Systems and methods for screening pharmaceutical chemicals

• US 20030064358A1
• Filed: 08/14/2002
• Published: 04/03/2003
• Est. Priority Date: 08/14/2001
• Status: Active Grant

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Wakatsuki, Tetsuro, McConnaughey, William B., Elson, Elliot
• Granted Patent: US 7,449,306 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: G01N 2203/0005 Repeated or cyclic
• CPC Class Codes: G01N 2203/0016 Tensile or compressive
• CPC Class Codes: G01N 2203/0073 Fatigue
• CPC Class Codes: G01N 2203/0282 Two dimensional, e.g. tapes, webs, sheets, strips, disks or membranes
• CPC Class Codes: G01N 3/38 generated by electromagnetic means
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/5061 Muscle cells
• CPC Class Codes: G01N 33/5064 Endothelial cells
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

A method for obtaining a response of a tissue model system to an activator includes contacting a bio-artificial tissue model system with an activator and measuring cellular mechanical response thereto of at least one of contractile force and tissue stiffness. A method for obtaining a response of a tissue model system to an activator includes contacting a bio-artificial tissue model system with an activator and measuring cellular mechanical response thereto of at least one of contractile force and hysteresis.

Claims

1. A method for screening pharmaceutical chemicals and for obtaining a response of a tissue model system to an agent comprises contacting a tissue model system with the agent and determining a parameter of at least one of contractile force and tissue stiffness.

2. A method in accordance with claim 1 wherein the parameter comprises contractile force and tissue stiffness.

3. A method in accordance with claim 1 wherein the tissue stiffness includes at least one of hysteresis, phase lag and dynamic stiffness.

4. A method in accordance with claim 1 wherein the tissue model system is a ring system.

5. A method in accordance with claim 1 wherein the activator is a pharmaceutical candidate or a known pharmaceutical.

6. A method in accordance with claim 1 wherein the tissue model system comprises cells in collagen.

7. A method in accordance with claim 6 wherein the cells are muscle cells.

8. A method in accordance with claim 7 wherein the cells are non-muscle cells.

9. A method in accordance with claim 7 wherein the tissue model system is a bio-artificial system.

10. A method in accordance with claim 9 wherein the system is a living system.

11. A method in accordance with claim 8 wherein cells are endothelial cells.

12. A method in accordance with claim 7 wherein the cell is selected from at least one of cardiac cells.

13. A method in accordance with claim 7 wherein the tissue model system comprises cells of extracellular matrix.

100. A method in accordance with claim 12 wherein said cardiac cells are selected from at least one of chicken and mouse.

101. A method in accordance with claim 100 wherein said cardiac cell is a chicken cardiac cell.

102. A method in accordance with claim 100 wherein said cardiac cell is a mouse cardiac cell.

14. A method for obtaining a mechanical response of a tissue model system to contact with an agent, said method comprising constructing a tissue model system, providing an agent in contact therewith, and measuring cellular mechanical properties in response to contact of the agent with the system of at least one parameter of contractile force and hysteresis.

15. A method in accordance with claim 14 wherein the cellular mechanical response thereof includes contractile force and hysteresis.

16. A method in accordance with claim 15 wherein the mechanical response is measured using a tissue indentation system.

17. A method in accordance with claim 16 wherein the tissue indentation system comprises an indentation probe coupled to a force transducer and a computer.

18. A method in accordance with claim 17 wherein the tissue model system is a bio-artificial system.

19. A method in accordance with claim 18 wherein the bio-artificial system is a living system.

20. A method in accordance with claim 19 wherein the tissue model system comprises cells in collagen.

21. A method in accordance with claim 20 wherein the cells comprise muscle and non-muscle cells.

22. A system for obtaining a mechanical response profile based on reconstituted model muscle and non-muscle tissue mechanical measurements of the response of a tissue model to contact with an agent, which comprises constructing a reconstituted tissue model comprising reconstituted cells in collagen, contacting said reconstituted cell matrix with an agent and measuring cellular mechanical response to contact with said agent in terms of at least one of contractile force and tissue stiffness.

23. A system in accordance with claim 22 wherein the cellular response comprises at least one of force development and tissue stiffness.

24. A system in accordance with claim 23 wherein the tissue stiffness includes at least one of hysteresis, phase lag and dynamic stiffness.

25. A system in accordance with claim 24 wherein the cellular response comprises force development, tissue stiffness and hysteresis.

26. A system in accordance with claim 23 wherein the cellular response is determined using a tissue indentation method.

27. A system in accordance with claim 23 wherein the mechanical response is measured using a tissue indentation system.

28. A system in accordance with claim 27 wherein the tissue indentation system comprises an indentation probe, a force transducer and a computer.

29. A system in accordance with claim 22 wherein the system is a bio-artificial system.

30. A system in accordance with claim 23 wherein the system is a living system.

31. A method for screening pharmaceuticals comprising contacting a tissue model comprising cells in collagen with an effective amount of a candidate pharmaceutical and measuring cellular response to the candidate pharmaceutical in terms of at least one of contractile force and tissue stiffness.

32. A method in accordance with claim 31 further comprising measuring cellular response to the pharmaceutical in terms of contractile force and tissue stiffness.

33. A method in accordance with claim 32 wherein the tissue stiffness includes at least one of hysteresis, phase lag and dynamic stiffness.

34. A method in accordance with claim 33 further comprising measuring cellular response to contact with said pharmaceutical by determining phase lag and dynamic stiffness.

35. A tissue model for measuring response data of a matrix comprising reconstituted cells in collagen to an activator exposure comprising:

(a) cells

(b) collagen; and

(c) ECM.

36. A tissue model in accordance with claim 35 wherein the cells comprise muscle and non-muscle cells.

37. A method of managing a library of one or more pharmaceuticals comprising obtaining a profile of a mechanical response to contact of an agent pharmaceutical with a tissue model system wherein a tissue model has been contacted with the pharmaceutical, storing that profile in a database, storing at least one additional profile of another pharmaceutical in a tissue model system in the database and comparing one profile with another profile.

38. A method in accordance with claim 37 further comprising comparing the profile of the first pharmaceutical with the profile of a second pharmaceutical based on a pre-established or ordered standard of comparison.

39. A method in accordance with claim 38 further comprising ranking the pharmaceuticals in an order of pharmaceutical activity based upon the corresponding profiles of each pharmaceutical with respect to a mechanical effect on a tissue model system.

40. A method in accordance with claim 37 wherein the tissue model system is a ring system or a tissue indentation system.

41. A method in accordance with claim 39 further comprising advancing a pharmaceutical to a secondary level of testing based upon the ranking.

42. A method in accordance with claim 37 wherein said pharmaceutical is a group or a class of chemicals.

43. A method in accordance with claim 37, wherein said comparison of profile is based on contracted force or tissue stiffness.

44. A method in accordance with claim 37 wherein the cellular matrix system uses a tissue indentation system.

45. A method in accordance with claim 37 wherein comparing comprises establishing baseline mechanical response data comprising contractile force and stiffness.

46. A method in accordance with that of claim 37 wherein the system is a bio-artificial system.

47. A method in accordance with claim 37 wherein the system is a living system.

48. A method in accordance with claim 37 wherein the cells are muscle cells.

49. A method in accordance with claim 37 wherein the cells are non-muscle cells.

50. A method of obtaining a mechanical response profile for a tissue model comprising living cells contacted with a pharmaceutical, said method comprising contacting a tissue model with the pharmaceutical and measuring the cellular response thereof.

51. A method in accordance with claim 50 wherein the tissue model system is a ring system.

52. A method in accordance with claim 50 wherein the tissue model system uses a tissue indentation system.

53. A method in accordance with claim 50 wherein the profile is obtained from a ring system.

54. A method in accordance with claim 50 wherein the profile is obtained using an indentation system.

55. A method in accordance with claim 50 wherein the profile of a candidate pharmaceutical is compared to the profile of a known pharmaceutical and a ranking is created based on that comparison.

56. A method in accordance with claim 50 wherein the profile is obtained using a tissue indentation system.

57. A method in accordance with claim 56, wherein the candidate pharmaceutical is evaluated against a cell system known to be involved in a disease, said method comprising obtaining a profile of a candidate pharmaceutical to provide an indication of the probable activity of the candidate pharmaceutical to a particular cell and comparing the profile of the candidate pharmaceutical to a profile of a known active pharmaceutical.

58. A method in accordance with claim 57 wherein a comparison is made of the profile of the known pharmaceutical known to provide effective treatment against a known disease to the profile of a candidate pharmaceutical whose efficiency to a disease is desired to be known.

59. A method in accordance with claim 50, wherein a comparison is made of the profile of a pharmaceutical known to be effective against cells which are known to be involved in a particular disease.

60. A tissue model system, comprising tissue assembled in the form of a ring mounted on a system comprising an isometric force transducer electrically coupled to a computer controlled stepping motor wherein the tissue spans the isometric force transducer and the computer-controlled stepping motor provides an application of force and strain to the tissue.

61. A system in accordance with claim 60 wherein the application of force is linear.

62. A system in accordance with claim 60 wherein the application of force is a ramp force.

63. A system in accordance with claim 60 wherein the force is applied sinusoidally.

64. A system in accordance with claim 60 wherein the force is applied to achieve a 20% stretch of tissue.

65. A method in accordance with claim 60 wherein tissue stiffness is measured by subjecting the tissue in the tissue model system to a stretch and observing the change in force as the strain increases.

66. A system in accordance with claim 60 wherein the tissue is muscle tissue.

67. A tissue model system, comprising a membrane of reconstituted tissue provided with a support and having a bar attached to an isometric force transducer coupled to a probe.

68. A system in accordance with claim 67 further comprising a computer, said computer directing the probe to indent the membrane and measure the force resisting the indentation.

69. A system in accordance with claim 67 wherein a contractile force is measured as peak force along with tissue stiffness which is registered as a resistance to indention of the tissue model by the probe contacting the tissue.

70. A system in accordance with claim 67 wherein tissue stiffness is measured by subjecting the tissue in the system to a steady stretch and observing the change in force as the strain increases.

71. A method of preparing a tissue model system, said method comprises growing tissue in the form of a ring around a mandrel, removing the ring shaped tissue from the mandrel and thereafter subjecting said ring shaped tissue to an application of stretch force by application of a standard force.

72. A method in accordance with claim 71, wherein the force is applied in a constant manner.

73. A method in accordance with claim 71 wherein the tissue is a living tissue.

74. A method in accordance with claim 71 wherein the tissue is muscle cells.

75. A method in accordance with claim 71 wherein the cells are cardiac cells.

76. A high throughput screening system comprising at least one unit comprising a tissue model system comprising cells and ECM arranged in a membrane adapted to receive a probe, a probe adapted for receiving instructions as to advancing or retracting to/from said membrane, a transducer coupled to said probe having electrical transmission capability to an electronic recording system wherein advancement instructions are provided to said probe.

77. A system in accordance with claim 76 wherein a multi-well plate system is engaged to house the membranes.

78. A system in accordance with claim 76 wherein the number of units is from about 2 to about 10,000 units.

79. A system in accordance with claim 76 wherein the number of units is from about 2 to about 96.

80. A system in accordance with claim 76 wherein the cells are muscle cells.

81. A system in accordance with claim 76 wherein the cells are non-muscle cells.

82. A system in accordance with claim 76 wherein the muscle cells are cardiac cells.

83. A diagnostic kit comprising a tissue model system and cell activator, said activator contacts the tissue model system and a response is measured therefrom.

84. A kit in accordance with claim 83 wherein said tissue model system is a living system and said kit is portable.

85. A kit in accordance with claim 83 wherein the response determined is contractile force or tissue stiffness.

86. A kit in accordance with claim 83 wherein the response determined is hysteresis.

87. A tissue indentation system comprising a tissue, one end of said tissue is connected to a fixed position and another other end of said tissue is connected to a transmitter probe which is connected to a computer, wherein the probe is advanced and contacted with a tissue sample to indent the tissue.

88. A system in accordance with claim 87 wherein the tissue is living tissue.

89. A system in accordance with claim 87 wherein the tissue is muscle tissue.

90. A system in accordance with claim 87 wherein the tissue is muscle cells.

92. A method in accordance with claim 87 further comprising associating said profile with a cell present in the tissue model.

93. A method in accordance with claim 87 wherein a profile is obtained using a ring system.

94. A method in accordance with claim 87 wherein a profile is obtained using a tissue indentation system.

95. A method in accordance with claim 87 wherein a profile is determined which reflects the contribution of cells and cytoskeleton to the overall profile.

91. A method for establishing a mechanical response profile of a pharmaceutical compound comprising contacting the pharmaceutical with a tissue model and determining the mechanical response(s) of the tissue model comprising a bio-artificial system of living cells utilizing at least one of contractile force and stiffness.

96. A method of culturing a cardiac tissue model, said method comprising using a medium conditioned by fibroblasts.

97. A method in accordance with claim 96 wherein fibroblasts are cardiac fibroblasts.

98. A method in accordance with claim 97 wherein said medium is DMEM with 1% FBS.

99. A method in accordance with claim 98 wherein a condition of said method is selected from coculture with feeding fibroblast monolayer, coculture with artificial tissue with fibroblasts and mixing fibroblasts in the tissue.

103. A method and apparatus to identify the effect of a drug upon a tissue which comprises treating a tissue using this invention and measuring the effect so resulting and thereafter comparing such measured effect with a standard drug effect on the same or similar tissue.

104. A method and apparatus to data mine a library of pharmaceutical moieties for activity in tissue, which comprises treating a tissue using this invention and measuring the effect so resulting. Optionally the measured effect may be compared with a correspondingly measured effect of a standard drug on the same or similar tissue.

105. A method of optimizing tissue culture conditions for constructing implantable artificial cardiac tissue, with additional different factors influencing cardiac tissue development including growth factors, and matrix proteins and hormones to a tissue model of the invention.