Bioreactors with an array of chambers and a common feed line

• US 8,129,179 B2
• Filed: 08/27/2003
• Issued: 03/06/2012
• Est. Priority Date: 08/27/2002
• Status: Expired due to Fees

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Wikswo, John F., Cliffel, David, Leboeuf, Eugene J., Reiserer, Randall S.
• Primary Examiner(s): Bowers, Nathan
• Assistant Examiner(s): EDWARDS, LYDIA E
• Application Number:
  US10/525,538
• Publication Number:
  US 20060099705A1
• Time in Patent Office: 3,114 Days
• Field of Search: 4352831-3031
• US Class Current: 435/305.2
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/163 Biocompatibility
• CPC Class Codes: B01L 2400/0472 Diffusion
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 41/00 Means for regulation, monitoring, measurement or control, e.g. flow regulation controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus

Abstract

A bioreactor (200) for cultivating living cells in a liquid medium. In one embodiment, the bioreactor (200) has a substrate (230) having a first surface and an opposite second surface, defining a channel (202) therein, and a plurality of chambers (204) formed in the substrate, wherein each of the plurality of chambers (204) is adapted for receiving cells in a liquid medium and formed with an open end (262), an opposite closed end (264) and side walls, the open end (262) and the closed end (264) defining a depth, d, therebetween for the corresponding chamber (206), the sidewalls defining a width, w, therebetween for the corresponding chamber (206). As such formed, each chamber (206) is in fluid communication with the channel (202) through the open end of the chamber (206), and at least two of the plurality of chambers (206) have depths or widths the same or different form each other.

Claims

1. A bioreactor comprising:

(a) a substrate having a first surface and an opposite second surface, defining a channel therein;

(b) a plurality of chambers formed in the substrate, wherein each of the plurality of chambers is adapted for receiving and culturing at least one predetermined type of cells in a liquid medium and formed with an open end, an opposite closed end and side walls, the open end and the closed end defining a depth, d, therebetween for the corresponding chamber, the sidewalls defining a width, w, therebetween for the corresponding chamber, and the chamber being in fluid communication with the channel through the open end;

(c) means adapted for electrochemical measurements of the cells responsive to the liquid medium in at least one of the chambers; and

(d) a barrier for at least one of the chambers, wherein the barrier;

(i) is positioned at the open end of the corresponding chamber and proximate to the intersection of the channel and the corresponding chamber; and

(ii) has a porosity to allow the corresponding chamber and the channel to be in fluid communication, and to allow at least one predetermined type of cells to permeate between the corresponding chamber and the channel and at least another predetermined type of cells not to permeate between the corresponding chamber and the channel,

wherein the corresponding chamber defined between the open end and opposite closed end is formed substantially perpendicular to the channel and configured not to allow a liquid medium to pass through the open end and opposite closed end through the channel, and

wherein the means for electrochemical measurements comprises a plurality of electrically individually addressable working electrodes arranged along the corresponding chamber between the open end and opposite closed end such that none of the electrodes are arranged along the channel.

2. The bioreactor of claim 1, wherein at least two of the plurality of chambers have depths or widths same or different from each other.

3. The bioreactor of claim 1, further comprising an inlet port in fluid communication with the channel, and an outlet port in fluid communication with the channel, wherein the inlet port and the outlet port are apart from each other along the channel.

4. The bioreactor of claim 1, wherein the cells are in the form of a biofilm.

5. The bioreactor of claim 1, wherein the cells comprise bacteria.

6. The bioreactor of claim 1, wherein the cells comprise protozoa.

7. The bioreactor of claim 1, further comprising a biocompatible coating layer applied to the channel walls.

8. The bioreactor of claim 7, wherein the biocompatible coating layer comprises a material that may inhibit cell adhesion to the biocompatible coating layer.

9. The bioreactor of claim 1, further comprising a biocompatible coating layer applied to the sidewalls of each chamber.

10. The bioreactor of claim 9, wherein the biocompatible coating layer comprises a material that may inhibit cell adhesion to the biocompatible coating layer, enhance cell adhesion to the biocompatible coating layer, or function as a fluorescent marker or indicator of the state of cells.

11. The bioreactor of claim 1, further comprising at least one auxiliary port and an auxiliary channel in fluid communication with the auxiliary port and a corresponding one of the chambers for allowing individual control of the environment of the corresponding chamber.

12. The bioreactor of claim 11, wherein the individual control of the environment of the corresponding chamber includes delivery or removal of the cells, fluids or chemicals to the corresponding chamber or flushing the corresponding chamber.

13. The bioreactor of claim 11, further comprising at least one sample chamber and a plurality of sample channels in fluid communication with the sample chamber and a corresponding chamber, wherein the sample chamber is in fluid communication with at least one corresponding auxiliary channel that is in fluid communication with at least one corresponding auxiliary port, for allowing individual control of the environment of the corresponding sample chamber.

14. The bioreactor of claim 13, wherein the individual control of the environment of the corresponding sample chamber includes delivery or removal of the fluids or chemicals to the corresponding sample chamber.

15. The bioreactor of claim 13, wherein the sample chamber is adapted for receiving a sample of host material that provides exudates affecting the cells or biofilm in the corresponding chamber.

16. The bioreactor of claim 15, wherein the sample chamber is formed with a closed end and an opposite open end through which the host material can be received into or removed from the sample chamber.

17. The bioreactor of claim 16, further comprising a lid adapted for slidably covering or opening the open end of the sample chamber.

18. The bioreactor of claim 16, wherein the host material comprises soil.

19. The bioreactor of claim 1, further comprising at least one sample chamber in fluid communication with a corresponding chamber.

20. The bioreactor of claim 19, wherein the sample chamber is adapted for receiving a sample of host material that affects the cells or biofilm in the corresponding chamber.

21. The bioreactor of claim 20, wherein the sample chamber is formed with a closed end and an opposite open end through which the host material can be received into or removed from the sample chamber.

22. The bioreactor of claim 21, further comprising a lid adapted for slidably covering or opening the open end of the sample chamber.

23. The bioreactor of claim 1, wherein the substrate is fabricated from glass, Mylar, PDMS, silicon, a polymer, a semiconductor, or any combination of them.

24. The bioreactor of claim 1, wherein the means for electrochemical measurements comprises:

(i) a counter electrode;

(ii) a reference electrode; and

(iii) a plurality of electrically conductive leads, where a first electrically conductive lead electrically couples the reference electrode to a corresponding edge connector pad, and a second electrically conductive lead electrically couples the counter electrode to a corresponding edge connector pad.

25. The bioreactor of claim 24, wherein the means for electrochemical measurements further comprises:

(iv) a plurality of corresponding amplifiers, wherein each individually addressable working electrode is electrically coupled to a corresponding amplifier through a corresponding electrically conductive lead.

26. The bioreactor of claim 25, wherein the liquid medium comprises at least one analyte, and wherein the plurality of individually addressable working electrodes are adapted for capable of sensing the concentration of a single analyte of the liquid medium at multiple locations in a corresponding chamber or the concentrations of a plurality of analytes of the liquid medium at multiple locations in the corresponding chamber at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

27. The bioreactor of claim 26, wherein the plurality of individually addressable working electrodes are further adapted for capable of measuring the metabolic variables related to the cells responsive to the liquid medium at multiple locations in the corresponding chamber at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

28. The bioreactor of claim 25, further comprising a plurality of electrically conductive output leads, each electrically coupling a corresponding amplifier to an output device.

29. The bioreactor of claim 28, wherein the output device comprises a multiplexed potentiostat.

30. The bioreactor of claim 25, further comprising a plurality of controlling ports and a plurality of connection channels, wherein each of the connection channels is in fluid communication with a corresponding controlling port and the chamber.

31. The bioreactor of claim 30, further comprising a fluid control valve adapted for controlling the fluid communication between the plurality of controlling ports and the chamber.

32. The bioreactor of claim 31, wherein the fluid control valve comprises a pneumatic or mechanical valve.

33. The bioreactor of claim 32, further comprising a control port adapted for controlling the fluid control valve.

34. The bioreactor of claim 32, wherein the counter electrode and the reference electrode are positioned between the fluid control valve and the plurality of controlling ports.

35. The bioreactor of claim 34, wherein the liquid medium comprises at least one analyte, and wherein the plurality of individually addressable working electrodes are positioned between the fluid control valve and the plurality of controlling ports and adapted for capable of sensing the concentration of a single analyte of the liquid medium corresponding to multiple locations in a corresponding chamber, or the concentrations of a plurality of analytes of the liquid medium corresponding to multiple locations in the corresponding chamber, at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

36. The bioreactor of claim 35, wherein the plurality of individually addressable working electrodes are further adapted for capable of measuring the metabolic variables related to the cells responsive to the liquid medium at multiple locations corresponding to the corresponding chamber, at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

37. The bioreactor of claim 24, wherein the reference electrode is adapted for electrochemical measurements of the cells responsive to the liquid medium in the plurality of chambers.

38. The bioreactor of claim 24, wherein in each of the plurality of chambers, a counter electrode is adapted for electrochemical measurements of the cells responsive to the liquid medium in a corresponding chamber to allow the plurality of chambers to be operated individually and the means for electrochemical measurements for the plurality of chambers to be activated for one or more chambers at a time sequentially.

39. The bioreactor of claim 1, further comprising means positioned in the channel and adapted for monitoring of the cells therein.

40. The bioreactor of claim 39, wherein the means for monitoring of the cells comprises at least one optical sensor and at least one lead in optical communication with a corresponding optical sensor.

41. The bioreactor of claim 40, wherein the optical sensor comprises at least one device selected from the group of an LED and photodiode pair, a fiber optic coupler, and an optical detecting head.

42. The bioreactor of claim 39, wherein the means for monitoring of the cells comprises at least one electrical sensor and at least one lead in electrical communication with a corresponding electrical sensor.

43. The bioreactor of claim 1, wherein the channel is adapted for allowing a bolus of selected chemicals to move along the channel.

44. A bioreactor comprising:

(a) a substrate having a first surface and an opposite second surface; and

(b) a plurality of arrays of chambers formed on the substrate, each array being adapted for receiving cells in a liquid medium and comprising a channel and a plurality of chambers formed in the substrate, wherein each of the plurality of chambers is adapted for receiving cells in a liquid medium and formed with an open end, an opposite closed end and side walls, the open end and the closed end defining a depth, d, therebetween for the corresponding chamber, the sidewalls defining a width, w, therebetween for the corresponding chamber, and the chamber being in fluid communication with the channel through the open end, wherein at least two of the plurality of chambers have depths same or different from each other, and wherein for at least one array, each of the chambers is adapted to receive and culture at least one predetermined type of cells;

(c) means adapted for electrochemical measurements of the cells responsive to the liquid medium in at least one of the chambers of at least one array; and

(d) a barrier for at least one of the chambers of at least one array, wherein the barrier;

(i) is positioned at the open end of the corresponding chamber proximate to the intersection of the channel and the corresponding chamber; and

(ii) has a porosity to allow the corresponding chamber and the channel to be in fluid communication, and to allow at least one predetermined type of cells to permeate between the corresponding chamber and the channel and at least another predetermined type of cells not to permeate between the corresponding chamber and the channel,

wherein the corresponding chamber defined between the open end and closed end is formed substantially perpendicular to the channel and configured not to allow a liquid medium to pass through the open end and opposite closed end through the channel, and

wherein the means for electrochemical measurements comprises a plurality of individually addressable working electrodes arranged along the corresponding chamber between the open end and opposite closed end such that none of the electrodes are arranged along the channel.

45. The bioreactor of claim 44, wherein for at least one array of chambers at least two of the plurality of chambers have widths same or different from each other.

46. The bioreactor of claim 44, for at least one array of chambers, further comprising an inlet port in fluid communication with the channel, and an outlet port in fluid communication with the channel, wherein the inlet port and the outlet port are apart from each other along the channel.

47. The bioreactor of claim 44, wherein the cells are in the forms of a plurality of biofilms.

48. The bioreactor of claim 47, wherein the cells comprise bacteria.

49. The bioreactor of claim 47, wherein the cells comprise protozoa.

50. The bioreactor of claim 44, for at least one array of chambers, further comprising a biocompatible coating layer applied to the channel walls.

51. The bioreactor of claim 50, wherein the biocompatible coating layer comprises a material that may inhibit cell adhesion to the biocompatible coating layer.

52. The bioreactor of claim 44, for at least one array of chambers, further comprising a biocompatible coating layer applied to the sidewalls of each chamber.

53. The bioreactor of claim 52, wherein the biocompatible coating layer comprises a material that may inhibit cell adhesion to the biocompatible coating layer, enhance cell adhesion to the biocompatible coating layer, or function as a fluorescent marker or indicator of the state of cells.

54. The bioreactor of claim 44, for at least one array of chambers, further comprising at least one auxiliary port and an auxiliary channel in fluid communication with the auxiliary port and a corresponding one of the chambers for allowing individual control of the environment of the corresponding chamber.

55. The bioreactor of claim 54, wherein the individual control of the environment of the corresponding chamber includes delivery or removal of the cells, fluids or chemicals to the corresponding chamber or flushing the corresponding chamber.

56. The bioreactor of claim 44, wherein the substrate is fabricated from glass, Mylar, PDMS, silicon, a polymer, a semiconductor, or any combination of them.

57. The bioreactor of claim 44, wherein the means for electrochemical measurements comprises:

(i) a counter electrode;

(ii) a reference electrode; and

(iii) a plurality of electrically conductive leads, where a first electrically conductive lead electrically couples the reference electrode to a corresponding edge connector pad, and a second electrically conductive lead electrically couples the counter electrode to a corresponding edge connector pad.

58. The bioreactor of claim 57, wherein the means for electrochemical measurements further comprises:

(iv) a plurality of corresponding amplifiers, wherein each individually addressable working electrode is electrically coupled to a corresponding amplifier through a corresponding electrically conductive lead.

59. The bioreactor of claim 58, wherein the liquid medium comprises at least one analyte, and wherein the plurality of individually addressable working electrodes are adapted for capable of sensing the concentration of a single analyte of the liquid medium at multiple locations in a corresponding chamber or the concentrations of a plurality of analytes of the liquid medium at multiple locations in the corresponding chamber at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

60. The bioreactor of claim 59, wherein the plurality of individually addressable working electrodes are further adapted for capable of measuring the metabolic variables related to the cells responsive to the liquid medium at multiple locations in the corresponding chamber at a time period shorter than a characteristic reaction time related to at least one of cellular physiological activities of the cells.

61. The bioreactor of claim 58, further comprises a plurality of electrically conductive output leads, each electrically coupling a corresponding amplifier to an output device.

62. The bioreactor of claim 61, wherein the output device comprises a multiplexed potentiostat.

63. The bioreactor of claim 44, for at least one array of chambers, further comprising means positioned in the channel and adapted for monitoring of the cells therein.

64. The bioreactor of claim 63, wherein the means for monitoring of the cells comprises at least one optical sensor and at least one lead in optical communication with a corresponding optical sensor.

65. The bioreactor of claim 64, wherein the optical sensor comprises at least one device selected from the group of an LED and photodiode pair, a fiber optic coupler, and an optical detecting head.

66. The bioreactor of claim 63, wherein the means for monitoring of the cells comprises at least one electrical sensor and at least one lead in electrical communication with a corresponding electrical sensor.

67. The bioreactor of claim 64, wherein the channel is adapted for allowing a bolus of selected chemicals to move along the channel.

68. A method for culturing a plurality of biofilms, each containing a predetermined type of cells or cell growth conditions, comprising the steps of:

(i) providing a bioreactor that has a substrate having a first surface and an opposite second surface and a plurality of arrays of chambers formed on the substrate, each array being adapted for receiving cells in a liquid medium and comprising a channel and a plurality of chambers formed in the substrate, wherein each of the plurality of chambers is adapted for receiving cells in a liquid medium and formed with an open end, an opposite closed end and side walls, the open end and the closed end defining a depth, d, therebetween for the corresponding chamber, the sidewalls defining a width, w, therebetween for the corresponding chamber, and the chamber being in fluid communication with the channel through the open end, wherein the corresponding chamber defined between the open end and opposite closed end is formed substantially perpendicular to the channel and configured not to allow a liquid medium to pass through the open end and opposite closed end through the channel, and wherein at least two of the plurality of chambers have depths different from each other; and

(ii) culturing at least two biofilms in at least two arrays of chambers of the bioreactor.

69. The bioreactor of claim 68, further comprising the step of providing a bolus of selected chemicals to move along the channel for each of the at least two arrays of chambers.