High throughput miniaturized assay system and methods
The present invention provides an apparatus for conducting biological assays which employs “virtual wells” in lieu of the physical wells of conventional array plates. Also provided are methods of processing a sample and/or culturing cells using the apparatus and systems described herein. In some embodiments, the apparatus includes a first structure having a sheet layer with a plurality of discrete through holes; and a second structure coupled to the first structure, the second structure including a base layer. At least a portion of a first surface of the sheet layer of the first structure is exposed from the second structure, and a second surface of the sheet layer, opposite to the first surface of the sheet layer, is embedded in the base layer of the second structure adjacent the first surface of the base layer.
1. A method of culturing cells, comprising:
providing a processing compartment comprising a reservoir with a base surface, the processing compartment including a hydrophobic material and a hydrophilic material that is distinct from the hydrophobic material, said base surface having a hydrophobic solid surface defined by the hydrophobic material and an array of hydrophilic areas, each of said hydrophilic areas defined by the hydrophilic material and surrounded by the hydrophobic solid surface, only the hydrophobic solid surface being further covered with an immiscible hydrophobic liquid that is distinct from the hydrophobic material, and a plurality of said hydrophilic areas collectively having sample droplets disposed thereon, the sample droplets comprising cells;
incubating said base surface at a temperature suitable for cell culture;
flooding the reservoir with a wash fluid so that the wash fluid is concurrently in contact with the plurality of said hydrophilic areas;
draining the wash fluid from the reservoir;
filling said reservoir with an immiscible hydrophobic liquid;
tilting said processing compartment to remove all but a thin layer of said immiscible hydrophobic liquid that is retained on said base surface;
adding a first reagent to said processing compartment; and
tilting said processing compartment to remove said first reagent.
2. The method of claim 1 , wherein said cells are non-adherent cells.
3. The method of claim 1 , wherein said cells are stem cells selected from embryonic stem cells and fetal stem cells.
4. The method of claim 1 , wherein only said hydrophilic areas are functionalized with a biomaterial prior to addition of the cells.
5. The method of claim 4 , wherein said biomaterial is selected from the group consisting of collagen I, collagen II, collagen IV, poly-D-lysine (PDL), gelatin, fibronectin, laminin, and combinations thereof.
6. The method of claim 1 , wherein the plurality of said hydrophilic areas is coated or covalently linked with a biological moiety selected from a small molecule, lipid, peptide, oligonucleotide, or oligosaccharide.
7. The method of claim 1 , further comprising filling the reservoir with a cell culture medium, thereby immersing said cells.
8. The method of claim 7 , further comprises replacing said cell culture medium by draining said cell culture medium and replacing with fresh cell culture medium.
9. The method of claim 8 , wherein said replacing is done using a pipette.
10. The method of claim 8 , wherein said replacing is done by tilting said surface to drain said cell culture medium and adding new cell culture medium.
11. The method of claim 7 , wherein the sample droplets are coated with the immiscible liquid, thereby encasing each of the sample droplets on the base surface.
12. The method of claim 1 , further comprising adding a transfection nucleic acid to a plurality of said hydrophilic areas and incubating at a suitable temperature.
13. The method of claim 12 , further comprising assaying said cells to determine whether transfection has occurred.
14. The method of claim 12 , wherein said transfection nucleic acid is DNA or siRNA.
15. The method of claim 1 , further comprising:
adding a compound to at least one of said hydrophilic areas;
incubating said surface at a temperature suitable for cell culture; and
detecting the effect of said compound on said cells.
16. The method of claim 1 , wherein said immiscible liquid is selected from a group consisting of a mineral oil, a silicone oil, a hydrocarbon compound, a hydroperflurocarbon compound, a perfluorocarbon compound, and a mixture thereof.
17. The method of claim 1 , wherein each sample droplet has a volume of no greater than 12 μl.
18. The method of claim 1 , wherein the array of said hydrophilic areas has a surface roughness in the range of 0.1-1000 μm.
19. The method of claim 18 , wherein the surface roughness of the array of said hydrophilic areas is in the range of 100-1000 μm.
20. The method of claim 1 , wherein draining the wash fluid from the reservoir includes tilting the reservoir at 90 degree or higher angle so that the wash fluid is drained from the reservoir.