CN113322181A - Cell lysis device and cell purification and/or cell lysis method based on cell lysis device - Google Patents
Cell lysis device and cell purification and/or cell lysis method based on cell lysis device Download PDFInfo
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- CN113322181A CN113322181A CN202110646975.1A CN202110646975A CN113322181A CN 113322181 A CN113322181 A CN 113322181A CN 202110646975 A CN202110646975 A CN 202110646975A CN 113322181 A CN113322181 A CN 113322181A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
Abstract
The invention provides a cell lysis device and a cell purification and/or cell lysis method based on the cell lysis device, belonging to the technical field of cell lysis and purification; the cell lysis device comprises a pulse power supply (1) and a microtube electrode (12) electrically connected with the pulse power supply (1), wherein the microtube electrode (12) comprises an electrode tube (1-1); the inner wall of the electrode tube (1-1) is attached with a liquid metal film. The invention forms a non-uniform electric field above impurity cells by controlling the voltage released by a pulse power supply (1) through an electrode tube (1-1). The peripheral voltage of the impurity cells is far higher than the transmembrane potential of the impurity cells, so that the impurity cells are quickly cracked, and the aim of cracking the impurity cells is fulfilled.
Description
Technical Field
The invention relates to the technical field of cell lysis and purification, in particular to a cell lysis device and a cell purification and/or cell lysis method based on the cell lysis device.
Background
The selective lysis of single cells is a key step in the analysis of rare cells or target populations in complex biological samples such as blood or biopsies by disrupting intact and complex membrane structures to collect cellular components and to facilitate the analysis of intracellular components such as DNA, RNA and proteins.
The current means for cell lysis are mainly chemical lysis, sonic lysis, electrical lysis, optical lysis. In the chemical lysis process, lysis reagents such as detergents, chaotropic salts, enzymes and alkalis are required, which can affect the subsequent analysis of cellular fluid molecules. The optical lysis is to focus laser micro-pulses near target cells, form cavitation bubbles around the focused laser pulses, the bubbles can rapidly expand to burst within tens of milliseconds, huge impact force can be generated at the moment of burst of the bubbles to shatter cell membranes, but the impact force of burst of the bubbles is not easy to control, and other cells around impurity cells are easy to damage. The ultrasonic cell lysis has a great disadvantage in efficiency, and under the condition that the cells are not specially treated, tens of seconds are often needed for cell lysis by adopting ultrasonic waves, and a large amount of joule heat is generated in the process, so that the protein in the cells is denatured. In contrast, the electric lysis applies a high-voltage electric field far exceeding the transmembrane potential of the cell around the cell through the microtube electrode to lyse the cell, and has the advantages of high lysis efficiency, small influence on the cell property, easy operation and the like. However, the existing electrode using microtube needs to make microarray electrode on the cell culture substrate for cell lysis. However, the microarray electrode is difficult to manufacture, the cells are not regularly arranged, the cells can not be guaranteed to grow on the microarray electrode, the impurity cells which are not above the microarray electrode can not be cracked, and the cracking precision is low.
Disclosure of Invention
The invention aims to provide a cell lysis device and a cell purification and/or cell lysis method based on the cell lysis device.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a cell lysis device, which comprises a pulse power supply 1 and a microtube electrode 12 electrically connected with the pulse power supply 1, wherein the microtube electrode 12 comprises an electrode tube 1-1 and a liquid metal film attached to the inner wall of the electrode tube 1-1; the inner diameter of the tip of the electrode tube 1-1 is 4-20 mu m. The electrode tube 1-1 of the invention has small inner diameter which is only 4-20 μm. The pulse power supply 1 is controlled to release voltage, a non-uniform high-voltage electric field is formed above the impurity cells through the electrode tube 1-1, and after the voltage is applied, the impurity cells are quickly cracked because the peripheral voltage is far higher than the limiting value of the transmembrane potential of the impurity cells, so that the aim of cracking the impurity cells is fulfilled. Compared with the cell lysis method based on the microfluidic chip, the method for cell lysis by adopting the device disclosed by the invention has the advantages that the impurity cells are more accurately positioned, the loss of sample cells is avoided, and the inherent defects of the traditional lysis means and the large loss of the sample cells in the purification process based on the microfluidic technology are avoided; compared with other cracking means, the cell cracking device provided by the invention can be used for performing specified cracking on impurity cells, the leakage rate of the impurity cells is low, the purification rate is high, multiple purification is performed, and the purification rate can even reach 100%. In addition, the cell lysis device has high electric lysis speed and high efficiency, and can complete single cell lysis in about 1 s. In addition, the microtube electrode 12 of the present invention is simple to manufacture, low in cost, and simple to operate, and avoids the complicated manufacture of a microarray motor in the conventional electric cracking.
Drawings
FIG. 1 is a schematic structural diagram of the apparatus according to example 1, in which 1 is a pulse power supply, 2 is a pressure sensor, 3 is an injection pump, 4 is a stepping motor, 5 is a data acquisition card, 6 is a data processing terminal, 7 is a copper wire, 8 is a micro-tube channel, 9 is a robot arm, 10 is a micro-tube holder, 11 is an inverted microscope, 12 is a micro-tube electrode, 13 is a culture dish (cell sample), 14 is a mobile stage, and 15 is a camera;
FIG. 2 is a schematic diagram of cells looking for impurities;
FIG. 3 is an impurity cell lysis module;
FIG. 4 is a collection module for cell lysate;
in FIGS. 2 to 4, 1-1 is an electrode tube and 1-2 is a collecting tube.
Detailed Description
The invention provides a cell lysis device, which comprises a pulse power supply 1 and a microtube electrode 12 electrically connected with the pulse power supply 1, wherein the microtube electrode 12 comprises an electrode tube 1-1 and a liquid metal film attached to the inner wall of the electrode tube 1-1; the liquid metal film does not react with water chemically; the inner diameter of the tip of the electrode tube 1-1 is 4-20 mu m.
In the invention, the liquid metal film can be attached to the inner wall of the tip of the microtube with the tip caliber of several microns, thereby not only ensuring the conductivity, but also having simple manufacturing process and low cost.
In the present invention, the liquid metal thin film preferably includes a liquid metal gallium thin film.
In the present invention, the shape of the electrode tube 1-1 preferably includes a needle shape; a thin, preferably thin, film of liquid metal gallium is attached to the tip of the electrode tube 1-1.
In the present invention, the inner diameter of the tip of the electrode tube 1-1 is preferably 5 to 15 μm, and more preferably 8 to 10 μm.
In the present invention, the microtube electrode 12 preferably further includes a collection tube 1-2.
In the invention, the microtube electrode 12 preferably consists of 3 single microtubes, two are electrode tubes 1-1 for generating a high-voltage electric field to crack cells, and 1 is a collecting tube 1-2 without filler, and can realize the suction and collection of cells or cell sap by matching with a pressure control system; the shape of the cross section of the microtube electrode 12 is preferably a regular triangle; the microtube electrode 12 is preferably made of glass or acrylic.
The microtube electrode 12 is preferably prepared by the following method: drawing a raw material pipe of the microtube by using a needle drawing instrument to obtain the microtube;
filling liquid metal into the two microtubes, and centrifuging to move the liquid metal to the tips of the microtubes; and cutting the micro-tube to ensure that the inner diameter of the tip of the micro-tube is 4-20 mu m. The invention has no special limit on the amount of the filled liquid metal and can realize the electric conduction.
In the present invention, the inner diameter of the tip of the microtube is preferably 5 to 15 μm, and more preferably 10 μm.
In the invention, the speed of centrifugation is preferably 3000 r/min-1200 r/min (corresponding to 5-15 μm); the time of the centrifugation is preferably 2 min; the equipment used for the centrifugation is preferably a centrifuge.
In the present invention, the device for cutting the microtube is preferably a short needle device.
In the present invention, the liquid metal exposed to the air is rapidly oxidized to form a thin film on the surface of the tip of the microtube, contributing to stabilization.
In the invention, the needle drawing instrument is preferably a multi-tube needle drawing instrument PMP-107 with the following product number: 20JYSH/920740 HK.
In the present invention, the pulse power source 1 is preferably connected to the electrode tube 1-1 of the microtube electrode 12 through a copper wire. In the specific implementation process of the invention, two copper wires are respectively inserted into two microtubes filled with liquid metal, and the other ends of the copper wires are connected with a pulse power supply and used for applying a high-voltage electric field to the electrodes of the microtubes.
In the invention, the device preferably further comprises a pressure sensor 2, an injection pump 3, a data acquisition card 5 and a data processing terminal 6;
the pressure sensor 2 is connected with the collecting pipe 1-2 and used for receiving a pressure signal in the collecting pipe 1-2 and converting the pressure signal into an electric signal;
the data acquisition card 5 is respectively connected with the pressure sensor 2 and the data processing terminal (6) and is used for acquiring the electric signals and transmitting the electric signals to the data processing terminal 6 in real time;
the data processing terminal 6 is connected with the injection pump 3, and outputs a control signal according to the acquired electric signal, wherein the control signal controls the injection pump 3 to inject or suck;
the injection pump 3 is connected with the collecting pipe 1-2, and positive pressure or negative pressure is generated inside the collecting pipe 1-2 by injection or suction of the injection pump 3, so that cell sap is sucked or discharged.
In the present invention, the connection between the syringe pump 3 and the collection tube 1-2 is preferably a pipe connection.
In the present invention, it is preferable to further include a stepper motor 4; the step motor 4 is respectively and electrically connected with the data processing terminal 6 and the injection pump 3; the data processing terminal 6 controls the injection or suction of the injection pump 3 by controlling the stepping motor 4.
In the specific implementation process of the invention, the data processing terminal 6 is used for controlling the stepping motor 4, so that the internal pressure and the external pressure of the tip of the microtube are balanced, the injection and the suction are not performed, and the real-time pressure acquired by the sensor is assumed to be Xpa. When the lysis of the impurity cells is completed, the data processing terminal 6 inputs an electric signal to the stepping motor 4, the electric signal controls the stepping motor 4 to further control the injection pump 3 to suck, so that the tip of the microtube generates a negative pressure of 10pa (namely, the pressure acquired by the sensor is X-10pa), and the cell lysate is sucked into the microtube under the action of the negative pressure. After the suction is finished, the data processing terminal 6 rapidly controls the stepping motor 4, so that the pressure of the micro-pipeline is balanced again, namely the real-time pressure intensity acquired by the sensor is stabilized at Xpa again.
In the present invention, the data processing terminal 6 is preferably a computer.
The cell lysis device of this application can also collect the cell lysate when the schizolysis is purified.
In the present invention, the cell lysis device is used in combination with an inverted microscope 11; the inverted microscope 11 is provided with a camera 15, and the camera 15 is electrically connected with the data processing terminal 6 and used for collecting images of the field of view of the inverted microscope 11 in real time. The culture dish (cell sample) 13 is placed on the stage 14 of the inverted microscope 11.
In the present invention, the microtube electrode 12 is preferably placed on the microtube holder 10, and the microtube electrode 12 is moved by controlling the microtube holder 10 by the robot arm 9.
The invention also provides a cell lysis method based on the cell lysis device, which comprises the following steps:
applying 10-50V pulse voltage to the position 2-5 mu m above the impurity cells of the impurity cells through the electrode tube 1-1 until the impurity cells are cracked.
The invention distinguishes impure cells and non-impure cells under a microscope depending on the obvious difference of the shapes and the sizes of different cells in an adherent state.
In the invention, the voltage of the pulse power supply is preferably 10-50V.
In the present invention, the electrode tube 1-1 is preferably located 3 μm above the impurity cells of the impurity cells.
In the invention, the method also comprises the step of washing the lysed cell sample by using a DPBS buffer solution (Du's phosphate buffer solution, pH7.4) to obtain a purified cell sample. In the present invention, the number of times of the washing is preferably 3 to 4 times.
In the present invention, after the contaminating cells are lysed, it is preferable to further collect the cell sap after the contaminating cells are lysed. Control signals are applied to the stepping motor 4 through the data processing terminal 6, negative pressure of 10pa is generated at the tip of the collecting pipe 1-2, the pressure intensity collected by the pressure intensity sensor is stabilized at X-10pa (the stable voltage of the tip of the collecting pipe 1-2 is measured as Xpa), and cell sap collection can be completed within seconds. After the collection is finished, the data processing terminal 6 controls the stepping motor 4 to apply a control signal, so that the voltage of the tip of the collecting pipe 1-2 is stabilized at Xpa again, and the tip of the microtube is not continuously sucked.
In the present invention, the steps of lysing the impurity cells and collecting the cell fluid after lysing the impurity cells in the above-mentioned protocol are preferably repeated until all the impurity cells in the cell sample are lysed and all the impurity cell fluid is collected.
Conventionally, a microfluidic chip is adopted, cells are mixed together and cannot be separated individually, the position of an electrode in the microfluidic chip is fixed, and only liquid flow in the chip can be controlled to enable the cells to reach a lysis area. Meanwhile, as the impurity cells and the cells to be purified are both in the microfluidic pipeline, the two cell lysates can be mixed inevitably and cannot be collected separately. And this application adopts the microtube schizolysis, and the electrode can be along with the arm removes, and the cell is in adherence fixed state, can control the arm removal or the objective table removes accurate location impurity cell.
In the specific implementation process of the invention, the objective table 14 is moved, the culture dish (cell sample) 13 under the inverted microscope 11 is observed through the display of the data processing terminal 6 by the objective table 14, impurity cells in the culture dish (cell sample) 13 are searched, the mechanical arm 9 is controlled to move, the microtube holder 10 is driven to move, and then the tip of the microtube electrode 12 is focused at a position 2-3 μm above the impurity cells and is displayed on the display screen of the data processing terminal 6. Control signals are input through the data processing terminal 6, the pulse power supply 1 is controlled to release corresponding voltage, and a non-uniform high-voltage electric field is formed at the tip of the electrode tube 1-1, namely above impurity cells. The contaminating cells lyse rapidly because the ambient voltage is much higher than the self transmembrane potential limit. And (3) repeatedly cracking the impurity cells until the impurity cells are completely eliminated, then washing the sample for 3-4 times by using a DPBS buffer solution, and removing cell fragments to complete sample purification.
The invention also provides a cell purification method based on the cell lysis device, which comprises the following steps:
applying 10-50V pulse voltage to the position 2-5 mu m above the impurity cells of the impurity cells through an electrode tube (1-1) until the impurity cells are cracked;
and (3) washing the obtained lysed cell sample by adopting a DPBS buffer solution to obtain a purified cell sample. In the present invention, the impurity cell debris is washed out, and the adherent cells remaining in the culture vessel are the purified cells.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in FIG. 1, the cell lysis apparatus of the present embodiment comprises a pulse power supply 1, a pressure sensor 2, an injection pump 3, a stepping motor 4, a data acquisition card 5, a computer 6, a copper wire 7, a micro-tube channel 8, a mechanical arm 9, a micro-tube holder 10, an inverted microscope 11, a micro-tube electrode 12, a culture dish (cell sample) 13, a movable stage 14, and a camera 15.
The whole system is mainly divided into two modules, namely an impurity cell lysis module in figure 3 and a cell lysate collection module in figure 4. FIG. 3 shows a partial schematic diagram of the high voltage electric field generated by the tip of the microtube electrode during cell lysis. The microtube electrode 12 in the system consists of three single microtubes, two of which are filled with liquid metal gallium and are used as electrodes. The two electrodes are connected to a pulse power supply 1 by copper wires 7, and during lysis, the pulse power supply applies pulse voltage to the electrodes under the control of a computer, so that a non-uniform electric field is generated at the upper ends of the impurity cells.
FIG. 4 is a schematic diagram showing the aspiration of the cell fluid by the negative pressure generated at the tip of the microtube during the collection of the cell lysate. After the lysis is finished, cell lysate can be collected according to the requirement, the computer 6 controls the stepping motor 4 to suck so that the tip of the microtube 13 generates negative pressure of about 10pa, and the cell lysate is sucked in, wherein the suction process is shown in figure 4.
The microtube device and the method for purifying the cell sample by cracking the impurity cells in the cell sample are specifically realized by the following steps (for example, retinal photoreceptor cells are taken as an example, when an ophthalmic hospital researches the retinal photoreceptor cells of a patient, the separated retinal pigment epithelial cells are mixed with the retinal pigment epithelial cells):
a. preparation of cells: a cell sample (sample: retinal photoreceptor cells, impurity cells: retinal pigment epithelial cells, sample source: obtained by culturing after being extracted from a patient body by an ophthalmic hospital in cooperation with a project group) to be purified is prepared in advance (adherent state), washed for 2-3 times by Phosphate Buffer Solution (PBS), and then added with sucrose solution with the concentration of 0.3 mol/L.
b. Preparation of the device before the experiment: the injection pump 3, the micro-tube channel 8, the internal channel of the micro-tube holder 10 are thoroughly cleaned by rinsing with 70 vol% ethanol aqueous solution and deionized water. The micro-tube system is installed, and the mechanical arm 9 is controlled to move, so that the tip of the micro-tube electrode 12 is focused at the center of the visual field of the microscope 11, namely, the center of the display screen of the data processing terminal 6.
c. During operation, the stepping motor 4 is controlled by the data processing terminal 6, so that the pressure of the pipeline collected by the sensor 2 is stabilized at Xpa, and at the moment, the pressure inside and outside the tip of the microtube electrode 12 is balanced, and neither suction nor injection is performed (here, the initial balance pressure is replaced by Xpa).
d. The control stage 14 drives the culture dish 13 to move, the retinal pigment epithelial cells in the searched sample are observed on the display screen, and the retinal pigment epithelial cells are moved to the position 5 mu m away from the tip of the microtube electrode 12.
e. A control signal is input to the pulse power supply 1 through the data processing terminal 6, then the 10-50V voltage pulse power supply is released, a high-voltage electric field is formed at the tip of the microtube electrode 12, namely around the retinal pigment epithelial cells, because the electric potential is far higher than the transmembrane voltage (about 1V) of the retinal pigment epithelial cells, the retinal pigment epithelial cell membranes are completely destroyed within about 1 second, and then cell sap is released, because the cells usually gather and grow, a group of retinal pigment epithelial cells can be cracked each time by moving on the stage.
f. After cell lysis, the retinal pigment epithelial cell sap may be collected. Signals are applied to the stepping motor 4 through the data processing terminal 6, the injection pump 3 is controlled to suck, and the tip of the microtube electrode 12 generates negative pressure of 10 pa. At the moment, the pressure intensity collected by the sensor is stabilized at X-10pa, and the collection of the cell sap can be completed within seconds.
g. And immediately after the collection is finished, the data processing terminal controls the stepping motor to feed so that the voltage collected by the sensor is stabilized at Xpa again, and the tip of the microtube is not continuously sucked.
h. And d, repeating the steps e, f and g until all the retinal pigment epithelial cells in the cell sample are cracked. And (4) determining whether the steps f and g are needed according to actual requirements and whether the collection of cell sap is needed.
And after the whole cracking process is finished, sucking the sucrose solution and the retinal pigment epithelial cell fragments in the culture dish by using a pipette, washing the cells for 2-3 times by using Phosphate Buffer Solution (PBS), adding 4ml of culture medium into the culture dish with the diameter of 35mm, and finishing the whole process of purifying the cell sample.
As a result: the samples before and after purification are respectively counted by a visual inspection method and a cell counter, wherein about 115 ten thousand retinal photoreceptor cells, about 15 ten thousand impurity cells, namely retinal pigment epithelial cells, in the samples before purification are counted, and the total number of the cells is about 130 ten thousand. After purification, about 102 ten thousand of retinal photoreceptor cells and about 2.3 ten thousand of retinal pigment epithelium are obtained. The loss rate of the retina photoreceptor cells is 11.3 percent, and the purification rate is 97.8 percent.
The method is single purification, and can reach more than 99% after multiple purifications, ideally can approach 100%, but the corresponding cell loss rate can be increased.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A cell lysis device comprises a pulse power supply (1) and a microtube electrode (12) electrically connected with the pulse power supply (1), and is characterized in that the microtube electrode (12) comprises an electrode tube (1-1) and a liquid metal film attached to the inner wall of the electrode tube (1-1); the liquid metal film does not react with water chemically;
the inner diameter of the top end of the electrode tube (1-1) is 4-20 mu m.
2. The cell lysis device of claim 1, wherein said liquid metal film comprises a liquid metal gallium film.
3. The cell lysis device according to claim 1 or 2, wherein the shape of the electrode tube (1-1) comprises a needle shape; the liquid metal film is attached to the inner wall of the tip of the electrode tube (1-1).
4. The cell lysis device according to claim 1, wherein the microtube electrodes (12) further comprise a collection tube (1-2).
5. The cell lysis device according to claim 4, further comprising a pressure sensor (2), a syringe pump (3), a data acquisition card (5) and a data processing terminal (6);
the pressure sensor (2) is connected with the collecting pipe (1-2) and is used for receiving a pressure signal in the collecting pipe (1-2) and converting the pressure signal into an electric signal;
the data acquisition card (5) is respectively connected with the pressure sensor (2) and the data processing terminal (6) and is used for acquiring the electric signals and transmitting the electric signals to the data processing terminal (6) in real time;
the data processing terminal (6) is connected with the injection pump (3), and outputs a control signal according to the acquired electric signal, wherein the control signal controls the injection or suction of the injection pump (3);
the injection pump (3) is connected with the collecting pipe (1-2), and positive pressure or negative pressure is generated inside the collecting pipe (1-2) through injection or suction of the injection pump (3), so that cell sap is sucked or discharged.
6. A cell lysis method based on the cell lysis device of any one of claims 1 to 5, comprising the following steps:
applying 10-50V pulse voltage to the position 2-5 mu m above the impurity cells of the impurity cells through an electrode tube (1-1) until the impurity cells are cracked.
7. The method according to claim 6, wherein the voltage of the pulse voltage is 10-50V.
8. A method for purifying cells based on the cell lysis device of any one of claims 1 to 5, comprising the following steps:
applying 10-50V pulse voltage to the position 2-5 mu m above the impurity cells of the impurity cells through an electrode tube (1-1) until the impurity cells are cracked;
and (3) washing the obtained lysed cell sample by adopting a DPBS buffer solution to obtain a purified cell sample.
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| WO2023083255A1 (en) * | 2021-11-11 | 2023-05-19 | 中南大学 | Microelectrode sensor for detecting staphylococcus aureus, and preparation method and application method therefor |
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| CN111849768A (en) * | 2020-07-14 | 2020-10-30 | 苏州大学 | Egg cell multi-efficiency accurate electrical stimulation device and method |
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| WO2023083255A1 (en) * | 2021-11-11 | 2023-05-19 | 中南大学 | Microelectrode sensor for detecting staphylococcus aureus, and preparation method and application method therefor |
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