PAPER-BASED CELLULAR ARRAYS

• US 20110105360A1
• Filed: 03/27/2009
• Published: 05/05/2011
• Est. Priority Date: 03/27/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Derda, Ratmir, Laromaine Sague, Anna, Whitesides, George M.
• Granted Patent: US 9,829,488 B2
• Time in Patent Office: Days
• US Class Current: 506/10
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/126 Paper
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/548 Carbohydrates, e.g. dextran

Abstract

Three-dimensional cellular arrays, methods of making three-dimensional cellular arrays, and methods of identifying agents using the arrays are disclosed.

Claims

1. -43. (canceled)

44. A cellular system, comprising:

a plurality of porous, hydrophilic substrates, wherein the hydrophilic substrates are stacked over one another and at least one of the porous hydrophilic substrates comprises at least one defined region comprising a cell-containing hydrogel.

45. The cellular system of claim 44 wherein the substrate is one or more substrates selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.

46. The cellular system of claim 44, wherein the cells are one or more cells selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells and mixtures thereof.

47. The cellular system of claim 44, wherein the hydrogel comprises a temperature sensitive hydrogel.

48. The cellular system of claim 44, wherein the hydrogel comprises an ionotropic hydrogel.

49. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises hydrogels.

50. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises a cell-containing hydrogel.

51. The cellular system of claim 44, wherein one or more of the plurality of substrates comprises a test agent.

52. The cellular system of claim 44, further comprising living tissue in contact with the cell-containing hydrogel.

53. The cellular system of claim 44, wherein there are a plurality of porous regions positioned in the porous hydrophilic substrate in the form of an array.

54. The cellular system of claim 53, further comprising:

a bottomless microtiter plate having a plurality of wells disposed over the porous hydrophilic substrate;

wherein the wells of the microtiter plate and the plurality of porous regions of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned and sealingly joined to the plurality of porous regions to form an individual chamber for each porous region, wherein the wells and porous regions define an array.

70. A method of identifying an agent that modifies cellular activity, the method comprising:

contacting the cellular system of claim 44 or 54 with one or more test agents; and

detecting one or more cellular activities in the presence of the one or more test agents.

71. The method of claim 70, wherein the cellular system is contacted with the one or more test agents at a plurality of defined regions.

72. The method of claim 71, wherein each defined region is contacted with a different test agent.

73. The method of claim 70, wherein the cellular activity is one or more selected from the group consisting of proliferation, migration, apotosis, differentiation, viability, upregulation of gene transcription, or downregulation of gene transcription.

74. The method of claim 70, wherein the test agent is selected from the group consisting of one or more of a small molecule, amino acid, polypeptide, nucleic acid, carbohydrate, polysaccharide, and metabolite.

75. The method of claim 70, wherein the cells are selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells and mixtures thereof.

76. A method of claim 70, further comprising:

cutting the cellular system into a plurality of segments, each segment comprising at least one porous region comprising cells and hydrogel;

contacting each segment with a test agent or a control; and

detecting one or more cellular activities in the presence of the test agent.

55. A cellular system comprising:

a porous, hydrophilic substrate, wherein the substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary, wherein at least one of the porous regions comprises a hydrogel comprising cells.

56. The cellular system of claim 55, wherein the liquid impervious boundary is selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, poly(methyl methacrylate), polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax and a fat and mixtures thereof.

57. The cellular system of claim 55, further comprising:

a bottomless microtiter plate having a plurality of wells disposed over the porous hydrophilic substrate;

wherein the wells of the microtiter plate and the liquid impervious boundaries of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned and sealingly joined to the plurality of liquid impervious boundaries to form an individual chamber for each porous region, wherein the wells and porous regions define an array.

58. A kit comprising:

one or more porous, hydrophilic substrates, wherein the substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary; and

a bottomless microtiter plate having a plurality of wells equal to the number of porous regions in the substrate, wherein the wells of the microtiter plate and the liquid impervious boundaries of the porous hydrophilic substrate are positioned so that the plurality of wells are aligned.

59. The kit of claim 58, further comprising a hydrogel.

60. The kit of claim 58 wherein the porous hydrophilic substrate is selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.

61. A method of making a cellular system, comprising:

providing a porous, hydrophilic substrate; and

in any order,

contacting a plurality of defined regions of the substrate with a suspension comprising cells and a hydrogel precursor, wherein the cells and the hydrogel precursor saturate the plurality of defined regions of the substrate; and

contacting the hydrogel precursor with a gelling agent, wherein the gelling agent induces the formation of a hydrogel embedded in the plurality of defined regions of the substrate.

62. The method of claim 61, wherein the hydrogel precursor is a temperature-sensitive hydrogel or an ionotropic hydrogel precursor.

63. The method of claim 61, wherein cells are introduced into the hydrogel precursor before contacting the hydrogel precursor to the substrate.

64. The method of claim 61, wherein the gelling agent is temperature, a solution containing salt, or a chemical cross-linking agent.

65. The method of claim 61, wherein the substrate is one or more substrates selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film.

66. The method of claim 61, wherein the porous, hydrophilic substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary.

67. The method of claim 66, wherein the liquid impervious boundary is one or more selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy, polystyrene, a polyether, a polyamide, poly(methyl methacrylate), polycarbonate, polyethylene, polypropylene, a photoresist precursors, a wax and a fat and mixtures thereof.

68. The method of claim 61, wherein the cells are one or more cells selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells.

69. The method of claim 61, further comprising contacting the system with living tissue.

77. A method of measuring cell migration activity, comprising:

providing a plurality of porous, hydrophilic substrates, wherein the hydrophilic substrates are stacked over one another;

introducing a first hydrogel comprising cells within the first defined region in a first substrate;

introducing a second hydrogel free of the cells within the second defined region in a second substrate; and

allowing the cells migrate from the first defined region to the second defined region, wherein a measure of cell migration activity is determined.

78. The method of claim 77, wherein cell migration activity is determined by destacking the substrates and characterizing the cells in each of the first and second defined regions.

79. The method of claim 77, wherein the cells are bacterial cells, insect cells, yeast cells, mammalian cells or mixtures thereof.

80. The method of claim 77, further comprising introducing a hydrogel comprising cells into defined regions located in a plurality of substrates.

81. The method of claim 80, wherein the cells of the hydrogel are different in different defined regions.

82. A method of patterning a porous, hydrophilic substrate, the method comprising:

contacting a porous, hydrophilic substrate with an aqueous solution comprising a water-soluble compound, the solution infiltrating the substrate to form a first region of the substrate that is saturated with the solution and a second region that is not contacted with the solution;

contacting the substrate with a hydrophobic material, the hydrophobic material saturating the second region; and

removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.

84. The method of claim 82 or 83, wherein the water-soluble compound is selected from the group consisting of sucrose, trehalose, glucose, fructose, xylitol, ribose, threitol, mannose, and glycerol and mixtures thereof.

85. The method of claim 82 or 83, wherein the hydrophobic material is selected from the group consisting of poly(dimethylsiloxane), poly(lactic-co-glycolic acid), epoxy and polystyrene and mixtures thereof.

86. The method of claim 82 or 83, wherein the solution is printed using an inkjet printers.

83. A method of patterning a porous, hydrophobic substrate, the method comprising:

contacting a porous, hydrophilic substrate with a hydrophobic material, the hydrophobic material saturating the substrate;

contacting a region of the substrate with an aqueous solution comprising a water-soluble compound, the solution displacing the hydrophobic material from the region; and

removing the water-soluble compound, resulting in a hydrophilic porous region that is bounded by the hydrophobic material.