Droplet-based cell culture and cell assays using digital microfluidics

• US 20090203063A1
• Filed: 09/26/2008
• Published: 08/13/2009
• Est. Priority Date: 02/11/2008
• Status: Active Grant

• Current Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Original Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Inventors: Barbulovic-Nad, Irena, Wheeler, Aaron R.
• Granted Patent: US 8,367,370 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 25/01 Drops
• CPC Class Codes: C12M 25/08 electrically charged
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• 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: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: G01N 33/54386 Analytical elements

Abstract

We introduce a new method for implementing cell-based assays and long-term cell culture. The method is based on digital microfluidics (DMF) which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. DMF method is sutable for assaying and culturing both cells in suspension and cells grown on surface (adherent cells). This method is advantageous for cell culture and assays due to the automated manipulation of multiple reagents in addition to reduced reagent use and analysis time. No adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. These results suggest that DMF has great potential as a simple yet versatile analytical tool for implementing cell-based assays and cell culture on the microscale.

Claims

1. A digital microfluidic based method of performing any one or both of cell assays and cell cultures, comprising the steps of:

a) providing a digital microfluidic device having an array of actuating electrodes formed on a substrate surface, a coating having a working surface coating the substrate surface and array of actuating electrodes, an actuating electrode controller for exciting or de-exciting said actuating electrodes for translating liquid droplets over said working surface;

b) dispensing one or more first droplets containing a suspension of at least one kind of cells onto one or more first positions on a working surface of the digital microfluidic device above the array of actuating electrodes and substrate surface, and dispensing one or more second droplets containing any one of at least one chemical reagent, at least one biochemical reagent, at least one biological reagent, and any combination thereof onto one or more second positions on the working surface;

c) translating each of the one or more first and second droplets to a corresponding third position on the working surface such that they substantially mix to form one or more secondary droplets;

d) incubating the one or more secondary droplets; and

e) analyzing the one or more secondary droplets to identify products produced by incubation of the one or more secondary droplets.

2. The method according to claim 1 wherein some or all of said one or more secondary droplets is split into at least two smaller secondary droplets either after step c) or after step d), and wherein step e) is conducted on the at least two smaller secondary droplets.

3. The method according to claim 1 wherein any one or combination of said one or more first and second droplets is split into at least two smaller droplets during any one of steps b) and c) of the method.

4. The method according to claim 1 wherein said one or more first droplets contain a single cell.

5. The method according to claim 1 wherein step e) of analyzing said one or more secondary droplets is performed by detecting signals emitted from the secondary droplets using a device capable of detecting a signal from the secondary droplets selected from the group consisting of optical sensors, optical detectors comprising a light source and a photodetector, optical detectors that measure any one or combination of absorbance, fluorescence, epifluorescence, and chemiluminescence, UV light detectors, radiometric detectors, any one of scanning, imaging, and confocal microscopy detectors, CCD cameras, microplate readers.

6. The method according to claim 1 wherein in the step e) said one or more secondary droplets are translated to one or more selected positions on said working surface for analysis or the said one or more secondary droplets are removed from the working surface and analyzed externally.

7. The method according to claim 1 wherein prior to step b) including modifying one or more pre-selected positions on said working surface to produce one or more cell culture sites wherein said one or more pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array, and wherein adherent cells adhere to said plurality of cell culture sites.

8. The method according to claim 7 wherein in step c) said one or more first droplets optionally mixed with one or more second droplets contain adherent cells and are translated to said one or more cell culture sites and incubated there to allow cell attachment thereto, after which said one or more second droplets are translated onto said one or more cell culture sites such that they substantially mix with droplets with submersed adhered cells.

9. The method according to claim 7 wherein in step c) said one or more first droplets optionally mixed with one or more second droplets contain adherent cells and are translated onto said one or more cell culture sites and incubated there to allow cell attachment thereto, after which said one or more second droplets are translated onto said one or more cell culture sites such that the droplet on the cell culture site is replaced by the second droplet such that the cells attached to the cell culture site are submersed in said second droplet.

10. The method according to claim 7 wherein said one or more pre-selected positions are modified by depositing a bio-substrate thereon.

11. The method according to claim 10 wherein said bio-substrate is deposited using any one of microprinting and microstamping.

12. The method according to claim 10 wherein said bio-substrate is produced from cell specific constituents.

13. The method according to claim 12 wherein said cell specific constituents are extracellular matrix proteins.

14. The method according to claim 13 wherein said extracellular matrix proteins include any one of fibronectin, laminin, collagen, elastin and any combination thereof.

15. The method according to claim 12 wherein said cell specific constituents are synthetic molecules comprised of one of poly-L-lysine, poly-D-lysine and any combination thereof.

16. The method according to claim 7 wherein said one or more pre-selected positions on said working surface include any one of a hydrophobic layer and a dielectric layer, and wherein said cell culture sites are produced using any one or combination of plasma treatment, hydrophobic layer etching, dielectric layer etching, electrode etching and stamping.

17. The method according to claim 1 wherein said at least one second droplet contains said biological agent only, said biological agent being a cell culture medium, and wherein said incubated secondary droplet includes cells cultured in suspension or adherent cells, and wherein when said cells are adherent cells, including modifying one or more pre-selected positions on said working surface to produce one or more cell culture sites wherein said one or more pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array.

18. The method according to claim 17 including periodically dispensing additional droplets containing the cell culture medium and translating the additional droplets to said corresponding third positions to mix with, or displace and replace, said one or more secondary droplets.

19. The method according to claim 1 wherein said at least one second droplet contains each one of or any combination of said chemical agent and said biochemical agent only, said chemical agent and biochemical agent being cell assay reagents.

20. The method according to claim 1 wherein said at least one second droplet contains a combination of said biological agent and each one of, or any combination of, said chemical agent and said biochemical agent only, said chemical agent and biochemical agent being cell assay reagents.

21. The method of performing a cell assay as claimed in claim 1 wherein step b) is performed by dispensing said one or more first and second droplets from sources external to said digital microfluidic device.

22. The method of performing a cell assay as claimed in claim 21 wherein the external source is selected from the group consisting of pipettes, robotic dispensers, microprinters and microstamps.

23. The method of performing a cell assay as claimed in claim 1 wherein step b) is performed by dispensing said first and second droplets from sources integrated as part of said digital microfluidic device, said sources being in flow communication with said working surface.

24. The method of performing a cell assay as claimed in claim 23 wherein said sources integrated as part of said digital microfluidic device are liquid reservoirs.

25. The method of performing a cell assay as claimed in claim 24 wherein said liquid reservoirs are formed on said working surface above some of said actuating electrodes which are modified to act as said liquid reservoirs.

26. The method of performing a cell assay as claimed in claim 1 wherein step b) is performed by dispensing said one or more first and second droplets from sources integrated as part of a cartridge assembled with a said digital microfluidic device, said sources being in flow communication with said working surface.

27. The method according to claim 1 wherein said suspension of cells is a combination of cells, a suspension medium, and a non-ionic surfactant.

28. The method according to claim 27 wherein said suspension medium is selected to facilitate cell-containing droplet actuation by preventing non-specific adsorption of cells and proteins to device surfaces.

29. The method according to claim 27 wherein the suspension of cells is a combination of cells and a suspension medium selected from the group consisting of block copolymers formed from poly(propylene oxide) and poly(ethylene oxide), pluronic F68, pluronic F127, hydrophilic polymers, sodium bicarbonate, phosphate buffered saline (PBS), HEPES, and other biological buffers, and cell culture medium selected from the group consisting of balanced salt solutions, nutrient mixtures, basal media, complex media, serum free media, insect cell media, virus production media, serum, fetal bovine serum, serum replacements, antibiotics, antimycotics, and any combination thereof, and any combination thereof.

30. The method according to claim 27 wherein the suspension of cells is a combination of cells, phosphate buffered saline, and pluronic F68.

31. The method according to claim 1 wherein said chemical, biochemical and biological reagents are selected from the group consisting of chemicals, biochemicals, drugs, drug lead compounds, toxins, surfactants, transfection reagents, supplements, anti-clumping agents, streptavidin, biotin, antibody production enhancers, antibodies, antibody ligands, nucleic acids, nucleic acid binding molecules, enzymes, proteins, viruses, cell process agonists or antagonists, labeling agents, fluorescent dyes, fluorogenic dyes, viability dyes, calcein AM, quantum dots, nano particles, Tween 20, and ethidium homodimer-1, block copolymers formed from poly(propylene oxide) and poly(ethylene oxide), pluronic F68, pluronic F127, hydrophilic polymers, sodium bicarbonate, phosphate buffered saline (PBS), HEPES, and other biological buffers, and cell culture medium selected from the group consisting of balanced salt solutions, nutrient mixtures, basal media, complex media, serum free media, insect cell media, virus production media, serum, fetal bovine serum, serum replacements, antibiotics, antimycotics, and any combination thereof, and any combination thereof.

32. The method according to claim 1 wherein the cells in the suspension of cells include primary/isolated or transformed/cultured cells selected from the group consisting of prokaryotic and eukaryotic cells, animal cells (blood cells, human leukemia cells, lymphocytes, beta cells, oocytes, eggs, primary cells, primary bone marrow cells, stem cells, neuronal cells, endothelial cells, epithelial cells, fibroblasts), insect cells, plant cells, bacterial cells, archebacterial cells.

33. The method according to claim 1 wherein the cells in said suspension of cells have a density less than about 1×

10⁸ cells/mL.

34. The method according to claim 1 wherein said working surface includes one or more hydrophilic areas and including translating a primary droplet over said one or more hydrophilic areas, said primary droplet having a base area larger than said one or more hydrophilic areas whereby a first smaller droplet is removed from said primary droplet and remains behind on said one or more hydrophilic areas, and wherein said primary droplet is any one of said one or more first, second and secondary droplets.

35. The method according to claim 34 including translating additional one or more primary droplets over said one or more hydrophilic areas already containing a said first smaller droplet, whereby the first smaller droplet is replaced by a second smaller droplet from said additional primary droplet.

36. The method according to claim 34 wherein said one or more hydrophilic areas are cell culture sites.

37. The method according to claim 9 wherein after incubating said secondary droplet in step d), including translating one or more droplets containing a washing solution over one or more cell culture sites to replace an incubation media and wash cells from the incubation media, then translating one or more droplets containing a cell dissociation agent to said one or more cell culture sites, and incubating in order to detach the cells adhered to said one or more cell culture sites, then mixing one or more droplets containing the dissociation agent and said detached cells with one or more droplets containing any one of at least one dissociation-blocking agent, a biological reagent, a chemical reagent, a biochemical reagent, and any combination thereof, wherein a biological reagent can be cell culture media, whereby forming a secondary droplet with re-suspended cells, and wherein said re-suspended cells can be optionally further assayed.

38. The method according to claim 37 including mixing said secondary droplet with re-suspended cells or any droplet split therefrom with one or more droplets containing cell culture media and translating at least some cells mixed with the cell culture media to at least one new cell culture site to seed a new generation of cells.

39. The method according to claim 1 conducted in a substantially sterile chamber.

40. The method according to claim 39 including controlling and regulating conditions in the sterile chamber including humidity, temperature and atmosphere.

41. The method according to claim 1 performed in a multiplexed mode, wherein

i) said step of dispensing one or more first droplets includes dispensing a plurality of first droplets each containing a suspension of at least one kind of cells onto a plurality first positions on the working surface,

ii) and wherein said step of dispensing one or more second droplet includes dispensing a plurality of second droplets onto a plurality of second positions on the working surface,

iii) and wherein step c) includes translating the plurality of first droplets to a corresponding plurality of third positions on said working surface, and translating the plurality of second droplets to said corresponding plurality of third positions in such a way that at least one second droplet is translated to each of said third positions such that they substantially mix with the first droplets at each third position to form a corresponding plurality of secondary droplets.

42. The method according to claim 41 wherein said plurality of second droplets is equal to said plurality of first droplets so each secondary droplet includes a first and second droplet mixed together.

43. The method according to claim 41 wherein said plurality of second droplets is a multiple of N times said plurality of first droplets so each secondary droplet includes N second droplets mixed with a first droplet.

44. The method according to claim 41 wherein each of said plurality of second droplets includes a plurality of reagents.

45. The method according to claim 41 wherein step b) is performed simultaneously for each of said plurality of first and second droplets.

46. The method according to claim 41 wherein step b) is performed sequentially for each of said plurality of first and second droplets in a certain order defined by a cell assay protocol.

47. The method according to claim 41 wherein step c) is performed simultaneously for each of said plurality of first and second droplets.

48. The method according to claim 41 wherein step c) is performed sequentially for each of said plurality of first and second droplets in a certain order defined by a cell assay protocol.

49. The method according to claim 41 wherein step d) is performed simultaneously for each of said secondary droplets.

50. The method according to claim 41 wherein step d) is performed sequentially for each of said secondary droplets in a certain order defined by a cell assay protocol.

51. The method according to claim 41 wherein step e) is performed simultaneously for each of said secondary droplets.

52. The method according to claim 41 wherein step e) is performed sequentially for each of said secondary droplets in a certain order defined by a cell assay protocol.

53. The method according to claim 41 wherein each of said plurality of first droplets include cell suspensions identical to the cell suspensions in the rest of said first droplets, and wherein step i) is performed by dispensing the plurality of first droplets from one source.

54. The method according to claim 41 wherein each of said plurality of first droplets include cell suspensions different to the cell suspensions in the at least one of said first droplets, and wherein step i) is performed by dispensing the plurality of first droplets from a corresponding plurality of sources, each of said plurality of sources having a cell suspension different to the rest of the cell suspensions.

55. The method according to claim 41 wherein each of said plurality of second droplets include reagents identical to the reagents in the rest of said second droplets, and wherein step ii) is performed by dispensing the plurality of second droplets from one source.

56. The method according to claim 41 wherein each of said plurality of second droplets include reagents different to the reagents in the at least one of said second droplets, and wherein step ii) is performed by dispensing the plurality of second droplets from a corresponding plurality of sources, each of said plurality of sources having a reagent different to the rest of the reagents in the other sources.

57. The method according to claim 41 wherein prior to step b) including modifying a plurality of pre-selected positions on said working surface to produce a plurality of cell culture sites wherein said plurality of pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array, and wherein adherent cells adhere to said plurality of cell culture sites.

58. The method according to claim 57 wherein in step c) said plurality of first droplets optionally mixed with plurality of second droplets contain adherent cells and are translated to said plurality of cell culture sites and incubated there to allow cell attachment thereto, after which said plurality of second droplets are translated onto said plurality of cell culture sites such that they substantially mix with droplets with submersed adhered cells.

59. The method according to claim 57 wherein in step c) said aid plurality of first droplets optionally mixed with said plurality of second droplets contain adherent cells and are translated onto said aid plurality of cell culture sites and incubated there to allow cell attachment thereto, after which said aid plurality of second droplets are translated onto said aid plurality of cell culture sites such that the droplet on the cell culture site is replaced by the second droplet such that the cells attached to the cell culture site are submersed in said second droplet.

60. The method according to claim 1 wherein steps b), c), d) and e) are conducted according to a selected cell assay or cell culture protocol under control of a computer controller interfaced to said digital microfluidic device.

72. The method according to claim 41 wherein said plurality of second droplets contain said biological agent only, said biological agent being a cell culture medium, and wherein said plurality of incubated secondary droplets include cells cultured in suspension or adherent cells, and wherein when said cells are adherent cells including modifying a plurality of pre-selected positions on said working surface to produce a plurality of cell culture sites wherein said plurality of pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array.

73. The method according to claim 72 including periodically dispensing a plurality of additional droplets containing the cell culture medium and translating the plurality of additional droplets to said corresponding plurality of third positions to mix with, or displace and replace, said plurality of secondary droplets.

61. A digital microfluidic device for conducting one or both of cell assays and cell culture, comprising:

a first substrate having a first substrate surface;

an array of actuating electrodes formed on the first substrate surface;

at least one dielectric layer formed on the first substrate surface covering each actuating electrode such that the actuating electrodes are electrically insulated from one another; and

at least one reference electrode, wherein each actuating electrode is proximal to at least one of the reference electrodes;

an electrode controller capable of selectively exciting or de-exciting actuating electrodes for translating liquid droplets across a surface of the dielectric layer;

one or more first reservoirs in flow communication with the surface of said dielectric layer for holding at least one suspension of cells and one or more reagent reservoirs in flow communication with the surface of said dielectric layer for holding one or more cell assay reagents, cell culture reagents; and

dispensing means for dispensing droplets of said at least one suspension of cells and droplets of said at least one cell assay reagents, cell culture reagents onto said surface of said dielectric layer; and

a computer controller interfaced to said dispensing means and said electrode controller and being programmed to dispense droplets of the suspension of cells and droplets of said one or more cell assay reagents, cell culture reagents onto said surface of said dielectric layer and translating them over said array of actuating electrodes for mixing and optionally splitting said droplets in selected positions on said array of actuating electrodes to form one or more secondary droplets in a selected order defined by a selected cell assay protocol or cell culture protocol for which said computer controller is programmed.

62. The device according to claim 61 wherein said surface of said dielectric layer includes one or more pre-selected positions having cell culture sites located thereon, and wherein said one or more pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array, and wherein adherent cells adhere to said plurality of cell culture sites.

63. The device according to claim 61 wherein the surface of the dielectric layer is hydrophobic.

64. A device according to claim 61 further comprising a first hydrophobic layer formed on said surface of the dielectric layer, and wherein all droplets are dispensed onto said hydrophobic layer for translation.

65. The device according to claim 61 including detection and analyzing means for detecting and analyzing signals from said one or more secondary droplets to identify products produced by incubation of the one or more secondary droplets.

66. The device according to claim 65 wherein said detection and analyzing means is selected from the group consisting of optical sensors, optical detectors comprising a light source and a photodetector, optical detectors that measure absorbance, fluorescence, epifluorescence, chemiluminescence, UV light detector, radiometric detector, scanning, imaging, and confocal microscopy detectors, CCD cameras, and microplate readers.

67. The device according to claim 61 further including a second substrate and a hydrophobic layer on a surface thereof, wherein the second substrate is in a spaced relationship to the first plate thus defining a space between the first and second substrates capable of containing droplets between the hydrophobic layer of the second substrate and the dielectric layer on the first substrate.

68. A device according to claim 67 wherein the second substrate is substantially transparent.

69. The device according to claim 61 wherein said array of actuating electrodes is an array of substantially coplanar actuating electrodes formed on the first substrate surface.

70. The device according to claim 69 wherein said at least one reference electrode is a grid of generally elongate reference electrodes formed in-between, and electrically insulated from, the actuating electrodes in the array of substantially coplanar actuating electrodes.

71. The device according to claim 61 further including a second substrate and a hydrophobic layer on a surface thereof, wherein the second substrate is in a spaced relationship to the first plate thus defining a space between the first and second substrates capable of containing droplets between the hydrophobic layer of the second substrate and the dielectric layer on the first substrate, and wherein said hydrophobic layer includes one or more pre-selected positions having cell culture sites located thereon, and wherein said one or more pre-selected positions are located such that each cell culture site is accessible to droplets being translated by said electrode array, and wherein adherent cells adhere to said plurality of cell culture sites.