Device and method for cell-based drug screening

Abstract

Provided are methods and compositions for cell-based drug screening with high-density arrays of conical microwells and slow-drug-release beads.

What is claimed is: 
     
         1 . A device comprising (i) a multiwell plate; (ii) a microwell array comprising a plurality of microwells, the microwell array located at the bottom of each well of the multiwell plate, wherein the diameter and depth of each microwell is in the range of about 7 μm to about 1 mm. 
     
     
         2 . The device of  claim 1 , wherein the size of the plurality of microwells in the microwell array are of a uniform depth and diameter. 
     
     
         3 . The device of  claim 1 , wherein the plurality of microwells are arranged in a hexagonal array. 
     
     
         4 . The device of  claim 1 , wherein each microwell in the plurality of microwells has a depth of about 150 μm and a top diameter in the range of about 100 μm to about 400 μm. 
     
     
         5 . The device of  claim 1 , wherein the microarray comprises a plurality of microwells comprising conical frustums with opening angles in the range from 0 to 400°. 
     
     
         6 . The device of  claim 5 , wherein each microwell in the plurality of microwells comprises an opening angle of 34°, a depth of about 150 μm, a diameter at the bottom of about 50 μm, and a diameter at the top of about 130 μm. 
     
     
         7 . The device of  claim 5 , wherein each microwell in the plurality of microwells comprises an opening angle of 17°. 
     
     
         8 . The device of  claim 7 , wherein each microwell in the plurality of microwells comprises a depth of about 40 μm, a diameter at the bottom of about 7 μm, and a diameter at the top of about 24 μm. 
     
     
         9 . The device of  claim 1 , wherein the microarray is made of polydimethylsiloxane (PDMS) or a hydrogel. 
     
     
         10 . The device of  claim 1 , wherein the multiwell plate is a 192-well plate, a 96-well plate, a 48-well plate, or a 24-well plate. 
     
     
         11 . The device of  claim 1 , further comprising cells at the bottom of the microwells. 
     
     
         12 . The device of  claim 11 , further comprising a plurality of drug-eluting beads, wherein one bead is nestled into or sealed into a microwell of the microwell array. 
     
     
         13 . The device of  claim 12 , wherein the drug-eluting beads are made of a hydrogel. 
     
     
         14 . The device of  claim 13 , wherein the hydrogel is one of: polyacrylamide (PAA), dextran, polyethylene oxide, UV-cross-linkable polyethylene glycol (PEG), gelatin or poly(D,L)-lactide-co-glycolide (PLGA). 
     
     
         15 . The device of  claim 12 , wherein the drug-eluting beads are made of a hydrocarbon polymer. 
     
     
         16 . The device of  claim 15 , wherein the drug-eluting beads are made of polystyrene. 
     
     
         17 . The device of  claim 12 , wherein each of the drug-eluting beads comprise a drug bound to the surface of the bead. 
     
     
         18 . The device of  claim 11 , wherein each drug-eluting bead is uniquely labeled with one or more fluorescing nanoparticles. 
     
     
         19 . The device of  claim 18 , wherein the one or more fluorescing nanoparticles is one or more qDots. 
     
     
         20 . The device of  claim 1 , wherein the microwell array comprises at least 8,000 microwells. 
     
     
         21 . The device of  claim 1 , wherein the microwell array comprises at least 80,000 microwells. 
     
     
         22 . The device of  claim 1 , wherein the top surface of the microwells is not flat. 
     
     
         23 . A method of screening for drug binding or activation of a target cell, the method comprising:
 a) providing a device of  claim 1 ;   b) coating target cells in isotonic solution on the bottom of the plurality of microwells;   c) capping one or more of the microwells with a drug-eluting bead under conditions that allow drug to diffuse from the bead to the target cell; and   d) detecting drug binding or activation of the target cell.   
     
     
         24 . The method of  claim 23 , wherein the isotonic solution is cell culture medium. 
     
     
         25 . The method of  claim 23 , further comprising reducing the ionic strength of the isotonic medium, thereby causing the drug eluting beads to swell in the microwell. 
     
     
         26 . The method of  claim 23 , wherein the drug-eluting bead is uniquely labeled with one or more fluorescing nanoparticles. 
     
     
         27 . The method of  claim 26 , wherein the one or more fluorescing nanoparticles is one or more qDots. 
     
     
         28 . The method of  claim 23 , wherein the drug-eluting bead is made of a hydrogel. 
     
     
         29 . The method of  claim 28 , wherein the hydrogel is of: polyacrylamide (PAA), dextran, polyethylene oxide, UV-cross-linkable polyethylene glycol (PEG), gelatin or poly(D,L)-lactide-co-glycolide (PLGA). 
     
     
         30 . The method of  claim 23 , wherein the drug-eluting beads are made of a hydrocarbon polymer. 
     
     
         31 . The method of  claim 30 , wherein the drug-eluting beads are made of a polystyrene. 
     
     
         32 . The device of  claim 23 , wherein each of the drug-eluting beads comprise a drug bound to the surface of the bead. 
     
     
         33 . The method of  claim 23 , wherein target cell activity is determined by measuring:
 i) drug binding and/or drug intake into cell;   ii) intracellular calcium release;   iii) G protein-coupled receptor activation;   iv) intracellular signaling cascades; and/or   v) transcriptional activity.   
     
     
         34 . The method of  claim 23 , further comprising: e) after (c) and before (d), exposing the microwells to fluorescence excitation and collecting fluorescence emission from the beads in the microwells. 
     
     
         35 . The method of  claim 34 , further comprising: f) after (e), correlating the detected fluorescence emission from the beads in the microwells with the location of each drug associated with the beads in the microwells. 
     
     
         36 . The method of  claim 35 , further comprising: g) after (d), correlating the detected drug binding or activation of the target cells in the microwells with the drug of the bead positioned in each of microwells of the detected cells. 
     
     
         37 . The method of  claim 34 , wherein the exposing further comprises exposing the microwells to fluorescence excitation derived from a laser.