KR102609307B1 - Gene transfection apparatus - Google Patents

Abstract

The gene transfection device according to an embodiment of the present invention includes a porous filter in which a plurality of micro channels are formed in the thickness direction, a filter cartridge through which a mixture of genetic material and cells passes through the plurality of micro channels, and the filter cartridge. One side of the combination is provided as an inlet through which the mixture is injected, and the other side is provided as an outlet through which the mixture passing through the plurality of micro channels flows out. When an electric current is applied, cells in the mixture are electroporated. It may include an electroporation electrode unit including an electrode that forms a hole and allows the genetic trait of the cell to be infected by the genetic material.

Description

Gene transfection device {GENE TRANSFECTION APPARATUS}

The present invention relates to a gene transfection device, and more specifically, is capable of controlling the flow rate of a mixture of genetic material and cells, as well as optimally controlling the current density for gene transfection, thereby enabling gene transfection of cells. It relates to a gene transfection device that can uniformly create an environment for, through which gene transfection of many cells can be carried out efficiently and accurately, and that can minimize damage to cells during this process.

Gene therapy is the treatment of congenital or acquired diseases caused by defects in the gene itself. Since cure is impossible with existing treatments, genes are injected into the body for fundamental treatment to treat genetic defects or to promote the expression of new proteins. Thus, while expecting a therapeutic effect, it shows superior selectivity and treatment efficiency compared to general drug treatment by treating the cause of the disease, not the symptoms.

Gene delivery methods required for gene therapy can be broadly classified into gene delivery vehicles and gene transfer technologies. Gene delivery vehicles are classified into viral and non-viral types. Among these, viral delivery vehicles have excellent advantages in terms of delivery efficiency and persistence, but have safety problems due to immunotoxicity, and gene expression rates decrease upon repeated administration due to induction of host immune responses. It has the disadvantage of decreasing and not having specific cell specificity.

On the other hand, non-viral carriers are made of cationic lipids or polymers, which are relatively safe materials, and are delivered into cells after forming a complex with anionic DNA by ionic bonding. Compared to viral carriers, non-viral carriers are biodegradable and have low molecular weight. Although it has the advantages of toxicity and ease of use, it has the limitation that the delivery efficiency of therapeutic genes is greatly reduced.

Therefore, in order to increase the efficiency of non-viral delivery vehicles without side effects related to immunotoxicity, transfection agents such as Lipofectamine and JetPEI, which are reagents that easily bind to the genetic material by charge and can be easily separated, are used, or nano-carriers are used to transfer the genetic material. can be transferred to the cytoplasm.

However, in this case, it is limited to specific cells capable of phagocytosis, and in the case of primary cells without phagocytosis, various methods are used, for example, physical stimulation such as electric field, magnetic field, mechanical force, ultrasound, or light. Membrane disruption approaches of the plasma membrane are being used.

Currently, the most commonly used transfection methods are cell squeezing and electroporation. Electroporation is a method of creating a hole in the cell membrane with an electric shock and then inserting DNA into the cytoplasm. Compared to the injection method, the efficiency is more than 1,000 times, but gene transfer can be expressed only at the cell level, and its use is limited due to serious damage to cells caused by electric shock, low cell survival rate, and the charge of the target delivery material.

Meanwhile, the cell squeezing method induces controllable partial damage to the nuclear membrane, enabling rapid direct delivery of DNA to the nucleus in large numbers of cells. However, by using a microfluidic chip to instantaneously cause mechanical deformation of cells, substances present in the surrounding buffer, such as antibodies and genes, can be generally transfected regardless of the cell type, but this method is also relatively simple. There is a limitation in that only a small number of cells can be processed.

Therefore, there is a need for the development of a new microchannel electroporation device that allows gene transfection of a large number of cells while minimizing damage to cells.

Embodiments of the present invention can control the flow rate of the mixture of genetic material and cells, as well as optimally adjust the current density for gene transfection, thereby creating a uniform environment for gene transfection of cells. , through which gene transfection of many cells can be carried out efficiently and accurately, and provides a gene transfection device that can minimize cell damage during this process.

The problem to be solved by the present invention is not limited to the problem(s) mentioned above, and other problem(s) not mentioned will be clearly understood by those skilled in the art from the description below.

The gene transfection device according to an embodiment of the present invention includes a porous filter in which a plurality of micro channels are formed in the thickness direction, a filter cartridge through which a mixture of genetic material and cells passes through the plurality of micro channels, and the filter cartridge. One side of the combination is provided as an inlet through which the mixture is injected, and the other side is provided as an outlet through which the mixture passing through the plurality of micro channels flows out. When an electric current is applied, cells in the mixture are electroporated. It may include an electroporation electrode unit including an electrode that forms a hole and allows the genetic trait of the cell to be infected by the genetic material.

According to one side, the porous filter may be a membrane filter.

According to one side, the filter cartridge may further include a filter support body surrounding the porous filter and a plurality of support members supporting multiple positions of the porous filter within the filter support body.

According to one side, the filter support is made of a net-shaped polymer material, and the plurality of support members are four support members arranged at 90-degree intervals along the circumference of the porous filter and may be made of plastic or metal. .

According to one side, it further includes a device housing surrounding the filter cartridge and surrounding at least a portion of the electroporation electrode portion, and the filter cartridge and the electroporation electrode portion can be fixed by the device housing.

According to one side, the electroporation electrode unit may further include a current applicator that applies a current to the electrode.

According to one side, the current and voltage exposed to each cell can be automatically calculated by inputting the distance, speed, and number of micro channels through which the mixture passes through the current applicator to ensure that the optimal current is applied.

According to one side, the electrodes of the electroporation electrode unit are provided in a symmetrical pair on both sides of the filter cartridge, and the electrodes have a fixed socket for fixing the electrodes and transmitting the current applied from the current applicator to the electrodes. This can be detachably coupled.

According to one side, the electrode of the electroporation electrode unit is coated with metal so that the current applied from the current applicator can be transmitted to the electrode.

According to one side, the gap between the electrodes is 100 to 1000 micrometers, and the voltage generated between the gaps between the electrodes when the current applied through the current application unit is applied may be 1 to 100 volts.

According to one side, an inlet syringe for injecting the mixture is detachably coupled to the inlet of the electroporation electrode part, and the outlet of the electroporation electrode part is a cell infected with the gene by the action of the porous filter and the electrode. The outlet cylinder in which the mixture is collected may be detachably coupled.

According to one side, the ends of the inlet syringe and the outlet syringe may be coupled to the inlet port and outlet port provided in the porous filter in the form of a lure lock.

According to one side, a pump connector for connection with a pump may be connected to the inlet port and the outlet port of the porous filter.

According to one side, it may further include a collection container connected to the outlet syringe to collect transfected cells introduced into the outlet syringe.

According to one side, the electroporation electrode unit may further include a flow rate control unit for controlling the flow rate of the mixture moving along the path formed by the electrode and the microchannel of the porous filter.

According to one side, the flow rate control unit may include a flow rate control step motor for controlling the injection speed of the mixture according to the operation of the inlet syringe coupled to the inlet of the electroporation electrode unit.

According to one side, an actuator is coupled to the flow rate control step motor so that the mixture can be provided to the inlet of the electroporation electrode unit.

According to one side, the genetic material for gene transfection of the cell includes proteins, small interfering RNA (siRNA), small hairpin RNA (shRNA), endoribonuclease-prepared siRNAs (esiRNA), antisense oligonucleotides, DNA, single-stranded RNA, It may include at least one of double-stranded RNA, DNA-RNA hybrid, and ribozyme.

According to one side, the porous filter is made of an inorganic or polymer material, the diameter of the microchannel is 1 to 200 micrometers, and the diameter of the microchannel is 80 to 120% of the size of the cell to be transfected. You can have it.

According to one side, the plurality of micro channels provided in the porous filter may be 10 to 10,000.

According to one side, the thickness of the porous filter may be 10 to 5000 micrometers.

According to an embodiment of the present invention, it is possible to control the flow rate of the mixture of genetic material and cells, as well as to optimally control the current density for gene transfection, thereby creating a uniform environment for gene transfection of cells. This allows gene transfection of many cells to be carried out efficiently and accurately, and damage to cells during this process can be minimized.

Figure 1 is a diagram schematically showing a gene transfection device according to an embodiment of the present invention.
FIG. 2 is a diagram illustrating a state in which the inlet syringe and the outlet syringe are coupled to the filter cartridge and the electroporation electrode unit in FIG. 1.
Figure 3 is an enlarged view of the filter cartridge and electroporation electrode part in Figure 2.
FIG. 4 is a diagram schematically showing the configuration of the filter cartridge shown in FIG. 3.
FIG. 5 is a diagram schematically showing the fixed socket shown in FIG. 3.

The advantages and/or features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms, but the present embodiments only serve to ensure that the disclosure of the present invention is complete and are within the scope of common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings.

Figure 1 is a diagram schematically showing a gene transfection device according to an embodiment of the present invention, and Figure 2 shows a state in which the inlet syringe and the outlet syringe are coupled to the filter cartridge and the electroporation electrode part in Figure 1. It is a drawing, FIG. 3 is an enlarged view of the filter cartridge and the electroporation electrode part in FIG. 2, FIG. 4 is a view schematically showing the configuration of the filter cartridge shown in FIG. 3, and FIG. 5 is a fixed view shown in FIG. 3. This is a diagram schematically showing a socket.

As shown in these figures, the gene transfection device 100 according to an embodiment of the present invention includes a device housing 110 and a plurality of devices mounted on the device housing 110 and through which a mixture of genes and cells passes. A filter cartridge 120 including a porous filter 121 with a micro channel 122, and a filter cartridge 120 provided on both sides of the filter cartridge 120 to enable gene transfection of cells in the mixture by electroporation. An electroporation electrode unit 130 having an electrode 131, a current applicator 160 for applying a current, a flow rate controller 170 for controlling the flow rate of the mixture, and a monitoring unit 180 for monitoring. may include.

With this configuration, gene transfection of a large number of cells can be performed while minimizing damage to the cells.

To describe each configuration, first, the device housing 110 of this embodiment is an external housing that surrounds the filter cartridge 120 and the electroporation electrode unit 130, which will be described later, and includes the filter cartridge 120 and the electroporation electrode unit. It may be provided in a shape corresponding to the external shape of (130).

Through this device housing 110, the combined structure of the filter cartridge 120 and the electroporation electrode unit 130 can be firmly maintained. The device housing 110 is made of, for example, a non-conductive plastic material, so that safety can be ensured even if current is applied to the electrode 131 of the electroporation electrode unit 130.

Referring to FIG. 4, the device housing 110 does not completely surround the electroporation electrode unit 130, but has a structure that partially surrounds the electroporation electrode unit 130 so that the end is exposed, and thus the fixing socket 140 is removable from the electrode 131. They can be combined, which will be described later.

Meanwhile, the filter cartridge 120 of this embodiment includes a porous filter 121 in which a plurality of fine channels 122 are formed in the thickness direction in the entire area, as shown in FIGS. 3 and 4, and a porous filter 121 ) may include a filter support 125 surrounding the filter support 125 and a plurality of support members 127 supporting multiple positions of the porous filter 121 within the filter support 125.

The porous filter 121 is provided with a plurality of micro channels 122, and the number may be 10 to 10,000. Preferably, between 100 and 1000 microchannels 122 may be provided, which allows gene transfection of a larger number of cells to be carried out more efficiently than before. In other words, gene transfection can be performed on a large number of cells at once.

In detail, in this example, genetic materials for gene transfection of cells include proteins, siRNA (small interfering RNA), shRNA (small hairpin RNA), esiRNA (endoribonuclease-prepared siRNAs), antisense oligonucleotides, DNA, and single-stranded RNA. , it may include at least one of double-stranded RNA, DNA-RNA hybrid, and ribozyme. However, it is not limited to this.

The diameter of the fine channel 122 provided in the porous filter 121 may be 1 to 200 micrometers. In detail, the diameter of the microchannel 122 is related to the size of the cell passing through it, and may have a size of 80 to 120% of the cell size.

If the diameter of the microchannel 122 is small compared to the size of the cell, a perforation may occur in the cell membrane as the cell barely passes through the microchannel 122, and genetic material may penetrate, thereby reducing the genetic traits of the cell. You can.

If the diameter of the microchannel 122 is large compared to the size of the cell, the cell can penetrate smoothly, and as will be described later, the gene transfection of the cell is performed by the electroporation electrode unit 130, so the cell's gene Transfection can be carried out smoothly.

The thickness of the porous filter 121 may be 10 to 5000 micrometers, and preferably 400 micrometers.

The porous filter 121 of this configuration may be made of an inorganic or polymer material, and may be provided as a membrane filter. Because it has a membrane structure, a plurality of microchannels 122 can be provided, through which gene transfection of a large number of cells can be performed.

In other words, in the conventional case, it took a long time to transfect the gene into the cell, but in the case of this embodiment, not only does it have a membrane structure, but, as will be described later, the current density can be adjusted by adjusting the applied current and flow rate. Gene transfection of cells can be accomplished in as little as 0.01 to 5 seconds.

Meanwhile, the filter support 125 supporting the porous filter 121 of this embodiment may be made of a net-shaped polymer material.

And, as schematically shown in FIG. 4, the plurality of support members 127 are provided in four numbers and are arranged at 90-degree intervals along the circumference of the porous filter 121, thereby forming a porous filter within the filter support 125. The position of (121) can be firmly fixed. These plurality of support members 127 may be made of plastic or metal.

However, the material and structure of the filter support body 125 and the support member 127 are not limited to the above-described contents.

Meanwhile, the electroporation electrode unit 130 of the present embodiment is provided with electrodes 131 provided symmetrically on both sides of the filter cartridge 120, as shown in FIGS. 1 to 3, with genetic material and cells on one side. An inlet 135 through which the mixed mixture is injected may be provided, and an outlet 137 through which the mixture penetrating the plurality of micro channels 122, that is, the mixture of cells transfected with genetic material, may flow out. .

With this configuration, when current is applied to the electrode 131 of the electroporation electrode unit 130 by the current application unit 160 shown in FIG. 1, holes (perforations) are made in the cell membranes of cells in the mixture by electroporation. It is possible to form a cell so that the genetic material of the cell can be infected by the genetic material.

For this purpose, as shown in FIGS. 3 and 5, a fixing socket 140 for transmitting the current applied from the current applying unit 160 while fixing the electrode 131 is detachably coupled to the electrode 131. You can.

This fixed socket 140 may be made of a conductive metal material, and therefore, the current applied from the current applicator 160 can be transmitted to the electrode 131 through the fixed socket 140, and through this, the electrode 131 ) can perform electroporation of cells that pass through the path formed by the cell, thereby enabling gene transfection of the cells.

However, it is not limited to this, and it is natural that the electrode 131 of the electroporation electrode unit 130 is coated with metal so that current can be transmitted from the current applicator 160 to the electrode 131. In other words, the application of current from the current applicator 160 to the electrode 131 can be achieved through various methods and structures.

As described above, the application of current to the electrode 131 is performed by the current applicator 160, which is a mixture of cells and genetic material flowing in through the inlet 135. By inputting the passing distance, speed, number of micro channels 122, etc., the current or voltage exposed to each cell is automatically calculated, and based on this, the optimal current can flow and voltage can be applied. And the results can be confirmed through the monitoring unit 180.

The gap between the electrodes 131 of the electroporation electrode unit 130 of this embodiment may be 100 to 1000 micrometers, and preferably 400 micrometers. Conventionally, for example, the gap between the electrodes was about 1 centimeter, so a voltage of about 400 volts was required. However, in the present embodiment, the gap between the electrodes 131 was significantly reduced to about 400 micrometers, so the voltage was 1 to 1 cm. The desired current density can be generated with only a voltage of 100 volts, preferably 1.5 to 3 volts.

Meanwhile, as shown in FIGS. 1 and 2, the inlet syringe 10 is detachably coupled to the inlet 135 of the electroporation electrode unit 130, and the outlet syringe 20 is attached to the outlet 137. Can be detachably coupled.

The mixture contained in the inlet syringe 10 may flow into the electroporation electrode unit 130 and the filter cartridge 120 through the inlet 135 by operating the inlet syringe 10. And the outlet syringe 20 can be filled with cells infected with genetic material while passing through the path formed by the plurality of micro channels 122 of the porous filter 121 and the electroporation electrode unit 130. .

Although not shown, the ends of the inlet syringe 10 and the outlet syringe 20 may be coupled to the inlet port and outlet port provided in the porous filter 121 in the form of a lure lock.

In addition, a pump connector for connection with a pump may be connected to the inlet port and outlet port of the porous filter 121. In other words, a pump other than a syringe can be detachably coupled to the filter cartridge 120 or the electroporation electrode unit 130.

In addition, although not shown, the gene transfection device 100 of this embodiment may further include a collection container connected to the outlet syringe 20 to collect the transfected cells introduced into the outlet syringe 20. Through this, the process of separately transferring gene-transfected cells can be omitted, thereby increasing efficiency.

Meanwhile, the syringes 10 and 20 may be made of plastic, glass, or a sterile material usable in biomedicine. A micro pump can be connected to these syringes (10, 20), through which the mixture can be introduced into the electroporation inlet at a constant speed.

Meanwhile, the flow rate control unit 170 of the present embodiment controls the operation of the inlet syringe 10 or any component replacing the inlet syringe 10 to control the operation of the electroporation electrode unit 130 and the porous filter 121. The flow rate of the mixture passing through the plurality of micro channels 122 can be adjusted. For example, the inflow speed of the mixture contained in the barrel 15 can be adjusted by adjusting the forward speed of the plunger (push bar) 11 of the inlet syringe 10 by the flow rate control unit 170.

This flow rate control unit 170 may include a flow rate control step motor for controlling the injection speed of the mixture, and an actuator is coupled to the flow rate control step motor to allow the mixture to flow into the inlet of the electroporation electrode unit 130. .

On the other hand, when large-scale gene transfection is required, the electrode that creates the electric field can be directly bound to the filter cartridge 120, and a silicone tube is connected to the inlet 135 of the electroporation electrode unit 130 and linked thereto. Genetic infection can be carried out in large quantities of cells using a pump.

In this way, the flow rate of the mixture can be adjusted by the flow rate control unit 170, and the current density can be adjusted by the above-described current application unit 160, thereby creating a uniform environment for gene transfection into cells. Through this, gene transfection of many cells can be carried out efficiently and accurately, and physical damage to cells can be minimized during this process.

Although specific embodiments according to the present invention have been described so far, it goes without saying that various modifications are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined by the scope of the patent claims described below as well as equivalents to the claims of this patent.

As described above, although the present invention has been described with reference to limited embodiments and drawings, the present invention is not limited to the above embodiments, and those skilled in the art can make various modifications and modifications from these descriptions. Transformation is possible. Accordingly, the spirit of the present invention should be understood only by the scope of the claims set forth below, and all equivalent or equivalent modifications thereof shall fall within the scope of the spirit of the present invention.

10: Inlet syringe
20: Outlet syringe
100: Gene transfection device
110: device housing
120: Filter cartridge
121: Porous filter
122: fine channel
125: Filter support
127: Support member
130: Electroporation electrode unit
131: electrode
135: inlet
137: Outlet
140: fixed socket
160: Current applicator
170: Flow rate control unit
180: monitoring unit

Claims (21)

A filter cartridge having a porous filter with a plurality of microchannels formed in the thickness direction, and through which a mixture of genetic material and cells passes through the plurality of microchannels; and
One side coupled to the filter cartridge is provided as an inlet through which the mixture is injected, and the other side is provided as an outlet through which the mixture passing through the plurality of micro channels flows out, and the mixture flows through the micro channels. An electroporation electrode unit including an electrode that applies an electric current when passing through the mixture to form holes on the surface of the cells by electroporation, thereby allowing the genetic material of the cells to be infected by the genetic material; and
It includes a current applicator that applies a current to the electrode of the electroporation electrode unit,
The gap between the electrodes is 100 to 1000 micrometers, and the voltage generated between the gaps between the electrodes when the current applied through the current application unit is applied is 1 to 100 volts. According to paragraph 1,
A gene transfection device, characterized in that the porous filter is a porous membrane filter. According to paragraph 1,
The filter cartridge is,
a filter support surrounding the porous filter; and
Gene transfection device further comprising a plurality of support members supporting multiple positions of the porous filter within the filter support. According to paragraph 3,
The filter support is made of a net-shaped polymer material,
The plurality of support members are four support members arranged at 90 degree intervals along the circumference of the porous filter and are made of plastic or metal. According to paragraph 1,
further comprising a device housing surrounding the filter cartridge and surrounding at least a portion of the electroporation electrode portion,
A gene transfection device, characterized in that the filter cartridge and the electroporation electrode unit are fixed by the device housing. delete According to paragraph 1,
A gene transfection device that automatically calculates the current and voltage exposed to each cell by inputting the distance, speed, and number of microchannels through which the mixture passes through the current application unit to ensure that the optimal current is applied. According to paragraph 1,
The electrodes of the electroporation electrode unit are provided in a symmetrical pair on both sides of the filter cartridge, and the electrodes have a detachably fixed socket for fixing the electrodes and transmitting the current applied from the current applicator to the electrodes. A gene transfection device characterized in that it is combined. According to paragraph 1,
A gene transfection device, characterized in that the electrode of the electroporation electrode unit is coated with metal so that the current applied from the current application unit is transmitted to the electrode. delete According to paragraph 1,
An inlet syringe for injecting the mixture is detachably coupled to the inlet of the electroporation electrode unit,
A gene transfection device, wherein an outlet syringe through which a mixture of genetically infected cells is collected by the action of the porous filter and the electrode is detachably coupled to the outlet of the electroporation electrode unit. According to clause 11,
A gene transfection device, characterized in that the ends of the inlet syringe and the outlet syringe are coupled to the inlet port and outlet port provided in the porous filter in the form of a lure lock. According to clause 12,
A gene transfection device, characterized in that a pump connector for connection with a pump is connected to the inlet port and the outlet port of the porous filter. According to clause 11,
Gene transfection device, characterized in that it further comprises a collection container connected to the outlet syringe to collect the transfected cells introduced into the outlet syringe. According to paragraph 1,
The gene transfection device further comprises a flow rate control unit for controlling the flow rate of the mixture moving along the path formed by the electrode of the electroporation electrode unit and the microchannel of the porous filter. According to clause 15,
The flow rate control unit is a gene transfection device characterized in that it includes a flow rate control step motor for controlling the injection speed of the mixture according to the operation of the inlet syringe coupled to the inlet of the electroporation electrode unit. According to clause 16,
A gene transfection device, characterized in that an actuator is coupled to the flow rate control step motor to provide the mixture to the inlet of the electroporation electrode unit. According to paragraph 1,
The genetic material for gene transfection of the cell includes proteins, small interfering RNA (siRNA), small hairpin RNA (shRNA), endoribonuclease-prepared siRNAs (esiRNA), antisense oligonucleotides, DNA, single-stranded RNA, double-stranded RNA, A gene transfection device comprising at least one of a DNA-RNA hybrid and a ribozyme. According to paragraph 1,
The porous filter is made of inorganic or polymer material,
The diameter of the microchannel is 1 to 200 micrometers,
A gene transfection device, wherein the microchannel has a diameter of 80 to 120% of the size of the cell to be transfected. According to paragraph 1,
Gene transfection device, characterized in that the plurality of micro channels provided in the porous filter are 10 to 10,000. According to paragraph 1,
A gene transfection device, characterized in that the thickness of the porous filter is 10 to 5000 micrometers.