Paper-based cellular arrays

• US 9,829,488 B2
• Filed: 03/27/2009
• Issued: 11/28/2017
• Est. Priority Date: 03/27/2008
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Derda, Ratmir, Sague, Anna Laromaine, Whitesides, George M.
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US12/934,192
• Publication Number:
  US 20110105360A1
• Time in Patent Office: 3,168 Days
• 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. A cellular system comprising:

a porous, hydrophilic substrate that wicks fluids by capillary action selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film, wherein the substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary, and wherein at least one of the porous regions comprises a temperature-sensitive hydrogel comprising cells.

2. The cellular system of claim 1, 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.

3. The cellular system of claim 1, 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.

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

contacting the cellular system of claim 1 with one or more test agents; and

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

13. The method of claim 12, wherein the cellular system is contacted with the one or more test agents at a plurality of defined regions.

14. The method of claim 13, wherein each defined region is contacted with a different test agent.

15. The method of claim 12, wherein the cellular activity is one or more selected from the group consisting of proliferation, migration, apotosis apoptosis, differentiation, viability, upregulation of gene transcription, or downregulation of gene transcription.

16. The method of claim 12, 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.

17. The method of claim 12, wherein the cells are selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells and mixtures thereof.

18. A method of claim 12, 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.

19. The cellular system of claim 1, 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.

20. The cellular system of claim 1, wherein there are a plurality of porous regions positioned in the porous hydrophilic substrate in the form of an array.

4. A kit comprising:

one or more porous, hydrophilic substrates selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film, 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.

5. The kit of claim 4, further comprising a temperature-sensitive hydrogel.

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

providing a porous, hydrophilic substrate selected from the group consisting of paper, nitrocellulose, cellulose acetate, cloth, and porous polymer film, wherein the porous, hydrophilic substrate comprises at least one porous region, each porous region bounded at least in part by a liquid impervious boundary; and

in any order,

contacting a plurality of defined regions of the substrate with a suspension comprising cells and a temperature-sensitive 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.

7. The method of claim 6, wherein cells are introduced into the hydrogel precursor before contacting the hydrogel precursor to the substrate.

8. The method of claim 6, wherein the gelling agent is temperature, a solution containing salt, or a chemical cross-linking agent.

9. The method of claim 6, 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.

10. The method of claim 6, wherein the cells are one or more cells selected from the group consisting of bacterial cells, insect cells, yeast cells, and mammalian cells.

11. The method of claim 6, further comprising contacting the system with living tissue.