High throughput screen

• US 20050221282A1
• Filed: 05/20/2005
• Published: 10/06/2005
• Est. Priority Date: 06/12/1998
• Status: Active Grant

• Current Assignee: Sophion Bioscience A/S
• Original Assignee: CeNeS Limited (PAION AG), Xention Discovery Ltd. (Xention Pharma Ltd.)
• Inventors: Byrne, Nicholas Gerard, Owen, David Geraint
• Granted Patent: US 7,846,389 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 27/28 Electrolytic cell components
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: Y10T 29/49826 Assembling or joining

Abstract

The present invention relates to a structure comprising a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.

Claims

1. A structure which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate for use in a high throughput screen wherein the biological membrane comprises an ion channel or transporter.

2. A structure according to claim 1 wherein the biological membrane comprises a continuous layer of cells which is capable of adhering to a substrate with a high resistance seal wherein each cell forms a tight junction with adjacent cells and expresses an ion channel or transporter which is localised in the cell membrane.

3. A structure according to claim 1 which comprises cells having an ion channel or transporter which naturally resides in the cell membrane thereof or, it can be inserted by transfection with cDNA and/or cRNA encoding the ion channel or transporter.

4. A structure according to claim 1 which comprises a plurality of ions channels or transporters which are predominantly preselected ion channels or transporters of interest.

5. A structure according to claim 1 which comprises genetically engineered cells which have been engineered to predominantly express an ion channel or transporter.

6. A structure according to claim 1 which comprises voltage gated ion channels.

7. A structure according to claims 2 wherein the cells are selected from the group which comprises HEK-293 cells, genetically modified Chinese hamster ovary (CHO) cells, primary neuronal tissue such as hippocampus, dorsal root ganglia, superior cervical ganglia etc.;

skeletal muscle;

smooth muscle;

cardiac muscle;

immune cells;

epithelia;

endothelia.

8. A structure according to claim 1 which comprises an ion channel having rapid activation and inactivation kinetics.

9. A structure according to claim 1 having an ion channel which shows specificity for an ion selected from the group which comprises sodium, potassium, calcium, chloride.

10. A structure according to claims 2 wherein the contiguous layer of cells comprises cells is capable of adhering with a high resistance seal to a substrate selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP).

11. A structure according to claim 1 which comprises a pseudo-epithelium wherein one face of a contiguous layer of cells is permeabilized thereby providing access to the interior of the cells.

12. A structure according to claim 11 which comprises a pseudo-epithelium wherein one face of the contiguous layer of cells is permeabilized by an antibiotic selected from the group which comprises amphotericin and nystatin;

or detergent selected from the group which comprises digitonin and saponin;

or physical disruption using a high voltage field;

or by enzymatic digestion of a part of the membrane using an appropriate enzyme.

13. A structure according to claim 1 wherein the substrate is perforated.

14. A structure according to claim 1 which comprises a perforated coverslip.

15. A structure according to claim 1 wherein the substrate has pores of diameters between 0.5 μ

m and 10 μ

m.

16. A structure according to claim 15 wherein the pores are of diameter between 1 μ

m and 7 μ

m.

17. A structure according to claim 15 wherein the pores are of diameter 1-2 μ

m.

18. A structure according to claim 1 which comprises a coverslip having a grid of pores.

19. A structure according to claim 1 which comprises a perforated substrate which is manufactured of a material selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP).

20-46. -46. (canceled)