Device and method for electroporation-based delivery of molecules into cells and dynamic monitoring of cell responses

• US 20060121446A1
• Filed: 11/23/2005
• Published: 06/08/2006
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: ACEA Biosciences, Inc (Agilent Technologies, Inc.)
• Inventors: Wang, Xiaobo, Xu, Xiao, Abassi, Yama A., Xu, Junquan, Atienza, Josephine
• Granted Patent: US 8,206,903 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: G01N 15/12 by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10

Abstract

The present invention includes devices and methods for transfecting a cell or cell population and dynamic monitoring of cellular events. A variety of microelectronic devices are provide that incorporate functions such as electroporation, modulation of a transmembrane potential and dynamic monitoring of cellular functions and mechanisms.

Claims

1. A method of monitoring a cellular response in real time comprising:

a. transfecting a cell or cell population with a molecule; and

b. monitoring impedance of said cell or said cell population.

2. The method according to claim 1, wherein said cell or cell population is transfected using a chemical transfection method.

3. The method according to claim 1, wherein said cell or cell population is transfected using electroporation.

4. The method according to claim 3, wherein said cell or cell population is electroporated by applying a sine wave or a square wave, or waveform following an exponential decay.

5. The method according to claim 1, wherein said cell or cell population is transfected using a thermal transfection method.

6. The method according to claim 1, wherein said cell or cell population is infected using a viral transfection method.

7. The method according to claim 1, wherein said cell is a eukaryotic cell.

8. The method according to claim 1, wherein said cell is a mammalian cell.

9. The method according to claim 1, wherein said molecule is selected from the group consisting of a DNA molecule, an RNA molecule, an oligopeptide, a polypeptide, a protein and a compound.

10. The method according to claim 8, wherein said DNA molecule is a recombinant DNA molecule, a native DNA molecule, a plasmid, a cDNA, anti-sense DNA strand or oligonucleotides.

11. The method according to claim 8, wherein said RNA molecule is a siRNA molecule, a microRNA molecule, native RNA molecule, ribozyme RNA or an aptamer.

12. The method according to claim 1, wherein said molecule is a capable of affecting transcription or affecting translation or affecting RNA splicing or affecting RNA editing.

13. The method according to claim 1, wherein said molecule is capable of affecting a cellular function selected from the group consisting of cell proliferation, cell adhesion, and cell spreading.

14. The method according to claim 1, wherein said molecule is capable of affecting cell morphology.

15. The method according to claim 1, wherein said molecule is capable of affecting a cellular receptor or signal transduction pathway activated by the said receptor.

16. The method according to claim 1, further comprising, transfecting a cell or cell population with a control molecule and monitoring the impedance of the cell or cell population transfected with the control molecule.

17. The method according claim 16, wherein the control molecule is the meolcule that has no direct effects on cellular functions.

18. The method according to claim 16, wherein further comprising the comparison between impedances of cell transfected with a molecule and control molecule.

19. The method according to claim 1, wherein said monitoring impedance comprises performing a series of impedance measurements.

20. The method according to claim 1, wherein said transfecting said cell or cell populations and said monitoring impedance are performed on the same device.

21. The method according to claim 1, wherein said transfecting said cells or cell population and said monitoring impedance are performed on different devices.

22. The method according to claim 1, further comprising monitoring at least one impedance prior to transfecting said cell or cell population.

23. The method according to claim 22, further comprising determining said cell population is to be transfected if said at least one impedance measurement prior to transfecting said cell or cell population is within a predetermined range.

24. The method according to claim 1, further comprising visually monitoring said cell population while monitoring impedance of said cell population.

25. The method according to claim 1, further comprising detecting the fluorescence of said cell or cell population.

26. The method according to claim 1, further comprising determining a cell index.

27. The method according to claim 26, further comprising determining a change in said cell index.

28. The method according to claim 1, futher comprising using impedance measurements in conjunction with end point assays such as cell viability assay, apotosis assay, enzymatic activity assay, signal transduction analysis assay, reporter assays.

29. The method according to claim 27, further comprising where impedance measurement is used as a guide to determine the time for performing the end-point assays.

30. A device for monitoring a cell or cell population comprising:

i. a nonconductive substrate;

ii. a plurality of electrode arrays positioned on said substrate, wherein each electrode array comprises at least two electrodes, further wherein each electrode is separated from at least one adjacent electrode by an area of non-conductive material; and

iii. at least one set of electroporation or electrostimulation electrodes, said electroporation electrodes capable of electroporating a cell or said electrostimulation electrodes capable of affecting cell membrane potential.

31. The device according to claim 30, wherein a first half of said set of electroporation or electrostimulation electrodes are in the plane of the substrate and a second half of said set of electrodes are not within the plane of the substrate.

32. The device according to claim 30, wherein the device comprises a plate comprising multiple wells, further wherein at least one of said multiple wells comprises a top, bottom and sidewall, further wherein said bottom is constructed from said nonconductive materials and said plurality of electrode arrays are positioned at said bottom, further wherein a first half of said at least one set of electroporation or electrostimulation electrodes are positioned at said bottom and a second half of said at least one set of electroporation or electrostimulation electrodes are positioned at said top.

33. The device according to claim 32, wherein said plate is a 96 well plate, 384 well plate or 1536 well plate.

34. The device according to claim 33, wherein said plate is capable of stacking on top of a second plate.

35. The device according to claim 32, wherein said multiple wells are capable of electroporating or electrostimulating cells together or separately.

36. The device according to claim 30, wherein said nonconductive substrate is a porous substrate that is located above one half of said at least one set of electroporation electrodes.

37. The device according to claim 30, wherein said affecting cell membrane potential results in opening or closing of a voltage gated ion channel in cell membrane.

38. A method of monitoring a cellular response to a test compound comprising:

i. providing a cell or cell population comprising a voltage gated ion channel to a device capable of inducing a change in cell membrane potential and capable of monitoring impedance of a cell or cell population, wherein said device is operably connected to an impedance analyzer;

ii. adding a cell or cell population comprising a voltage gated ion channel to the device;

iii. adding a test compound to the device;

iv. inducing a change in cell membrane potential;

v. and monitoring the impedance of said cell or cell population.

39. The method according to claim 38, wherein monitoring the impedance is conducted before, during, after, or before and during and after adding the test compound the device.

40. The method according to claim 38, wherein said inducing a change in cell membrane potential is capable of opening or closing voltage gated ion channels on the cell membrane.

41. The method according to claim 38, wherein monitoring the impedance is conducted before and after inducing a change in cell membrane potential.

42. The method according to claim 38, further comprising monitoring cell membrane potential or other ion channel activity associated parameters before and after said inducing a change in cell membrane potential.

43. The method according to claim 42, wherein monitoring cell membrane potential is performed using an optical detection method after an optically detectable compound is added to the device.

44. The method according to claim 43, wherein said optically detectable compound is a membrane-potential sensitive fluorescent dye.

45. The method according to claim 42, wherein monitoring cell membrane potential or other ion-channel activity-associated parameters is performed at the same time as said monitoring the impedance of said cell or said cell population.