Ink-jet printing of viable cells

• US 7,051,654 B2
• Filed: 09/17/2003
• Issued: 05/30/2006
• Est. Priority Date: 05/30/2003
• Status: Active Grant

• Current Assignee: Clemson University
• Original Assignee: Clemson University
• Inventors: Boland, Thomas, Xu, Tao, Wilson, William Crisp Jr.
• Primary Examiner(s): Eickholt, Eugene H.
• Application Number:
  US10/666,836
• Publication Number:
  US 20040237822A1
• Time in Patent Office: 986 Days
• Field of Search: 101/483, 347/46, 347/10, 347/12, 347/16, 347/19, 347/23, 347/58, 600/407, 435/7.2, 435/29, 435/395, 435/397, 463/164
• US Class Current: 101/483
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 3/0268 using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B33Y 70/00 Materials specially adapted for additive manufacturing
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/00168 Manufacturing or preparing test elements
• CPC Class Codes: G01N 2035/1041 Ink-jet like dispensers

Abstract

A method for forming an array of viable cells is provided. In one embodiment, the method comprises ink-jet printing a cellular composition containing cells onto a substrate. Upon printing, at least about 25% of the cells remain viable after incubation for 24 hours at 37° C. in a 5% CO₂/95% O₂ environment.

Claims

1. A method for forming an array of viable cells, said method comprising ink-jet printing a cellular composition containing cells onto a substrate, wherein at least about 25% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

2. A method as defined in claim 1, wherein an ink-jet printer containing at least one printer head is used to print said cellular composition onto said substrate.

3. A method as defined in claim 2, wherein said printer head defines at least one orifice through which said cellular composition is capable of flowing.

4. A method as defined in claim 3, wherein said orifice is positioned from about 0.1 to about 30 millimeters from said substrate.

5. A method as defined in claim 3, wherein said orifice is positioned from about 0.5 to about 3 millimeters from said substrate.

6. A method as defined in claim 3, wherein said orifice has a size sufficient to inhibit substantial clogging of said cellular composition within said printer head.

7. A method as defined in claim 2, wherein a pressurization actuator facilitates the formation of a droplet of said cellular composition.

8. A method as defined in claim 7, wherein said pressurization actuator receives a voltage pulse ranging from about 1 to about 50 volts.

9. A method as defined in claim 7, wherein said pressurization actuator receives a voltage pulse ranging from about 10 to about 20 volts.

10. A method as defined in claim 7, wherein said pressurization actuator is selected from the group consisting of piezoelectric crystals, acoustic devices, thermal devices, and combinations thereof.

11. A method as defined in claim 1, wherein said cellular composition contains procaryotic cells.

12. A method as defined in claim 1, wherein said cellular composition contains eucaryotic cells.

13. A method as defined in claim 1, wherein said cellular composition contains cell aggregates.

14. A method as defined in claim 1, wherein the concentration of said cells within said cellular composition is from about 1×

10³ to about 1×

10¹⁶ cells per milliliter.

15. A method as defined in claim 1, wherein the concentration of said cells within said cellular composition is from about 3×

10⁵ to about 1×

10⁹ cells per milliliter.

16. A method as defined in claim 1, further comprising depositing a support compound onto said substrate.

17. A method as defined in claim 16, wherein said support compound is a gel or a compound capable of forming a gel.

18. A method as defined in claim 17, wherein said support compound forms a gel after being deposited onto said substrate.

19. A method as defined in claim 17, wherein said support compound is crosslinked after being deposited onto said substrate.

20. A method as defined in claim 19, wherein the crosslinking is induced by immersing said substrate into a solution containing said support compound or a crosslinking agent for said support compound.

21. A method as defined in claim 16, wherein said support compound is printed onto said substrate.

22. A method as defined in claim 21, wherein said support compound is mixed with said cellular composition prior to being printed onto said substrate.

23. A method as defined in claim 16, wherein said support compound is selected from the group consisting of agar, collagen, hydrogel polymers, and combinations thereof.

24. A method as defined in claim 1, wherein a two-dimensional array of said cells is formed on said substrate.

25. A method as defined in claim 1, wherein a three-dimensional array of said cells is formed on said substrate.

26. A method as defined in claim 1, further comprising ink-jet printing multiple droplets of said cellular composition onto said substrate.

27. A method as defined in claim 26, wherein said multiple droplets fuse into a cohesive cellular assembly.

28. A method as defined in claim 26, wherein said multiple droplets are printed in multiple printing passes.

29. A method as defined in claim 1, wherein at least about 50% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

30. A method as defined in claim 1, wherein at least about 75% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

31. A method as defined in claim 1, wherein at least about 85% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

32. A method as defined in claim 1, wherein the density of said cells on said substrate is from about 0.1 to about 2 cells per square millimeter.

33. A method as defined in claim 32, wherein the density of said cells on said substrate is from about 0.25 to about 1 cell per square millimeter.

34. A method as defined in claim 1, wherein the density of said cells on said substrate is from about 0.0001 to about 1 cell per square micrometer.

35. A method as defined in claim 34, wherein the density of said cells on said substrate is from about 0.0004 to about 0.25 cells per square micrometer.

36. A method for forming an array of viable cells, said method comprising:

supplying a cellular composition containing cells to at least one printer head of an ink-jet printer, said printer head defining an orifice through which said cellular composition is capable of flowing;

forming one or more droplets from said cellular composition;

flowing the droplets through said orifice so that said cells are printed onto a substrate; and

depositing a support compound onto said substrate for supporting said cells, said support compound forming a gel after being deposited onto said substrate.

37. A method as defined in claim 36, wherein said cellular composition contains eucaryotic cells, procaryotic cells, or combinations thereof.

38. A method as defined in claim 36, wherein said support compound is crosslinked after being deposited onto said substrate.

39. A method as defined in claim 38, wherein the crosslinking is induced by immersing said substrate into a solution containing said support compound or a crosslinking agent for said support compound.

40. A method as defined in claim 36, wherein said support compound is printed onto said substrate.

41. A method as defined in claim 40, wherein said support compound is mixed with said cellular composition prior to being printed onto said substrate.

42. A method as defined in claim 36, wherein said support compound is selected from the group consisting of agar, collagen, hydrogel polymers, and combinations thereof.

43. A method as defined in claim 36, wherein a two-dimensional array of said cells is formed on said substrate.

44. A method as defined in claim 36, wherein a three-dimensional array of said cells is formed on said substrate.

45. A method as defined in claim 36, wherein multiple droplets are printed onto said substrate.

46. A method as defined in claim 45, wherein said multiple droplets fuse into a cohesive cellular assembly.

47. A method as defined in claim 36, wherein at least about 25% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

48. A method as defined in claim 36, wherein at least about 50% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

49. A method as defined in claim 36, wherein at least about 75% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

50. A method as defined in claim 36, wherein at least about 85% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

51. A method as defined in claim 36, wherein the density of said cells on said substrate is from about 0.1 to about 2 cells per square millimeter.

52. A method as defined in claim 36, wherein the density of said cells on said substrate is from about 0.0001 to about 1 cell per square micrometer.

53. An array formed on a substrate from viable printed cells, wherein a gel provides structural support for said viable printed cells, wherein the density of said cells when printed is from about 0.0001 to about 1 cell per square micrometer.

54. An array as defined in claim 53, wherein said gel is crosslinked.

55. An array as defined in claim 53, wherein said gel is selected from the group consisting of agar, collagen, hydrogel polymers, and combinations thereof.

56. An array as defined in claim 53, wherein at least about 50% of said cells remain viable after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

57. An array as defined in claim 53, wherein at least about 75% of said cells remain viable after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

58. An array as defined in claim 53, wherein at least about 85% of said cells remain viable after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

59. An array as defined in claim 53 wherein the density of said cells when printed is from about 0.0004 to about 0.25 cells per square millimeter.

60. An array as defined in claim 53, wherein said cells comprise procaryotic cells.

61. An array as defined in claim 53, wherein said cells comprise eucaryotic cells.

62. An array as defined in claim 53, wherein the array comprises cells of more than one cell type.

63. An array as defined in claim 53, wherein the array is two-dimensional.

64. An array as defined in claim 53, wherein the array is three-dimensional.

65. An array as defined in claim 53, wherein the printed cells form a cohesive cellular assembly.

66. An array as defined in claim 53, wherein the density of said cells when printed varies across at least a portion of the array.

67. An ink-jet printer configured to deposit viable cells onto a substrate, said printer comprising:

a reservoir for containing the cells;

a printer head in fluid communication with said reservoir, said printer head defining an orifice having a size of from about 2 to about 200 micrometers, wherein the cells are capable of flowing through said orifice without substantial clogging; and

a pressurization actuator that is capable of facilitating the formation of a droplet containing the cells for flowing through said orifice, wherein said pressurization actuator receives a voltage pulse that is sufficiently low to facilitate the survival of the cells.

68. An ink-jet printer as defined in claim 67, wherein said voltage pulse ranges from about 1 to about 50 volts.

69. An ink-jet printer as defined in claim 67, wherein said voltage pulse ranges from about 10 to about 20 volts.

70. An ink-jet printer as defined in claim 67, wherein said pressurization actuator is selected from the group consisting of piezoelectric crystals, acoustic devices, thermal devices, and combinations thereof.

71. An ink-jet printer as defined in claim 67, wherein said printer head is moveable in an −

x direction.

72. An ink-jet printer as defined in claim 67, further comprising a feed mechanism for receiving the substrate.

73. An ink-jet printer as defined in claim 72, wherein said feed mechanism is configured to move the substrate in a −

y direction.

74. A method for forming an array of viable cells, said method comprising:

supplying a cellular composition containing cells to at least one printer head of an ink-jet printer, said printer head defining an orifice through which said cellular composition is capable of flowing;

forming one or more droplets from said cellular composition;

flowing the droplets through said orifice so that said cells are printed onto a substrate; and

depositing a support compound onto said substrate for supporting said cells, said support compound including a gel or a compound capable of forming a gel, wherein at least about 25% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

75. A method as defined in claim 74, wherein said cellular composition contains eucaryotic cells, procaryotic cells, or combinations thereof.

76. A method as defined in claim 74, wherein said support compound forms a gel after being deposited onto said substrate.

77. A method as defined in claim 74, wherein said support compound is crosslinked after being deposited onto said substrate.

78. A method as defined in claim 77, wherein the crosslinking is induced by immersing said substrate into a solution containing said support compound or a crosslinking agent for said support compound.

79. A method as defined in claim 74, wherein said support compound is printed onto said substrate.

80. A method as defined in claim 79, wherein said support compound is mixed with said cellular composition prior to being printed onto said substrate.

81. A method as defined in claim 74, wherein said support compound is selected from the group consisting of agar, collagen, hydrogel polymers, and combinations thereof.

82. A method as defined in claim 74, wherein a two-dimensional array of said cells is formed on said substrate.

83. A method as defined in claim 74, wherein a three-dimensional array of said cells is formed on said substrate.

84. A method as defined in claim 74, wherein multiple droplets are printed onto said substrate.

85. A method as defined in claim 84, wherein said multiple droplets fuse into a cohesive cellular assembly.

86. A method as defined in claim 74, wherein at least about 50% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

87. A method as defined in claim 74, wherein at least about 75% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

88. A method as defined in claim 74, wherein at least about 85% of said cells remain viable on said substrate after incubation for 24 hours at 37°

C. in a 5% CO₂/95% O₂ environment.

89. A method as defined in claim 74, wherein the density of said cells on said substrate is from about 0.1 to about 2 cells per square millimeter.

90. A method as defined in claim 74, wherein the density of said cells on said substrate is from about 0.0001 to about 1 cell per square micrometer.