A flow electroporation device and temperature control method

Abstract

The application discloses a flow electroporation device, which comprises a flow electroporation modular device containing a shell and an electroporation assembly, the shell has an inner cavity, and the electroporation assembly is fixed in the inner cavity; the electroporation assembly comprises an electroporation shell, the inside of the electroporation shell forms an electroporation chamber for performing electroporation treatment on a cell suspension, and the flow electroporation modular device further comprises a cooling mechanism, the cooling mechanism comprises a refrigeration part, an airflow channel and a heat dissipation assembly arranged between the refrigeration part and the airflow channel, the airflow channel comprises an airflow inlet, a channel main body and an airflow outlet, the heat dissipation assembly is used for forming an airflow with a flow direction of the airflow inlet -> the channel main body -> the airflow outlet, and the airflow is used for conducting heat of the cell suspension in the electroporation chamber to the outside of the shell; the application further provides a temperature control method; and the flow electroporation device provided by the application can significantly reduce the temperature of the cell suspension in the electroporation chamber (nearly 30 DEG C of temperature reduction) and significantly reduce the cell damage rate in the electroporation process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a flow electroporation device capable of cooling and a temperature control method using the device. Background Art

[0002] Since the 1970s, electroporation (or electrotransfection) has been used to introduce molecules into animal and plant cells. Researchers have demonstrated that exposing cells to a brief, continuous, high-voltage electric field can form channels in the cell membrane, allowing macromolecules such as proteins and DNA to enter the cell. The technique of using an electric field to create micropores in the cell membrane is called electroporation, and electrofection is a technique that uses this technique to introduce exogenous molecules / substances into cells. Specifically, electrotransfection involves subjecting a cell suspension to a pulsed electric field. By precisely controlling the voltage, pulse width (i.e., duration, with microsecond precision), and pulse interval (with millisecond precision), the electric field displaces the phospholipid molecules in the cell membrane, forming micropores (approximately 20 nanometers in diameter). Driven by the electric field, these micropores allow polarized exogenous molecules / substances (such as DNA, RNA, and proteins) to enter the cell, achieving transfection / delivery.

[0003] The optimal temperature for in vitro culture of mammalian and avian cells is generally 37-38°C. Temperatures that are too high or too low can affect cell growth. Cells are more tolerant to low temperatures than to heat; at low temperatures, cell metabolic activity and nuclear division are reduced. While temperatures above 0°C may affect cell metabolism, they are not harmful. Cells maintained at 25-35°C can still survive and grow, albeit at a slower rate. After several hours at 40°C and then returned to 37°C, cells can continue to grow. However, prolonged exposure to 40°C can impair cell viability and function.

[0004] Cells cultured at 39-40°C for one hour can sustain some damage, but may still recover. However, they cannot tolerate a temperature increase of 2°C for several hours, i.e., culturing at 41-42°C for one hour will cause severe cell damage. Most cells will be killed when the temperature rises above 43°C. High temperatures primarily inactivate enzymes, destroy lipids, disrupt nuclear division, produce coagulase that causes cell coagulation, and denature proteins. Therefore, high temperatures must be avoided when culturing cells in vitro.

[0005] During electrofection, the cell fluid in the electroporation component can be regarded as a resistor. When the current passes through the resistor of the electroporation component, a thermal effect will be generated. The heat generated is Q = U 2t / R, where U is the initial pulse voltage applied across the electroporation assembly, t is the product of the pulse duration and the number of pulses, and R is the resistance of the cell fluid within the electroporation assembly. This indicates that as electrofection parameters (such as pulse voltage, pulse duration, and number of pulses) increase, heat generation also increases rapidly, leading to an increase in the ambient temperature of the cells during electrofection, causing cell damage. A device to lower the temperature of the cell fluid in the electrofection chamber is urgently needed to mitigate this damage.

[0006] In addition, the temperature of the environment in which the cells are located may change with the different stages of the electrofection process, and the existing technology lacks a system or method for real-time control of the cell fluid temperature. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies in the prior art and to provide at least a novel flow electroporation device.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A flow electroporation device includes a modular flow electroporation device comprising a housing and an electroporation assembly, wherein the housing has an inner cavity, and the electroporation assembly is fixedly disposed in the inner cavity; the electroporation assembly includes an electroporation housing, wherein the interior of the electroporation housing forms an electroporation chamber for electroporating a cell suspension; the modular flow electroporation device also includes a cooling mechanism, wherein the cooling mechanism comprises:

[0010] The air flow channel includes a channel body, an air flow inlet and an air flow outlet provided on the housing and respectively communicating with the outside of the housing.

[0011] a refrigeration unit, configured to absorb heat from the cell suspension in the electroporation chamber;

[0012] A heat dissipation component is disposed between the refrigeration unit and the air flow channel. The heat dissipation component is used to form an airflow flowing from the air flow inlet → the channel body → the air flow outlet. The airflow is used to conduct heat from the cell suspension in the electroporation chamber to the outside of the shell.

[0013] In some embodiments, the electroporation assembly includes two oppositely disposed flat electrodes, one of which constitutes at least a portion of a side wall of the electroporation housing, and the refrigeration unit is disposed adjacent to the flat electrode.

[0014] In some embodiments, the cooling mechanism is provided at a corresponding position of at least one of the flat electrodes; or,

[0015] The corresponding position of each of the flat plate electrodes includes a set of the cooling mechanisms.

[0016] In some embodiments, the heat dissipation assembly further includes a radiator and a fan, wherein the fan is used to generate the airflow, and the radiator comprising a fitting portion and a heat dissipation portion is arranged between the refrigeration portion and the fan, and the fitting portion is fitted with the refrigeration portion so that the heat is transferred from the refrigeration portion to the fitting portion through heat conduction; the heat dissipation portion fixedly connected to the fitting portion has a larger surface area than the fitting portion.

[0017] In some embodiments, the heat dissipation portion includes a substrate and a plurality of vertical plates arranged on the substrate, and a groove opening toward a side away from the substrate is formed between two adjacent vertical plates. The groove constitutes a part of the channel body, and the air inlet end of the fan is arranged at the position of the opening.

[0018] In some embodiments, the air flow inlet is a plurality of through holes opened on the shell at corresponding positions close to the fitting portion, and the air flow outlet is a plurality of through holes opened on the shell at corresponding positions close to the air outlet end of the fan.

[0019] In some embodiments, a blind hole is formed on each of the vertical plates, and the blind holes of a plurality of the vertical plates are connected to each other to form a groove intersecting with the groove between two of the vertical plates.

[0020] In some embodiments, the flow electroporation modular device further comprises a support plate, the fitting portion and the base plate extend in the same plane, the support plate is arranged on a side of the fitting portion away from the refrigeration portion, and a conductor is provided between the support plate and the flat plate electrode to supply power to the flat plate electrode.

[0021] In some embodiments, the flat electrode includes a back wall in contact with the refrigeration portion and a side wall intersecting with the back wall; wherein,

[0022] The conductor contacts the side wall of the plate electrode to achieve electrical connection; or,

[0023] The bonding portion and the cooling portion are respectively provided with conductive through holes that are connected to each other, and the conductor is inserted into the conductive through holes and contacts the back wall of the flat electrode to achieve electrical connection. In some embodiments, the support plate is further provided with a positioning hole, and the heat dissipation portion is inserted into the positioning hole.

[0024] In some embodiments, the cooling mechanism includes a cooling mechanism at the front and a cooling mechanism at the rear. The cooling mechanism at the rear includes a radiator and a fan, and the cooling mechanism at the front includes the radiator but does not include the fan.

[0025] The cooling mechanism at the front is arranged between the cooling mechanism at the rear and the air flow outlet. The radiator in the cooling mechanism at the front is provided with an air flow hole, which is adapted to the fan in the cooling mechanism at the rear. The air flow hole is located between the air flow outlet and the fan.

[0026] In some embodiments, the heat dissipation portion includes a substrate and a plurality of vertical plates disposed on the substrate, and the air flow holes penetrate the substrate and the vertical plates.

[0027] In some embodiments, the temperature reduction mechanism further includes a sealing member for enhancing the sealing performance of the channel body.

[0028] In some embodiments, the heat dissipation assembly further includes a heat sink and a fan, and the fan is used to generate the airflow;

[0029] The sealing member is disposed between the fan and the air flow outlet; and / or,

[0030] The seal is disposed between the fan and the heat sink.

[0031] In some embodiments, the cooling mechanism further includes a heat conducting member for improving heat conduction efficiency.

[0032] The heat conducting member is disposed between the refrigeration unit and the electroporation housing; and / or,

[0033] The heat conducting member is arranged between the refrigeration unit and the heat dissipation component.

[0034] In some embodiments, the flow electroporation device further comprises a control system, the control system comprising a temperature sensor and an analysis control unit, the temperature sensor and the cooling mechanism being electrically connected to the analysis control unit, respectively;

[0035] The temperature sensor is used to monitor the real-time temperature of the cell suspension in the electroporation chamber and transmit the real-time temperature to the analysis control unit; the analysis control unit is used to compare the relative sizes of the real-time temperature and the target temperature, and to activate the cooling mechanism when the real-time temperature is greater than the target temperature.

[0036] In some embodiments, the refrigeration unit is disposed adjacent to the electroporation housing; and the analysis control unit is configured to activate the refrigeration unit when the real-time temperature is greater than the target temperature.

[0037] In some embodiments, the opening is closed at a position deviated from the air inlet end so that the airflow flowing out of the opening of the groove enters the air inlet end, thereby increasing the speed of the entire airflow carrying heat flowing out of the shell through the air outlet as much as possible.

[0038] Another object of the present invention is to provide a temperature control method comprising the following steps:

[0039] S1, analyzing the preset target temperature T0 in the control unit, starting the flow electroporation device and starting electroporation of the cell suspension in the electroporation chamber, at which time the cooling mechanism is in a stopped state;

[0040] S2, the temperature sensor monitors the real-time temperature T1 of the cell suspension in the electroporation chamber and transmits the real-time temperature T1 to the analysis control unit;

[0041] S3, the analysis control unit compares the target temperature T0 and the real-time temperature T1. When T1>T0, the process goes to step S4; when T1≤T0, the process goes to step S2.

[0042] S4, the analysis control unit starts the cooling mechanism to start cooling the cell suspension in the electroporation chamber, and executes step S2.

[0043] Due to the application of the above technical solutions, the flow electroporation device provided by the present invention can significantly reduce the temperature of the cell suspension in the electroporation chamber (by nearly 30° C.), significantly reducing the cell damage rate during the electroporation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0045] Attachment Figure 1 This is an exploded schematic diagram of the single-sided, front-side powered flow electroporation device according to Example 1 of the present invention;

[0046] Attachment Figure 2 for Figure 1 A partial enlarged view of point A in the middle;

[0047] Attachment Figure 3 This is a side cross-sectional view of the single-sided, front-powered flow electroporation device of Example 1 of the present invention, wherein the housing is not shown;

[0048] Attachment Figure 4 This is an exploded schematic diagram of the single-sided, side-powered flow electroporation device according to Example 1 of the present invention;

[0049] Attachment Figure 5 This is a side cross-sectional view of the single-sided, side-powered flow electroporation device according to Example 1 of the present invention, wherein the housing is not shown;

[0050] Attachment Figure 6 This is an exploded schematic diagram of a double-sided, front-side powered flow electroporation device according to Example 2 of the present invention;

[0051] Attachment Figure 7 This is a side cross-sectional view of a double-sided, front-side powered flow electroporation device according to Example 2 of the present invention, wherein the housing is not shown;

[0052] Attachment Figure 8 This is an exploded schematic diagram of the double-sided, side-powered flow electroporation device according to Example 2 of the present invention;

[0053] Attachment Figure 9 This is a side cross-sectional view of a double-sided, side-powered flow electroporation device according to Example 2 of the present invention, wherein the housing is not shown;

[0054] Attachment Figure 10 Another exploded schematic diagram of the double-sided, side-powered flow electroporation device according to Example 2 of the present invention;

[0055] Attachment Figure 11 Another side cross-sectional view of the double-sided, side-powered flow electroporation device according to Example 2 of the present invention, wherein the housing is not shown;

[0056] Attachment Figure 12 This is an exploded schematic diagram of a single-sided, front-side powered, temperature-controlled flow electroporation device according to Example 3 of the present invention;

[0057] Attachment Figure 13 This is a side cross-sectional view of a single-sided, front-powered, temperature-controlled flow electroporation device according to Example 3 of the present invention, wherein the housing is not shown;

[0058] Attachment Figure 14 This is an exploded schematic diagram of a double-sided, front-side powered flow electroporation device with temperature control according to Example 3 of the present invention;

[0059] Attachment Figure 15 This is a side cross-sectional view of a double-sided, front-side powered, temperature-controlled flow electroporation device according to Example 3 of the present invention, wherein the housing is not shown;

[0060] Attachment Figure 16 This is an exploded schematic diagram of a single-sided, side-powered, temperature-controlled flow electroporation device according to Example 3 of the present invention;

[0061] Attachment Figure 17 A side cross-sectional view of a single-sided, side-powered, temperature-controlled flow electroporation device according to Example 3 of the present invention, wherein the housing is not shown;

[0062] Attachment Figure 18 This is an exploded schematic diagram of a double-sided, side-powered, temperature-controlled flow electroporation device according to Example 3 of the present invention;

[0063] Attachment Figure 19This is a side cross-sectional view of a double-sided, side-powered, temperature-controlled flow electroporation device according to Example 3 of the present invention, wherein the housing is not shown;

[0064] Attachment Figure 20 Schematic diagram of a temperature control method for a flow electroporation device according to Example 3 of the present invention;

[0065] Attachment Figure 21 Graph showing the liquid outlet temperature of the flow electroporation device of Example 3 of the present invention and the flow electroporation device of the comparative example;

[0066] Attachment Figure 22 This is an exploded schematic diagram of a flow electroporation device with single-sided, front-side power supply, sealing and heat conducting components, and temperature control according to Example 4 of the present invention;

[0067] Attachment Figure 23 A side cross-sectional view of a single-sided, front-side powered flow electroporation device having a seal and a heat conductor, and having temperature control according to Example 4 of the present invention;

[0068] Attachment Figure 24 This is an exploded schematic diagram of a double-sided, side-powered, single-fan, temperature-controlled flow electroporation device according to Example 5 of the present invention;

[0069] Attachment Figure 25 This is a side cross-sectional view of a double-sided, side-powered, single-fan, temperature-controlled flow electroporation device according to Example 5 of the present invention, wherein the housing is not shown;

[0070] Attachment Figure 26 This is a three-dimensional diagram of the radiator of Example 6 of the present invention.

[0071] Among them, 1-stream electroporation modular device; 11-housing; 111-inner cavity; 112-air flow inlet; 113-air flow outlet; 12-electroporation assembly; 121-electroporation housing; 122-electroporation chamber; 123-flat electrode; 124, liquid flow outlet; 125, liquid flow inlet; 1231-back wall; 1232-side wall; 13-cooling mechanism; 131-refrigeration unit; 2-support plate; 21-positioning hole; 3-conductor; 4-heat dissipation assembly; 41-radiator; 411-fitting part; 412-heat dissipation part; 4121-base plate; 4122-vertical plate; 4123-groove; 4124-air flow hole; 42-fan; 421-air inlet; 422-air outlet; 5-temperature sensor; 6, sealing member; 7, thermal conductor. DETAILED DESCRIPTION

[0072] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art.

[0073] Example 1

[0074] like Figure 1-5 As shown, this embodiment provides a flow electroporation device, which includes a flow electroporation modular device 1 including a housing 11, an electroporation assembly 12, and a support plate 2. The housing 11 has an inner cavity 111, and the electroporation assembly 12 is fixedly disposed in the inner cavity 111; the electroporation assembly 12 includes an electroporation housing 121, and the interior of the electroporation housing 121 forms an electroporation chamber 122 for electroporating a cell suspension. The electroporation chamber 122 is connected to a liquid outlet 124 and a liquid inlet 125, respectively, and the liquid inlet 125 is located below the liquid outlet 124. The cell suspension enters the electroporation chamber 122 through the liquid inlet 125 and flows out of the electroporation chamber 122 through the liquid outlet 124 after the electroporation treatment is completed. The electroporation assembly 12 also includes two oppositely arranged flat electrodes 123, one of which constitutes a portion of the side wall of the electroporation housing 121. In this embodiment, the electroporation housing 121 includes a bracket and two flat electrodes 123 fixed on the bracket, wherein the two flat electrodes 123 respectively constitute at least a portion of the side wall of the electroporation housing 121 .

[0075] The support plate 2 in this embodiment is a PCB board, which is used to realize the electrical connection between the flat electrode 123 and the external power supply; the two support plates 2 are respectively arranged on the outside of the two flat electrodes 123, and a conductor 3 is arranged between the support plate 2 and the flat electrode 123 to supply power to the flat electrode 123.

[0076] like Figure 1 As shown, the flow electroporation modular device 1 further includes a cooling mechanism 13 disposed in the inner cavity 111. The cooling mechanism 13 includes an air flow channel and a heat dissipation component 4. The heat dissipation component 4 can be electrically connected to an external power source through the support plate 2. Specifically,

[0077] The airflow channel includes an airflow inlet 112 and an airflow outlet 113 provided on the housing 11 and respectively communicating with the outside of the housing 11. The airflow channel also includes a channel body provided between the airflow inlet 112 and the airflow outlet 113. The heat dissipation component 4 is used to form an airflow in the direction of the airflow inlet 112 → the channel body → the airflow outlet 113. The airflow is used to transfer the heat of the cell suspension in the electroporation chamber 122 to the outside of the housing 11. In this embodiment, the cooling mechanism 13 also includes a refrigeration unit 131 for absorbing heat. The refrigeration unit 131 is provided adjacent to the electroporation housing 121. In this embodiment, as shown in FIG. Figure 3As shown, the cooling section 131 is a semiconductor cooler positioned adjacent to one of the flat electrodes 123. When energized, the temperature of one end of the semiconductor cooler decreases, forming a cold end surface, while the temperature of the other end increases, forming a hot end surface. This creates a large temperature difference between the cold and hot end surfaces, allowing heat to be rapidly transferred from the cold end surface to the hot end surface of the semiconductor cooler. In this embodiment, the cold end surface of the semiconductor cooler (TEC) is aligned with the flat electrode 123, while the hot end surface is aligned with the heat sink 41 in the heat dissipation assembly 4. In this embodiment, the projected surface of the cooling section 131, made of the semiconductor cooler, completely covers the sidewalls of the electroporation housing 121, which are composed of the flat electrode 123 and a portion of the support of the electroporation housing 121 (in this case, the area of ​​the cooling section 131 is greater than the area of ​​the flat electrode 123), thereby increasing the contact area between the cooling section 131 and the heat sink 41 and enhancing the cooling effect.

[0078] In this embodiment, Figure 1 As shown, the heat dissipation assembly 4 includes a radiator 41 and a fan 42. The fan 42 for generating airflow includes an air inlet end 421 and an air outlet end 422. In this embodiment, the cross-sections of the air inlet end 421 and the air outlet end 422 are both circular, and the generated airflow flows from the air inlet end 421 to the air outlet end 422. The radiator 41, which includes a fitting portion 411 and a heat dissipation portion 412, is arranged between the refrigeration portion 131 and the fan 42. The fitting portion 411 fits the refrigeration portion 131 so that heat is transferred from the refrigeration portion 131 to the fitting portion 411 in the form of heat conduction; the heat dissipation portion 412, which is fixedly connected to the fitting portion 411, has a larger surface area than the fitting portion 411, so as to improve the efficiency of heat being carried away in the form of convection conduction when the airflow passes through. The airflow inlet 112 is a plurality of through holes opened on the shell 11 at corresponding positions close to the fitting portion 411. For example, the airflow inlet 112 is opened on the rear wall of the shell 11 (with Figure 1 For example, the left side is the front, the right side is the rear, and the components are arranged in the front-to-back direction. In this embodiment, through holes are also provided in the side walls, top wall, bottom wall, and front wall of the housing 11, serving as airflow inlets 112 to ensure a sufficiently high cooling airflow. The airflow outlets 113 are multiple through holes provided in the housing 11 at corresponding positions near the air outlet end 422 of the fan 42. For example, the airflow outlets 113 are provided in the rear wall of the housing 11. The multiple airflow outlets 113 are combined to form a circular shape similar to the fan 42.

[0079] Specifically, if Figure 2As shown, in this embodiment, the heat dissipation portion 412 includes a base plate 4121 and a plurality of vertical plates 4122 disposed on the base plate 4121. A groove 4123 is formed between two adjacent vertical plates 4122, opening toward a side away from the base plate 4121. The groove 4123 constitutes a portion of the channel body, and the air inlet end 421 of the fan 42 is disposed at the opening of the groove 4123. The opening of the groove 4123 is closed at a position offset from the air inlet end 421, so that all airflow flowing out of the opening of the groove 4123 enters the air inlet end 421, thereby maximizing the speed at which the heat-carrying airflow flows out of the housing 11 through the air outlet 113. In this embodiment, the frame of the fan 42 blocks or seals the opening of the groove 4123 at a position offset from the air inlet end 421 of the fan 42 to achieve the above purpose. In other embodiments, the opening of the groove 4123 may also be partially blocked by a portion of the housing 11 or other component. In this embodiment, the base plate 4121, the upright plate 4122, and the fitting portion 411 are integrally formed. The fitting portion 411 and the base plate 4121 extend in the same plane. Each groove 4123 also has an opening at a corresponding position near the fitting portion 411, so that airflow from the fitting portion 411 can be smoothly guided into the groove 4123. The airflow inlet 112 is a plurality of through holes provided on the housing 11 at positions corresponding to the fitting portion 411. The airflow outlet 113 is a plurality of through holes provided on the housing 11 at positions corresponding to the fitting portion 411.

[0080] In this embodiment, Figure 3 As shown, the support plate 2 is arranged on the side of the fitting portion 211 away from the refrigeration portion 131, and the fitting portion 211 and the refrigeration portion 131 are respectively provided with conductive through holes connected to each other, and the conductor 3 is passed through the conductive through holes and is electrically connected to the flat electrode 123; wherein the flat electrode 123 includes a back wall 1231 fitted with the refrigeration portion 131 and a side wall 1232 intersecting with the back wall 1231, and the conductor 3 and the back wall 1231 (as shown in FIG. Figure 1 、 Figure 3 as shown) or sidewall 1232 (as shown Figure 4-5 As shown) contact to achieve electrical connection. Figure 1 、 Figure 3 As shown, when the conductor 3 contacts the back wall 1231, the straight-line conductor 3 can conveniently supply power to the flat electrode 123, thereby ensuring the integrity of the electroporation housing 121 and the sealing of the electroporation chamber 122. Figure 4-5 As shown, the bent conductor 3 can bypass the cooling portion 131 and the bonding portion 411 without the need to open conductive through holes on the cooling portion 131 and the bonding portion 411, thereby increasing the effective contact area between the cooling portion 131 and the flat electrode 123 and enhancing the cooling effect. Figure 1 、 Figure 3 As shown, the support plate 2 is further provided with a positioning hole 21 , and the plurality of vertical plates 4122 of the heat dissipation portion 412 are passed through and positioned in the positioning hole 21 .

[0081] In this way, Figure 1 As shown, when the fan 42 is started, the air flow enters the corresponding position of the inner cavity 111 near the fitting portion 411 from the outside of the shell 11 through the air flow inlet 112, and then enters the groove 4123 through the opening formed at the corresponding position of each groove 4123 near the fitting portion 411. At this time, the air flow will carry the heat that is conducted to the radiator 41 (especially the vertical plate 4122) in the form of heat conduction through the flat electrode 123 and the refrigeration portion 131 in sequence, and then pass through the air inlet end 421 of the fan 42 and the air outlet end 422 of the fan 42, and then be conducted to the outside of the shell 11 through the air flow outlet 113.

[0082] As shown in Tables 1-3, the flow electroporation device in this embodiment was used to perform electroporation on cells with a density of 5×10 7 / ml and 1×10 7 / ml Jurkat cells and the cell density was 1×10 7 ExpiCHO-S cells were electroporated at a concentration of 100 μg / ml to obtain data on the percentage of surviving positive cells and cell viability. The same electroporation treatment was also performed on these cells using a flow electroporation device without a cooling mechanism to serve as a control group. The following results indicate that, compared to the significant decrease in the percentage of positive cells and cell viability in the control group, the percentage of positive cells and cell viability of cells treated with the flow electroporation device provided in this example remained essentially unchanged or only slightly decreased with increasing treatment time.

[0083] Table 1 Cell density is 5×10 7 Comparison of Jurkat cells electroporated with and without cooling

[0084] Proportion of surviving positive cells (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 83.6 76.9 77.8 81 81.3 82.2 79.2 No cooling group 77.7 70.6 71 70.1 68.8 68.1 60.3 Cell viability (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 88.8 86.9 86.2 85.9 85.7 86.3 85.8 No cooling group 81.9 74.6 75.4 72.3 72.4 72.8 65.3

[0085] Table 2 Cell density is 1×10 7 Comparison of Jurkat cells electroporated with and without cooling

[0086] Proportion of surviving positive cells (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 88 65.3 59.1 70 70.7 73 70.6 No cooling group 84.1 59.4 55.1 58.3 43.1 42.6 44.1 Cell viability (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 89.9 68.1 62 73.6 77.6 82.9 83 No cooling group 85.9 63.2 59.9 67.8 59.5 62.9 66.7

[0087] Table 3 Cell density is 1×10 7 Comparison of the results of electroporation of ExpiCHO-S cells with and without cooling

[0088] Proportion of surviving positive cells (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 81.9 69.8 60.4 54.7 57 61.1 66.6 No cooling group 35.8 1.36 0.99 1.88 1.68 1.18 1.46 Cell viability (%) 0 minutes 1 minute 2 minutes 5 minutes 10 minutes 15 minutes 20 minutes Semiconductor refrigeration chip assembly 86.3 74 63.7 57.3 60.1 64.3 71.2 No cooling group 39.3 2.56 1.55 3.16 2.86 2.56 2.91

[0089] Example 2

[0090] The difference between this embodiment and embodiment 1 is that Figure 6-11 As shown, in the flow electroporation device provided in this embodiment, a set of cooling mechanisms 13 is provided for each of the two flat electrodes 123. Specifically, the cooling mechanisms 13 on both sides are symmetrically arranged, and form two air flow channels on both sides of the two flat electrodes 123 respectively.

[0091] like Figure 6-7 As shown, the cooling mechanisms 13 on each side each include a cooling unit 131, a heat sink 41, and a fan 42, arranged in sequence. The cooling unit 131 abuts against the side wall of the electroporation housing 121 (composed of the flat electrode 123 and a portion of the bracket of the electroporation housing 121). The heat sink 41 and the cooling unit 131 each have interconnecting conductive through-holes. The conductor 3 is disposed in the conductive through-holes and contacts the back wall 1231 of the flat electrode 123 to achieve electrical connection.

[0092] like Figure 8-9 As shown, the cooling mechanisms 13 on both sides each include a cooling unit 131, a heat sink 41, and a fan 42, arranged in sequence. The cooling unit 131 abuts against the sidewall of the electroporation housing 121 (composed of the flat electrode 123 and a portion of the frame of the electroporation housing 121). Neither the heat sink 41 nor the cooling unit 131 has conductive through-holes. Instead, conductive through-holes are provided in the bracket of the electroporation housing 121. The conductor 3 passes through the conductive through-holes and contacts the sidewall 1232 of the flat electrode 123 to achieve electrical connection.

[0093] Figure 10-11 Another flow electroporation device is provided, which is Figure 8-9 The difference of the flow electroporation device shown is that the projection surface of the cooling portion 131 only completely covers the flat electrode 123, and the cross-section of the fitting portion 411 of the heat sink 41 is the same as the cross-section of the cooling portion 131, so that the effective working area for cooling is concentrated at the position of the flat electrode 123 where heat generation is more obvious during the electrofection process.

[0094] Example 3

[0095] The difference between this embodiment and the previous embodiment is that the flow electroporation device provided in this embodiment also includes a control system. Figure 12-13, Figures 14-15, 16-17, and 18-19 respectively show the solutions for adding a control system to a flow-through electroporation device with a single cooling mechanism 13 and front power supply, a double cooling mechanism 13 and front power supply, a single cooling mechanism 13 and side power supply, and a double cooling mechanism 13 and side power supply; the control system includes a temperature sensor 5 and an analysis and control unit 6, and the analysis and control unit 6 can be arranged on a support plate 2 made of PCB material; the temperature sensor 5 and the cooling mechanism 13 are electrically connected to the analysis and control unit 6 respectively. In this embodiment, the temperature sensor 5 is a thermistor, which abuts against the flat electrode 123 and realizes the electrical connection between the temperature sensor 5 and the analysis and control unit 6 via another conductor passing through the conductive through-hole and the support plate 2.

[0096] The temperature sensor 5 is used to measure the real-time temperature of the cell suspension in the electroporation chamber 122 and transmit the real-time temperature to the analysis and control unit 6; the analysis and control unit 6 is used to compare the relative magnitudes of the real-time temperature and the target temperature, and start the cooling mechanism 13 when the real-time temperature is greater than the target temperature. In this embodiment, the analysis and control unit 6 is electrically connected to the refrigeration part 131 (a semiconductor refrigerator) and / or the fan 42, and the analysis and control unit 6 is used to start the refrigeration part 131 and / or the fan 42 when the real-time temperature is greater than the target temperature.

[0097] For the flow-through electroporation device in this embodiment, its temperature control method is as follows:

[0098] S1, preset the target temperature T0 in the analysis and control unit 6, start the flow-through electroporation device and start the electroporation treatment on the cell suspension in the electroporation chamber 122. At this time, the cooling mechanism 13 is in a stopped operation state;

[0099] S2, the temperature sensor 5 monitors the real-time temperature T1 of the cell suspension in the electroporation chamber 122 and transmits the real-time temperature T1 to the analysis and control unit 6;

[0100] S3, the analysis and control unit 6 compares the magnitudes of the target temperature T_{0} and the real-time temperature T_{1}. When T_{0}<T_{1}, execute step S4; when T_{0}≥T_{1}, execute step S2;

[0101] S4, the analysis and control unit 6 starts the cooling mechanism 13 to start cooling the cell suspension in the electroporation chamber 122, and execute step S2.

[0102] The analysis and control unit 6 can independently control the start or stop of the refrigeration part 131 and the fan 42 of the cooling mechanism 13, or can also control both to start or stop simultaneously.

[0103] The flow-through electroporation device provided in this embodiment and a comparison device without the cooling mechanism 13 are operated under the same conditions and the temperature of the liquid flow outlet 124 is monitored. As Figure 21As shown, the temperature of the liquid outlet 124 of the flow electroporation device in this embodiment (about 45° C.) is significantly lower than the temperature of the liquid outlet of the flow electroporation device without the cooling mechanism 13 (greater than 70° C.).

[0104] Example 4

[0105] like Figure 22-23 As shown, this embodiment provides a flow electroporation device including a control system, wherein the control system scheme is similar to that of Example 3. It also differs from Example 1 in that it further includes a sealing member 6 for enhancing the sealing performance of the channel body and a heat conducting member 7 for improving heat conduction efficiency.

[0106] like Figure 22 As shown, a seal 6 is provided between the fan 42 and the air outlet 113, and the seal 6 is used to improve the sealing performance of the channel body between the fan 42 and the air outlet 113; in addition, a seal 6 is also provided between the fan 42 and the radiator 41, and the seal 6 is used to improve the sealing performance of the channel body between the fan 42 and the radiator 41. The provision of the seal 6 can reduce or even prevent the high-temperature airflow in the channel body from flowing back to the vicinity of the electroporation housing 121 through the gap between the fan 42 and the air outlet 113 or the gap between the fan 42 and the radiator 41, thereby increasing the temperature of the electroporation housing 121. It should also be noted that in this embodiment, the air outlet 113 is only provided on the front wall of the housing 11 (with Figure 22 For example, the left side is the front, the right side is the rear, and the components are arranged along the front-to-back direction), and no air flow outlet 113 is provided on the rear wall.

[0107] like Figure 22 As shown, in this embodiment, a heat conducting member 7 for improving heat conduction efficiency is further provided between the cooling unit 131 and the electroporation housing 121 and between the cooling unit 131 and the heat sink 41, thereby improving the efficiency of heat conduction from the electroporation housing 121 to the heat sink 41 via the cooling unit 131.

[0108] In addition, if Figure 23 As shown, in this embodiment, no conductive through hole is provided on the bonding portion 211 or the cooling portion 131. The conductor 3 is located on one side of the bonding portion 211 or the cooling portion 131, with one end of the conductor 3 connected to the fixing plate 2 and the other end in contact with the back wall 1231 of the flat electrode 123 to achieve electrical connection. Figure 22As shown, two support plates 2 are fixedly connected and electrically connected to each other, respectively, outside the two flat electrodes 123. The volume of the support plate 2 on the left is smaller than that of the support plate 2 on the right. The left support plate 2 is electrically connected to the temperature sensor 5 and a corresponding flat electrode 123. The temperature sensor 5 is only located on the side of the electroporation housing 121 away from the cooling portion 131. The right support plate 2 has a positioning hole 21 for positioning the heat dissipation portion 412.

[0109] Example 5

[0110] like Figures 24-25 As shown, the flow electroporation device provided in this embodiment includes a control system, wherein the control system scheme is similar to that of embodiment 3, and the cooling mechanism 13 includes a cooling mechanism 13 located at the front and a cooling mechanism 13 located at the rear. Figure 18-19 The corresponding technical solutions also differ in that one of the two cooling mechanisms 13 does not include a fan 42, and the two cooling mechanisms 13 share a set of airflow outlets 113. Specifically, the rear cooling mechanism 13 includes a radiator 41 and a fan 42, while the front cooling mechanism 13 includes a radiator 41 but does not include a fan 42.

[0111] like Figure 24 As shown, the air outlet 113 is only opened on the front wall of the housing 11 (with Figure 24 For example, the left side is the front, the right side is the rear, and the components are arranged along the front-to-back direction), and no air flow outlet 113 is provided on the rear wall of the housing 11.

[0112] The heat dissipation portion 412 of the front radiator 41 is provided with airflow holes 4124 adapted to accommodate the fan 42. These airflow holes 4124 extend through the base plate 4121 and the vertical plate 4122, which is located between the base plate 4121 and the fan 42. The front cooling mechanism 13 is located between the rear cooling mechanism 13 and the airflow outlet 113. The airflow holes 4124 between the airflow outlet 113 and the fan 42 accommodate the fan 42 in the rear cooling mechanism 13. The rear radiator 41 does not have airflow holes 4124; the vertical plate 4122 is located between the base plate 4121 and the fan 42.

[0113] During use, driven by the fan 42, a rear airflow is formed through the rear radiator 41, and a front airflow is formed through the front radiator 41 only. The rear airflow sequentially flows through the fitting portion 411, the vertical plate 4122, the fan 42, the airflow holes 4124, and the airflow outlet 113 of the rear radiator 41, while the front airflow sequentially flows through the fitting portion 411, the vertical plate 4122, the airflow holes 4124, and the airflow outlet 113 of the front radiator 41.

[0114] Example 6

[0115] This embodiment provides a heat sink 41 that can be applied to any of the above embodiments. Figure 26 As shown, the difference between the radiator 41 in this embodiment and the radiator 41 in the aforementioned embodiment is that a blind hole is provided on each vertical plate 4122, and the blind holes of multiple vertical plates 4122 are connected to each other to form a groove 4123 that intersects with the groove 4123 between the two vertical plates 4122, so that the hot air from the inner cavity 111 away from the fitting portion 411 can also be smoothly guided to the groove 4123, avoiding the formation of airflow dead corners in the inner cavity 111.

[0116] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A flow electroporation device comprising a modular flow electroporation device including a housing and an electroporation assembly, wherein the housing has an inner cavity, and the electroporation assembly is fixedly disposed in the inner cavity; the electroporation assembly includes an electroporation housing, wherein the interior of the electroporation housing forms an electroporation chamber for electroporating a cell suspension, wherein: The flow electroporation modular device further includes a cooling mechanism, which includes: The air flow channel includes a channel body, an air flow inlet and an air flow outlet provided on the housing and respectively communicating with the outside of the housing. a refrigeration unit, configured to absorb heat from the cell suspension in the electroporation chamber; a heat dissipation assembly disposed between the refrigeration unit and the air flow channel, the heat dissipation assembly being configured to form an airflow in the direction of the air flow inlet → the channel body → the air flow outlet, the airflow being configured to conduct the heat to the outside of the housing; The heat dissipation assembly further includes a radiator and a fan, the fan is used to generate the airflow, the radiator including a bonding portion and a heat dissipation portion is arranged between the refrigeration portion and the fan, and the bonding portion is bonded to the refrigeration portion so that the heat is transferred from the refrigeration portion to the bonding portion by heat conduction; The heat dissipation portion includes a base plate and a plurality of vertical plates arranged on the base plate, a groove having an opening facing away from the base plate is formed between two adjacent vertical plates, the groove constituting a part of the channel body, and the air inlet end of the fan is arranged at the position of the opening; The fitting portion and the substrate are integrated, the air flow inlet is opened at a position of the shell close to the fitting portion, the air flow inlet includes a through hole opened on the side wall of the shell and opposite to the end of the groove, and the air flow outlet is opened on the shell close to the air outlet end of the fan.

2. The flow electroporation device according to claim 1, characterized in that The electroporation assembly includes two oppositely arranged flat electrodes, one of which constitutes at least a portion of a side wall of the electroporation housing, and the refrigeration unit is disposed adjacent to the flat electrode.

3. The flow electroporation device according to claim 2, characterized in that The cooling mechanism is provided at a corresponding position of at least one of the flat electrodes; or, A set of cooling mechanisms is provided at the corresponding position of each flat electrode.

4. The flow electroporation device according to claim 2 or 3, characterized in that: The heat dissipation portion fixedly connected to the fitting portion has a larger surface area than the fitting portion.

5. The flow electroporation device according to claim 1, characterized in that The air flow outlets are a plurality of through holes provided on the housing at corresponding positions close to the air outlet end of the fan.

6. The flow electroporation device according to claim 1, characterized in that A blind hole is formed on each of the vertical plates, and the blind holes of the plurality of vertical plates are connected to each other to form a groove intersecting with the groove between two vertical plates.

7. The flow electroporation device according to claim 2, characterized in that The flow electroporation modular device also includes a support plate. The fitting portion and the base plate extend in the same plane. The support plate is arranged on a side of the fitting portion away from the refrigeration portion. A conductor is provided between the support plate and the flat electrode to supply power to the flat electrode.

8. The flow electroporation device according to claim 7, characterized in that The flat electrode includes a back wall attached to the refrigeration part and a side wall intersecting with the back wall; wherein, The conductor contacts the side wall of the plate electrode to achieve electrical connection; or, The fitting portion and the cooling portion are respectively provided with conductive through holes that are in communication with each other. The conductor is passed through the conductive through holes and contacts the back wall of the flat electrode to achieve electrical connection.

9. The flow electroporation device according to claim 7, characterized in that The support plate is further provided with a positioning hole, and the heat dissipation portion is passed through the positioning hole.

10. The flow electroporation device according to claim 2, characterized in that The cooling mechanism includes the cooling mechanism at the front and the cooling mechanism at the rear. The cooling mechanism at the rear includes a radiator and a fan, and the cooling mechanism at the front includes the radiator but does not include the fan.

11. The flow electroporation device according to claim 10, characterized in that The cooling mechanism at the front is arranged between the cooling mechanism at the rear and the air flow outlet. The radiator in the cooling mechanism at the front is provided with an air flow hole, which is adapted to the fan in the cooling mechanism at the rear. The air flow hole is located between the air flow outlet and the fan.

12. The flow electroporation device according to claim 11, characterized in that The heat dissipation portion includes a substrate and a plurality of vertical plates arranged on the substrate, and the air flow holes penetrate the substrate and the vertical plates.

13. The flow electroporation device according to claim 1, wherein The temperature reduction mechanism further includes a sealing member for enhancing the sealing performance of the channel body.

14. The flow electroporation device according to claim 13, wherein: The heat dissipation assembly further includes a radiator and a fan, wherein the fan is used to generate the airflow; The sealing member is disposed between the fan and the air flow outlet; and / or, The seal is disposed between the fan and the heat sink.

15. The flow electroporation device according to claim 1, wherein The cooling mechanism also includes a heat conducting member for improving heat conduction efficiency. The heat conducting member is disposed between the refrigeration unit and the electroporation housing; and / or, The heat conducting member is arranged between the refrigeration unit and the heat dissipation component.

16. The flow electroporation device according to claim 1, wherein The flow electroporation device further includes a control system, which includes a temperature sensor and an analysis control unit, wherein the temperature sensor and the cooling mechanism are electrically connected to the analysis control unit respectively; The temperature sensor is used to monitor the real-time temperature of the cell suspension in the electroporation chamber and transmit the real-time temperature to the analysis control unit; the analysis control unit is used to compare the relative sizes of the real-time temperature and the target temperature, and to activate the cooling mechanism when the real-time temperature is greater than the target temperature.

17. The flow electroporation device according to claim 16, characterized in that The refrigeration unit is disposed adjacent to the electroporation housing; and the analysis control unit is configured to activate the refrigeration unit when the real-time temperature is greater than the target temperature.

18. The flow electroporation device according to claim 1, characterized in that The opening is closed at a position deviated from the air inlet end so that the airflow flowing out of the opening of the groove all enters the air inlet end.

19. A temperature control method for a flow electroporation device according to any one of the preceding claims, characterized in that: The steps include: S1, analyzing the preset target temperature T0 in the control unit, starting the flow electroporation device and starting electroporation of the cell suspension in the electroporation chamber, at which time the cooling mechanism is in a stopped state; S2, the temperature sensor monitors the real-time temperature T1 of the cell suspension in the electroporation chamber and transmits the real-time temperature T1 to the analysis control unit; S3, the analysis control unit compares the target temperature T0 and the real-time temperature T1. When T1>T0, the process goes to step S4; when T1≤T0, the process goes to step S2. S4, the analysis control unit starts the cooling mechanism to start cooling the cell suspension in the electroporation chamber, and executes step S2.

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