Method for deforming and/or fragmenting a cell, spore or virus with a vibrating plate

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 deforming and/or fragmenting a cell, a virus, or a mixture thereof, wherein the cell is a bacterial cell, yeast cell, mammalian cell, or blood cell, the method comprising: (i) bringing a liquid sample containing the cell, virus, or mixture thereof into contact with a first surface of a vibratable plate having at least one aperture, and causing the plate to vibrate; (ii) passing the liquid sample containing the cell and/or virus through the at least one aperture in the vibrating plate to cause a) deformation of the cell and/or virus by shear force, mechanical impact and/or ultrasonic effects, and/or b) fragmentation of the cell and/or virus; and (iii) collecting a liquid comprising the deformed or fragmented cell and/or virus.

2. The method according to claim 1, wherein the at least one aperture has an average diameter of 20 to 170% relative to the diameter of an average cell or virus in the liquid sample.

3. The method according to claim 1, wherein relaxation of the cell and/or virus after passing of the cell and/or virus through the at least one aperture causes the outer membrane and/or wall of the cell and/or virus to become more permeable to external molecular species.

4. The method according to claim 1, wherein the method is for fragmenting the cell and/or virus and wherein the at least one aperture has an average diameter of less than 70% relative to the average diameter of the cell or virus.

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

6. The method according to claim 1, wherein vibration of the plate causes the plate to be displaced perpendicular to the plane of the plate.

7. The method according to claim 1, wherein vibration of the plate is caused by a piezoelectric actuator.

8. The method according to claim 1, comprising deforming or fragmenting the cell.

9. The method according to claim 1, wherein beads are added to the liquid sample containing the cell, virus, or mixture thereof.

10. The method according to claim 1, wherein enzymes and/or salts are added to the liquid sample containing the cell, virus, or mixture thereof.

11. The method according to claim 1, wherein the vibration of the plate has an amplitude of 0.1 to 5 m and a frequency of 5 to 200 kHz.

12. The 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 to deform and/or fragment the cell or virus as compared to a different cell or virus, respectively.

13. The method according to claim 1, further comprising chemical-based transfection, non-chemical-based transfection, and/or particle-based transfection.

14. The method according to claim 1, wherein relaxation of the cell and/or virus following passing of the cell and/or virus through the at least one aperture causes the outer membrane and/or wall of the cell and/or virus to become permeable to external macromolecular species.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1

(2) Analytical, results of mammalian blood lysates at concentration levels of 2 l of 250 l/ml, 125 l/ml and 15 l/ml used to provide a DNA template for qRT-PCR. The cycle threshold (Ct) values are averages of two duplicates, and relate to the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceed the background level). The Ct values are inversely proportional to the amount of target nucleic acid in the sample, i.e. the lower the Ct value the greater the amount of nucleic acid in the sample. The results marked 9/10 and 10/10 indicate lysate duplicates were prepared independently. Key: +=cells lysed and DNA purified using a QIAamp DNA Mini Kit (QIAGEN) prior to qRT-PCR. =cells unlysed.

(3) The final bar with no fill represents the result of a no-template control.

(4) FIG. 2

(5) Analytical results of PCR reaction products obtained after a single pass of a sample containing mammalian blood cells through a vibratable membrane having an average aperture diameter of 4.6 m. Key: +=PCR reaction including purified gDNA. =No template for reaction. Unlys.=250 l whole blood diluted in water to a final volume of 1 ml (1/4 dilution), and 2 l added to PCR reaction. Others=indicated volumes of whole blood diluted in water to a final volume of 1 ml, lysed using the device of the invention, and 2 l of lysates added to 50 l PCR reactions.

(6) FIG. 3

(7) Analytical results of the amplification of DNA from mammalian blood lysates using alternative DNA polymerases. Key: +=PCR reaction including purified gDNA. =No template for reaction. In all cases, the indicated volumes of whole blood were diluted in water to a final volume of 1 ml, lysed using the device of the invention (or unlysed), and 2 l of lysates added to 50 l PCR reactions.

(8) FIG. 4

(9) Analytical results using greater volumes of blood in PCR reactions. The results show that PCR can be carried out even when large volumes of blood lysate are added to the reaction mix. Key: +=PCR reaction including purified gDNA. =No template for reaction. Unlys.=250 l whole blood diluted in wales to a final volume of 1 ml (1/4 dilution), and 2 l added to PCR reaction. Others=250 l whole blood diluted in water to a final volume of 1 ml (1/4 dilution), lysed using the device of the invention, and the indicated volumes added to 50 l PCR reactions.

(10) FIG. 5

(11) Analytical results of PCR using (i) DNA in blood lysate or (ii) DNA purified from blood lysate using a Nexttec sorbent column. The Nexttec sorbent filter was used to purify DNA from blood lysed using the device of the invention, with no inhibitory effects on subsequent PCR. Key: +=PCR reaction including purified gDNA. =No template for reaction. Purification of DNA using Nexttec cleanColumns was then used for PCR, with 2 l added to PCR reaction.

(12) FIG. 6

(13) Analytical results of DNA purified from PCR reactions performed on blood lysate, using Nexttec sorbent columns. The Nexttec sorbent filter effectively purifies PCR-amplifted DNA (prepared using lysate taken from the device of the invention as a source of template for PCR). Key: +=PCR reaction including purified gDNA. =No template for reaction.

(14) FIG. 7

(15) Microscopy of samples containing mammalian blood cells before (top) and after (bottom) being subjected to a single pass through the device according to the invention.

(16) FIG. 8

(17) Microscopy of samples containing yeast cells before (top) and after (bottom) being subjected to a single pass through the device according to the invention.

(18) FIG. 9

(19) Microscopy of samples containing, mammalian blood cells and bacterial cells before (top) and after (bottom) being subjected to a single pass through the device according to the invention. The images show that blood cells may be selectively lysed in favour of the bacterial cells. The inserts show fluorescent scans (Using a TTP Labtech Acument Explorer system) of the sample treated with a calcein (live/dead) dye, indicating that the bacterial cells survived the treatment and the blood cells were selectively lysed

(20) FIG. 10

(21) The data shown is a fluorescent readout of a sample (200 L) of a mammalian, Jurkat, cell line that has been incubated with FITC labelled antibody and then washed. The antibody cannot permeate the plasma membrane of viable cells. A1 and A2 show a sample that has not passed through the device. A3 and A4 show the output of cells that have been passed through a device with 13 m apertures and into a solution of the FITC labelled antibody before washing. The considerable difference in the fluorescence measurement suggests the device provides considerable poration of the cells and transfer of the antibody through the membrane.

(22) FIG. 11

(23) A schematic view of a device according to the invention. 1 is a liquid sample containing a cell, spore or virus. 2 is an actuator, such as a piezo ring. 3 is an area of plate containing at least one aperture. 4 is a system holder. 5 is a plate.

(24) FIG. 12

(25) A fluorescent image of a sample of a mammalian, Jurkat, cell line (containing approximately 50,000 cells/mL) passed through a device according to the invention in the presence of an Alexa Fluor-labelled IgG antibody. The image was measured using a TTP Labtech Mirrorball system (TTP Labtech, Melbourn, UK).

(26) FIG. 13

(27) The data shown compares the intracellular delivery (in nominal units of fluorescence) of Alexa Flour-labelled IgG labelled antibody into a sample of a mammalian, Jurkat, cell line containing approximately 50,000 cells/mL and having a cell size of approximately 20 m) following passage through the device according to the invention. Subsequent labelling was performed with calcein-AM and a Hoechst counter stain. The Hoechst counter stain labels all cells and the calcein is only converted in live cells to give a fluorescent entity, thereby providing a measurement of the live:dead ratio (i.e. viability) of the cells in the treated sample.

EXAMPLES

(28) A device according to the invention (see FIG. 11) was employed in the following examples.

Example 1

(29) Quantification of DNA Purified from Blood Lysates

(30) TABLE-US-00001 TABLE 1 DNA in lysates Theoretical maximum DNA Blood sample (g/ml) in lysates (g/ml) 250 l/ml () 0.5-3.sup. 11.3 125 l/ml () 0.2-1.2 5.6 15 l/ml ( 1/66) 0.05-0.2 0.7 250 l/ml (, unlysed) 0.01-0.04

(31) Table 1 above shows concentrations of DNA purified from lysates, as calculated using Picogreen reagent, Invitrogen. DNA purification procedure: QIAamp Mini Kit, QIAGEN. Results obtained using vibratable mesh having 4.6 m pore diameter, and flow rate of 150 l lysate per minute. Theoretical maximum yields of DNA were calculated by assuming, that there are 7000 white blood cells per l of blood, and that each cell contains 6.8 pg DNA. Therefore, quantification of DNA purified from blood lysates indicated that large quantities of DNA are released by the device of the invention.

Example 2

Levels of DATA in Lysates

(32) FIG. 1 shows concentration levels of 2 l of 250 l/ml, and 15 l/ml of the lysates which were used to provide a DNA template for qRT-PCR. The Ct values are averages of two duplicates. The results marked 9/10 and 10/10 indicate lysate duplicates were prepared independently. +=cells lysed and DNA purified using QIAamp DNA Mini Kit (QIAGEN) prior to qRT-PCR. =cells unlysed. The final bar with no till represents the result of a no-template control.

(33) Therefore, qRT-PCR suggests that levels of DNA in lysates produced by the device are least comparable to those produced by commercially-available kits.

Example 3

DNA Released from Cells During Lysis of Diluted Blood

(34) FIG. 2 shows that a single pass through the vibratable mesh (4.6 m pore diameter) releases more than enough DNA to be used as a template for PCR. No subsequent purification of DNA was required before PCR.

(35) The PCR reaction products (HatStarTaq, QIAGEN) were analysed on 2% agarose gel. +=PCR reaction including purified gDNA. =No template for reaction. Unlys.=250 l whole blood diluted in water to a final volume of 1 ml (1/4 dilution), and 2 l added to PCR reaction. Others=indicated volumes of whole blood diluted in water to a final volume of 1 ml, lysed using the device if the invention, and 2 l of lysates added to 50 l PCR reactions.

(36) Therefore, DNA released from cells during lysis of diluted blood can be used as a template for PCR without prior purification of the DNA.

Example 4

Amplification DATA from Blood Lysates

(37) FIG. 3 shows that the ability to PCR directly from blood lysate produced by the device of the invention is not limited to one DNA polymerase. The PCR reaction products were analysed on 2% agarose gel. +=PCR reaction including purified gDNA. =No template for reaction. In all cases, the indicated volumes of whole blood were diluted in water to a final volume of 1 ml, lysed using the device of the invention (or unlysed), and 2 l of lysates added to 50 l PCR reactions.

(38) Therefore, many DNA polymerases can be used to amplify DNA from blood lysate produced by the device of the invention.

Example 5

Assessment of Greater Volumes of Blood in PCR Reactions

(39) FIG. 4 shows that greater volumes of blood in PCR reactions do not significantly inhibit the process, PCR is still possible even when large volumes of blood lysate are added to the reaction mix, PCR reaction products (HotStarTaq, QIAGEN) analysed on 2% agarose gel. +=PCR reaction including purified gDNA. =No template for reaction. Unlys.=250 l whole blood diluted in water to a final volume of 1 ml (1/4 dilution), and 2 l added to PCR reaction. Others=250 l whole blood diluted in water to a final volume of 1 ml (1/4 dilution), lysed using the device of the invention, and the indicated volumes added to 50 l PCR reactions.

(40) Therefore, using greater volumes of blood in PCR reactions does not significantly inhibit the process.

Example 6

(41) FIG. 5 shows that PCR is possible using (i) DNA in blood lysate or (ii) DNA purified from blood lysate using a Nexttec sorbent column. A Nexttec sorbent filter was used to purify DNA from blood lysed using the device of the invention, with no inhibitory effects on subsequent PCR. The PCR reaction products (HotStarTaq, QIAGEN) were analysed on 2% agarose gel. +=PCR reaction including purified gDNA. =No template for reaction. Purification of DNA using Nexttec cleanColumns were then used for PCR, with 2 l added to PCR reaction.

(42) Therefore, PCR is possible using (i) DNA in blood lysate or (ii) DNA purified from blood lysate using a Nexttec sorbent column.

Example 7

(43) FIG. 6 shows that DNA can be efficiently purified from PCR reactions performed on blood lysate, using Nexttec sorbent columns. The Nexttec sorbent filter can effectively purify PCR-amplified DNA (prepared using lysate obtained from the device of the invention as a source of template for PCR). The PCR reaction products (HotStarTaq, QIAGEN) were analysed on 2% agarose gel. DNA purified from PCR reactions using Nexttec cleanColumns as indicated.

(44) Therefore, DNA can be efficiently purified from PCR reactions performed on blood lysate, using Nexttec sorbent columns.

Example 8

(45) Light microscopy was carried out on porcine blood before and after transition through the device of the invention. In this case, the system employed 3 micron apertures and the cells were effectively lysed. The resulting image is shown in FIG. 7.

Example 9

(46) Dark field microscopy was carried out on yeast cells before and after transition through the device of the invention. A considerable increase in the damaged and empty cells was seen. In this case, the system employed 3 micron apertures. The resulting image is shown in FIG. 8.

Example 10

(47) Light microscopy was carried out on a porcine blood/bacteria mix (Serratia marcescens) before and after passage through a 4 micron aperture device according to the invention. The resulting image is shown in FIG. 9.

Example 11

(48) Enhanced macromolecular delivery to cells by use of the method and device of the invention. A sample (200 l) of a mammalian cell line was incubated with FITC labelled antibody and then washed. The antibody could not permeate the plasma membrane of viable cells. Fluorescence data was obtained with samples that had not been passed through the device and with cells that had been passed through a device with 13 m apertures and into a solution of the FITC labelled antibody before washing (see FIG. 10). The considerable difference in the fluorescence measurement suggests the device provides considerable poration of the cells and transfer of the antibody through the membrane.

Example 12

(49) Mammalian Jurkat cells were prepared and diluted to approximately 50,000 cells/mL. The cell suspension was mixed with an Alexa Fluor-labelled antibody. The suspension was then passed through a device according to the invention (at ca. 80 kHz) in which the sizes of the apertures of the vibrating plate are detailed below. The material having passed through the device according to the invention was imaged on a TTP Labtech Mirrorball system and the extent of cell permeability analysed by cell fluorescence. The samples were then incubated with calcein-AM and a Hoechst counter stain, whereby the Hoechst counter stain labels all cells and the calcein is only converted in live cells to give a fluorescent entity. This enabled measurement of the live:dead ratio (i.e. viability) of the cells in the samples.

(50) In FIG. 12, A1 and B1 show a sample that has not passed through the device according to the invention, i.e. a negative control. A2 and B2 show cells that have been passed through a device according to the invention with 9 m apertures with a solution of the Alexa Fluor-labelled antibody. A3 and B3 show cells that have been passed through a device according to the invention with 27 m apertures. A4 and B4 show cells that have been passed through a device according to the invention with 12 m apertures. A5 and B5 show cells that have been passed through a device according to the invention with 15-18 m apertures. The enhanced fluorescence measurements for A2/B2, A4/B4 and A5/B5 suggest the device according to the invention provides considerable poration (enhanced permeability) of the cells and transfer of the antibody through the membrane, yet results in cells which remain viable.

(51) In FIG. 13, D1 shows a sample that has been passed through a device according to the invention with 9 m apertures, resulting in a high level of deformation and a low level of cell viability (i.e. a high level of cell lysis). D2 shows a sample that has been passed through a device according to the invention with 27 m apertures, resulting in a low level of deformation and approximately 50% cell viability. D3 and D4 show samples that have been passed through a device according to the invention with 12 m apertures and 15-18 m apertures, respectively, resulting in a good level of deformation and a good level of cell viability. D5 shows a sample that has been passed through a device according to the invention with >25 m apertures, resulting in a low level of deformation and a high level of cell viability. These results therefore collectively show that cells passing through apertures of a similar size to the size of the cells are deformed (e.g. so as to facilitate enhanced intracellular macromolecular delivery) to a significant level and that the cells remain viable. Thus, when the macromolecular species is a nucleic acid species, transfection can be enhanced due to the improved delivery of the nucleic acid species into the cell. In addition, the results show that apertures of a smaller size than the size of the cells tend more to result in fragmentation (and potentially lysis) of the cells.

(52) Given that a relatively high vibrational amplitude was employed in this experiment, it may be expected that lower vibrational amplitudes could lead to improved survival rates of the cells (i.e. enhanced viability) with similar levels of deformation.