METHOD AND DEVICE

• US 20160083687A1
• Filed: 05/16/2014
• Published: 03/24/2016
• Est. Priority Date: 05/16/2013
• Status: Active Grant

• Current Assignee: TTP PLC
• Original Assignee: The Technology Partnership PLC (TTP Group Ltd.)
• Inventors: Sanders, Giles Hugo William, Graham, Abi Clair, Stella, Rita
• Granted Patent: US 10,640,745 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12N 1/066 by physical methods
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention relates to a method for the deformation and/or fragmentation of a cell, spore or virus, the method comprising: (i) bringing a liquid sample containing the cell, spore or virus into contact with a first surface of a vibratable plate having at least one aperture, and causing the plate to vibrate; and (ii) passing the sample of the cell, spore or virus through the at least one aperture in the vibrating plate so as to cause deformation and/or fragmentation of the cell, spore or virus. It also concerns a device for carrying out the method.

Claims

1. A method for the deformation and/or fragmentation of a cell, spore or virus, the method comprising:

(i) bringing a liquid sample containing the cell, spore or virus into contact with a first surface of a vibratable plate having at least one aperture, and causing the plate to vibrate; and

(ii) passing the sample of the cell, spore or virus through the at least one aperture in the vibrating plate so as to cause deformation and/or fragmentation of the cell, spore or virus.

2. A method according to claim 1, further comprising:

(iii) collecting the deformed and/or fragmented products.

3. A method according to claim 1, wherein the method is for the deformation of a cell, spore or virus.

4. A method according to claim 3, wherein the at least one aperture has an average diameter of 20 to 170% of the average cell, spore or virus diameter in the sample.

5. A method according to claim 1, wherein relaxation of the cell, spore or virus following passing of the cell, spore or virus through the at least one aperture causes the outer membrane and/or wall of the cell, spore or virus to become more permeable to external molecular species, such as macromolecular species.

6. A method according to claim 1, wherein the method is for the fragmentation of a cell, spore or virus.

7. A method according to claim 5, wherein the average diameter is less than 70% of the average cell, spore or virus diameter in the sample.

8. A method according claim 1, wherein the vibratable plate is a vibratable mesh having a plurality of apertures.

9. A method according to claim 1, wherein vibration of the plate causes the plate to be displaced, at least partially, in a direction perpendicular to the plane of the plate.

10. A method according to claim 1, wherein vibration of the plate is caused by means of a piezoelectric actuator.

11. A method according to claim 1, wherein the cell, spore or virus is selected from mammalian cells, bacterial cells, fungal cells, plant cells, algal cells, spores or viruses, or mixtures thereof.

12. A method according to claim 1, wherein beads are added to the sample containing the cell, spore or virus, or mixtures thereof.

13. A method according to claim 1, wherein enzymes and/or salts are added to the sample containing the cell, spore or virus, or mixture thereof.

14. A method according to claim 1, wherein the vibration of the plate has a drive waveform which is sinusoidal, square, or triangular.

15. A method according to claim 1, wherein the vibration of the plate has an amplitude of 0.1 to 5 μ

m.

16. A method according to claim 1 wherein the vibration of the plate has a frequency of 5 to 200 kHz.

17. A method according to claim 16, wherein the vibration of the plate has a frequency of 35 to 120 kHz.

18. A method according to claim 1, wherein a drive waveform of the vibrating plate is pulsed with a duty factor of 0.01 to 1% and a duty period of 1 ms to 2 s.

19. A method according to claim 1, wherein the passage of the sample through the vibrating plate has an average flow rate of 1 nl/s to 25 ml/s.

20. A method according to claim 2, further comprising repeating steps (i)-(iii) by means of recycling the collected products back to the first surface of the vibrating plate.

21. A method according to claim 2, further comprising repeating steps (i)-(iii) by means of bringing the collected products into contact with a first surface of at least one further vibrating plate arranged downstream.

22. A method according to claim 1, wherein plate size, aperture size, positioning of the at least one aperture in the plate, vibration frequency and/or velocity of plate movement are selected such that one type of cell, spore or virus is preferentially deformed and/or fragmented compared to another type of cell, spore or virus.

23. A method according to claim 1, wherein at least one diameter of the at least one aperture in the plate is in the range 50 nm to 40 μ

m.

24. A method according to claim 1 further comprising at least one known method of cell lysis carried out before, during or after the steps defined above, such as a known method selected from mechanical lysis, liquid homogenisation, sonication, freeze-thaw cycling, manual grinding, and chemical lysis.

25. A method according to claim 1, further comprising at least one known method of cell deformation carried out before, during or after the steps defined above, such as a known method selected from chemical-based transfection, non-chemical-based transfection, and/or particle-based transfection.

26. A device for the deformation and/or fragmentation of a cell, spore or virus, the device comprising:

a plate comprising at least one aperture extending from a first surface of the plate to a second surface of the plate;

an actuator in operational connection with the plate so as to cause the plate to vibrate upon activation of the actuator; and

a reservoir containing a liquid sample of the cell, spore or virus in fluid connection with the first surface of the plate.

27. A device according to claim 26, wherein the plate is a mesh having a plurality of apertures.

28. A device according to claim 26, wherein the actuator is activated upon application of an alternating potential difference.

29. A device according to claim 26, further comprising a receptacle into which the sample of the deformed and/or fragmented cell, spore or virus can be delivered upon passage through the apertures in the plate.

30. A device according to claim 26, further comprising a resilient member, positioned in registration with the second surface of the plate.

31. A device according to claim 26, further comprising a valve, in fluid connection with the second surface of the plate.

32. A device according to claim 26, wherein the plate is composed of a metal, ceramic, glass, or polymer.

33. A device according to claim 26, wherein at least one diameter of the at least one aperture in the plate is in the range 50 nm to 40 μ

m.

34. A device according to claim 26, wherein the at least one aperture in the plate has a substantially non-circular shape.

35. A device according to claim 34, wherein the at least one aperture in the plate is substantially square, triangular, oval, or slot-shaped in cross-section.

36. A device according to claim 26, wherein the cell, spore or virus is selected from mammalian cells, bacterial cells, fungal cells, plant cells, algal cells, spores or viruses, or mixtures thereof.

37. A device according to claim 26, wherein the actuator is a piezoelectric actuator.

38. A container comprising a device according to claim 26, and a receptacle, wherein the device is detachably mounted on a surface of the receptacle, such that a fluid sample passed through the device passes into or out of the receptacle.

39. A container according to claim 38, wherein the device is detachably mounted on a wall, a floor, on or in a lid, or at a mouth of the receptacle.

40. A container according to claim 38, wherein the container is a bottle, cuvette, well plate, or vial.

41. Use of a device according to claim 26, for the deformation and/or fragmentation of a cell, spore or virus.

42. Use according to claim 41, for the lysis of mammalian cells, bacterial cells, fungal cells, plant cells, algal cells, spores or viruses, or mixtures thereof.

43. Use according to claim 41, for the transfection of mammalian cells, bacterial cells, fungal cells, plant cells, algal cells, spores or viruses with one or more polynucleotide species.