor array, wherein the system is configured to culture, electrically pace, and electrically monitor a beating cell; culturing immature cardiomyocytes in said system; electrically pacing immature cardiomyocytes using an electrical pulse profile until the cardiomyocyte function is mature; adding to functionally mature cardiomyocytes a compound suspected of having an effect on cardiomyocyte beating power or cardiomyocyte beating rate; electrical monitoring of cultured cardiomyocytes before and after compound addition; determining at least one parameter indicative of the stroke power or rate before and after the compound is added; and comparing at least one parameter determined before and after the addition of the compound, thereby identifying a difference in response to the addition of the compound. According to the method, a compound can be characterized as a positive inotropic compound if pulsatility force increases after addition of the compound, or as a negative inotropic compound if pulsatility force decreases after addition of the compound, and/or as a positive chronotropic compound if pulsatility rate increases after addition of the compound, or as a negative chronotropic compound if pulsatility rate decreases after addition of the compound.

As a proof of principle, the assay was performed in example VI using milrinone. As shown in FIGS. 7A-B, the pulse rate (B) was slightly increased for both spontaneously beating cardiomyocytes and chronically paced cardiomyocytes following exposure to the positive inotropic compound, milrinone. However, in electrically paced cardiomyocytes, the beat amplitude (a) is significantly increased. Data are expressed as mean ± SD (N ═ 5).

Further analysis was performed to assess whether pulsatile force would increase after administration of a set of additional inotropic compounds. Figure 8 shows a set of force frequency displays after administration of a set of inotropic compounds before and after electrical pacing of iPSC cardiomyocytes. Positive inotropic compounds bayK and digoxin were shown to have positive inotropic effect, whereas ISO had positive chronotropic effect.

As with the system described above, and with brief reference to FIGS. 1-2D, the electrode arrays 10A-D can include impedance monitoring electrode arrays 16A-D, 18A-D positioned on a substrate and operatively connected to an impedance analyzer 110B to monitor the cell-substrate impedance of a population of cells cultured in the system 100. Alternatively, the electrode arrays 10A-D may include extracellular recording electrode arrays 12A-D, 14A-D operatively connected to an extracellular recording amplifier 110C for extracellular recording of a population of cells cultured in the system 100. Still further, the system 100 may include impedance monitoring electrode arrays 16A-D, 18A-D on a substrate, extracellular recording electrode arrays 12A-D, 14A-D on a substrate, an impedance analyzer 110B, and an extracellular recording amplifier 110C, wherein the system 100 is configured to monitor cell-substrate impedance and perform extracellular recording of a population of cells cultured in the system 100.

By way of non-limiting example, the method may be implemented using an xCELLigence RTCA CardioECR system (as shown in fig. 1) in the following manner: seeding iPSC cardiomyocytes into the wells of CardioECR plates (E-plateau CardioECR 48) at appropriate densities; allowing cells to attach, grow and achieve spontaneous beating rhythm while continuously monitoring spontaneous beating activity using CardioECR system; then, after spontaneous synchronized beating of the cardiomyocyte monolayer was observed, iPSC cardiomyocytes were subjected to directed electrical pacing over a period of 4 weeks post-inoculation while continuously monitoring beating activity.

Compounds suspected of having an effect on the cardiomyocyte beating power or rate of functionally mature cardiomyocytes can be added directly to the device and monitored electrically over time. Preferably, the cardiac myocytes are monitored electrically before and after the addition of the compound. In some embodiments, the cardiomyocytes are compared to a control population that does not receive the compound. At least one parameter indicative of the pulsatility or rate is then determined and compared to identify whether the pulsatility parameter is affected by the addition of the compound.

It should be added that the methods are not limited to any particular species, but that the most commonly used cells for these methods are from the same species that is ultimately diagnosed or treated with a therapeutic agent. For this reason, when making measurements that may lead to human therapy such as treatment of heart disease, the human iPSC is most commonly used. When performing veterinary studies, ipscs or ESCs from the same species to be diagnosed or treated are most commonly used. Still further, when the objective is to diagnose or treat a human patient, the ipscs are most preferably derived from cells collected from the same patient to be treated or diagnosed.

Although the method is described as being ready for further differentiation of immature cardiomyocytes, the initial differentiation procedure can also be performed on a cell culture device and monitored electrically. In addition, a coating for differentiating ipscs into mature cardiomyocytes may also be applied to the cell culture device and a corresponding procedure performed. .

In some embodiments, maturation of the immature cardiomyocytes is performed by arranging the immature cardiomyocytes on a patterned substrate. By way of non-limiting example, culturing cardiomyocytes on fibronectin patterned islets separated by cell-repellent naked (bare) polyacrylamide showed improved spontaneous beating. Werley et al (2017). As such, the methods herein may include culturing immature cardiomyocytes on a patterned substrate, such as, but not limited to, a fibronectin pattern as taught or suggested by Werley et al, with the addition of electrodes for pulsing the cardiomyocytes and their electrical monitoring. That is, the iPSC maturation device can be patterned for further drive in maturation.

In some embodiments, the assay is performed on a substrate having an elastic modulus approaching natural myocardial stiffness of 1kPa to 50kPa, 5kPa to 15kPa, 8kPa to 12kPa, or 10 kPa. Such a substrate may be formed following the teachings of Jacot et al (2008), with the addition of electrodes for pulsing the cardiomyocytes and their electrical monitoring. That is, the iPSC maturation device may have an elastic modulus that mimics the myocardium.

In some embodiments, the assay is performed in the presence of a vascular-like network. As a non-limiting example, immature cardiomyocytes were seeded on the vascular network of Endothelial Cells (EC). Vascular ECs produce a variety of autocrine and paracrine factors, including VEGF, angiogenin, and nitric oxide, which affect the metabolism, growth, contractility, and rhythmicity of cardiac myocytes. Brustaert (2003). In other embodiments, immature cardiomyocytes are seeded on a vascular network of fibroblasts. In other embodiments, immature cardiomyocytes are seeded on the vascular network of Human Umbilical Vein Endothelial Cells (HUVECs) and human adipose basal cells (hascs). Adipose basal cells (hascs) have been shown to produce a number of angiogenic factors and cytokines, including VEGF, hepatocyte growth factor, and angiogenin. In other embodiments, immature cardiomyocytes are seeded on the vascular network of HUVECs and human foreskin fibroblasts. Each of the above has been shown to be beneficial for culturing cardiomyocytes. Vourenpaa et al (2004). Each of which may be used with the methods and systems herein.

Preferably, the population of cardiomyocytes undergoing synchronized beating is paced. Cell-substrate impedance monitoring of immature cardiomyocytes can be used to obtain pulsatility parameters (whether raw impedance values or cellular indices) and then compared over time to determine synchronized pulsatility. A population with out-of-sync beats will not generate a continuous and reproducible impedance-based curve. Pulsatile cultures also did not produce reproducible pulsatile peaks. That is, the curves will incorrectly overlap each other. Instead, the curve may shift or lack fineness. To this end, the cardiomyocyte beat resolution provided herein is sufficiently high to be sufficient to determine whether the cardiomyocyte beats are synchronized. The system herein typically achieves a resolution of 40ms, and reproducibly achieves a resolution of 20 ms. By "40 ms resolution" or "20 ms resolution" is meant that the system will make consecutive measurements 40ms or 20ms apart from each other. Higher resolutions of 10ms or 1ms are also included. Typically, the cell population is measured over a time period of about 20 seconds, during which pacing is preferably stopped. Achieving synchronized beats without electrical stimulation typically takes 6 days to 2 weeks.

It has been found that synchronized beats can be achieved more quickly by electrically pacing immature cardiomyocytes. That is, if the cardiomyocyte beats out of sync, the method can further include electrically pacing the immature cardiomyocyte until the cardiomyocyte beats in sync. Electrically pacing the cardiomyocytes to synchronize the beats generally maintains a slower pulse rate compared to the pulse profile used for differentiation. That is, immature cardiomyocytes can be paced slowly but stably until the beat is synchronized. Typically, pacing is at a constant rate. Most preferably, the pacing is at the same pace as the rate of beating of immature cardiomyocytes in culture. The frequency is typically about 0.5Hz to about 1 Hz. In some cases (e.g., diseased genotypes/phenotypes), the frequency may be 0.05Hz to 0.5 Hz. Pacing to achieve synchronized beats typically occurs within a few days.

Pacing the immature cardiomyocytes comprises electrically delivering a pulse profile to the immature cardiomyocytes, thereby inducing further differentiation. Most preferably, the pulse spectrum has a rectangular pulse shape and an intensity of about 0.7V to 1V or up to 2 volts. In some embodiments, the voltage is up to 3 volts. However, other pulse shapes may be used, such as sinusoidal, triangular, and/or sawtooth waveforms. In some cases, the intensity increases when the pacing rate increases, but in other cases, the intensity remains consistent. The pulse duration is typically about 0.1 to 0.2 milliseconds or as long as 10 milliseconds, and may also increase with increasing pacing rate. However, the pulse duration may also remain consistent while the pulse frequency increases over time.

Although pulse intensity and duration may increase over time, the acceleration of differentiation of immature cardiomyocytes is believed to be primarily due to an increase in the beating rate of the cell culture, which is achieved by increasing electrical pacing. In experiments, it was found that increasing the beating rate of immature cardiomyocyte cultures over time increases the differentiation rate. To this end, it is demonstrated herein that it can be accomplished within weeks, which would take months to around a year. Beneficial results were surprisingly achieved when the pacing rate was maintained at the same rate for one week, and then paced at an increased rate of about 0.5Hz per week until a pacing rate of 2Hz was reached. However, it is expected that shorter times (e.g., days) are acceptable as are longer durations (e.g., weeks).

Thus, inducing differentiation of immature cardiomyocytes into cardiomyocytes with an adult phenotype may comprise electrically pacing the cardiomyocytes by applying a pulse profile having a pulse frequency between 1Hz and 2 Hz. Most preferably, the pulse frequency of the pulse spectrum increases over time. In embodiments, where the immature cardiomyocytes are beating at a frequency of less than 1Hz, the applied pulse frequency may be doubled in frequency over time. Electrical pacing is continued until the cardiomyocytes have a positive force-frequency relationship. Specifically, functionally mature or adult cardiomyocytes have a positive force-frequency relationship; whereas immature cardiomyocytes have a negative force-frequency relationship.

As with monitoring of functional maturation of cardiomyocytes, high resolution myocardial beating reveals a shift in beating force and/or frequency when compounds are added that correspondingly affect beating force and/or frequency. That is, while these methods use electrical monitoring of cardiomyocytes to track differentiation into mature cardiomyocytes, the same electrical monitoring methods can measure and track the effect of potential therapeutic or toxic compounds on cardiomyocytes. In particular, electrical monitoring of the cardiomyocytes can reveal changes in the pulsatile power or amplitude, and can reveal changes in the pulsatile frequency in response to the addition of the compound.

Thus, by monitoring the pulsatile power or amplitude and the frequency of the pulsation, the inotropic and chronotropic effects can be evaluated. As a non-limiting example, a comparison of at least one parameter determined before and after addition of a compound can be used to characterize the compound as follows: a positive inotropic compound if the pulsatility force increases after addition of the compound, or a negative inotropic compound if the pulsatility force decreases after addition of the compound, and/or a positive chronotropic compound if the pulsatility rate increases after addition of the compound, or a negative chronotropic compound if the pulsatility rate decreases after addition of the compound.

D. Screening of compounds affecting cardiomyocyte maturation to a more adult-like phenotype using iPSC-or ESC-derived cardiomyocytes

The skilled person will appreciate that the methods may also be used to assess the effect of a compound on the maturation process. As an exemplary embodiment, there is provided a method of characterizing the effect of a compound on cardiomyocyte maturation, the method comprising: providing a system configured to culture a beating cell, electrically pace the beating cell, and monitor the beating of the beating cell; culturing immature cardiomyocytes in a system; electrically pacing immature cardiomyocytes according to a pulse profile that matures cardiomyocyte function; adding a compound suspected to have an effect on cardiomyocyte maturation; electrical monitoring of cultured cardiomyocytes before and after compound addition; determining at least one parameter indicative of a pulsatile force-frequency relationship before and after addition of the compound; comparing at least one parameter determined before and after the addition of the compound, thereby identifying a difference in response to the addition of the compound.

In connection therewith, there is provided a method of characterizing the effect of a compound on the beating of a cardiomyocyte, the method comprising: providing a system configured to culture a beating cell, electrically pace it, and monitor its beating; culturing two populations of immature cardiomyocytes in a system; adding a compound suspected of having an effect on cardiomyocyte maturation to a population of immature cardiomyocytes; electrically pacing the two populations of immature cardiomyocytes according to a pulse profile that functionally matures the immature cardiomyocytes until at least one of the two populations of cardiomyocytes is functionally mature; and, characterizing the compound as further driving maturation if the functional maturation of the cardiomyocyte population to which it is added precedes that of another cardiomyocyte population.

As described in the above disclosure, in some embodiments, the immature cardiomyocytes are derived from induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs). Preferably, the two populations of immature cardiomyocytes are cultured before the compound is added until the beats within the respective populations are synchronized. That is, each population of cardiomyocytes should have synchronized beats, but the beats between the two different populations need not be synchronized. Cell-substrate impedance monitoring of immature cardiomyocytes can be used to obtain a beating parameter (whether a raw impedance value or a cellular index) and then compared over time to determine synchronized beating. Populations with asynchrony of beats do not generate a continuous and reproducible impedance-based curve. Pulsatile cultures also did not produce reproducible pulsatile peaks. That is, the curves will incorrectly overlap each other. Instead, the curve will shift or lack fineness. To this end, the cardiomyocyte beat resolution provided herein is sufficiently high to determine whether the cardiomyocyte beats are synchronized. The system herein typically achieves a resolution of 40ms, and reproducibly achieves a resolution of 20 ms. A 40ms resolution or a 20ms resolution means that the system will make consecutive measurements 40ms or 20ms apart from each other. Higher resolutions of 10ms or 1ms are also included. Typically, the cell population is measured over a time period of about 20 seconds, during which pacing is preferably stopped. Achieving synchronized beats without electrical stimulation typically takes 6 days to 2 weeks.

Pacing the immature cardiomyocytes comprises electrically delivering a pulse profile to the immature cardiomyocytes, thereby inducing further differentiation. Most preferably, the pulse spectrum has a rectangular pulse shape and an intensity of about 0.7V to 1V or up to 2 volts. In some embodiments, the voltage is up to 3 volts. However, other pulse shapes may be used, such as sinusoidal, triangular, and/or sawtooth waveforms. In some cases, the intensity increases when the pacing rate increases, but in other cases, the intensity remains consistent. The pulse duration is typically about 0.1 to 0.2 milliseconds or as long as 10 milliseconds, and may also increase with increasing pacing rate. However, the pulse duration may also remain consistent, but the pulse frequency increases over time.

Although pulse intensity and duration may increase over time, acceleration of differentiation of immature cardiomyocytes is believed to be primarily due to the increasing pulse rate of maintenance cell cultures, which is achieved by means of electrical pacing of the cells. Experimentally, it was found that increasing the beating rate over time increased the differentiation rate. To this end, it is demonstrated herein that it can be accomplished within weeks, which would take months to around a year. Beneficial results were surprisingly achieved when the pacing rate was maintained at the same rate for one week, and then paced at an increased rate of about 0.5Hz per week until a pacing rate of 2Hz was reached. However, it is expected that shorter times (e.g., days) will be acceptable as will longer durations (e.g., weeks).

Thus, inducing differentiation of immature cardiomyocytes into cardiomyocytes with an adult phenotype may comprise electrically pacing the cardiomyocytes by applying a pulse profile having a pulse frequency between 1Hz and 2 Hz. Most preferably, the pulse spectrum increases the pulse frequency over time. In embodiments, where the immature cardiomyocytes are beating at a frequency of less than 1Hz, the applied pulse frequency may be doubled in frequency over time. Electrical pacing is continued until the cardiomyocytes beat in a positive force-frequency relationship. In particular, functionally adult cardiomyocytes have a positive force-frequency relationship; whereas immature cardiomyocytes have a negative force-frequency relationship.

One or more compounds may be added to the immature cardiomyocyte population during or prior to electrical pacing and their effect on maturation assessed by comparison to controls. That is, the force-frequency relationship between cell populations in response to the addition of a compound can be compared to determine whether the addition of a compound drives further maturation or slows maturation. The skilled artisan will appreciate that by providing different concentrations of such compounds to determine the dose effect on maturation, further studies can be made to determine changes in maturation in response to administration of the compounds.

As with monitoring maturation, the compound can be administered to one or more immature cardiomyocyte populations and the force-frequency relationship assessed over time. Changes in the force-frequency relationship in response to compound administration demonstrate differences in the degree or course of maturation.