Organ-like virus transfection method based on filter membrane mediation

Abstract

The invention relates to the technical field of biomedicine, in particular to an organoid virus transfection method based on filter membrane mediation, which comprises the following steps: providing an organoid suspension which is mildly digested and retains part of matrigel; centrifuging in a container containing a microporous filter membrane, so that the organoid is flatly laid on the surface of the filter membrane to form a thin film; transferring the filter membrane bearing the organoid to an electric transfer device to be in contact with a virus vector, and applying an electric field to drive viruses to penetrate through the filter membrane and residual matrigel to enter cells; geometric shielding is eliminated through centrifugal tiling, and diffusion obstruction of matrigel is overcome through active transportation of electric field force; according to the method, the transfection efficiency and uniformity of the virus vector are remarkably improved on the premise of maintaining the integrity of the three-dimensional structure and high cell activity of the organoid, and the problem that the efficiency and the structure cannot be obtained at the same time in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for organoid virus transfection mediated by a filter membrane. Background Technology

[0002] Organoids, as in vitro three-dimensional (3D) models capable of mimicking the spatial structure and physiological functions of human organs, have become a key tool for disease research and drug screening. However, as research progresses, achieving efficient and uniform gene modification (such as viral transduction) while maintaining the complete three-dimensional structure and microenvironment of organoids has become the biggest technical bottleneck restricting the development of this field; the limitations of existing technologies (three major pain points):

[0003] Pain Point 1: The physical barrier of Matrigel leads to "inefficiency" (regarding traditional co-incubation methods). Organoids must grow in a dense Matrigel to maintain their 3D structure. Existing technologies typically employ a passive co-incubation method, directly immersing the organoids in viral fluid. Due to the small (nanoscale) pore size and charged nature of Matrigel, it creates severe "steric hindrance" and "electrostatic adsorption" for larger viral particles (such as lentiviruses and adenoviruses). This makes it difficult for the virus to diffuse into the deeper layers of the organoid, resulting in transfection efficiency typically below 20%, and often exhibiting an "eggshell effect" where only the outer cells are infected.

[0004] Pain Point Two: Matrix Gel Removal Leads to "Structural Disintegration" (Regarding Digestion Methods) To improve transfection efficiency, another existing technology attempts to remove matrix gel through enzymatic digestion, dissociating organoids into single cells or small clumps before transfection. While this method increases contact area and efficiency, it severely disrupts the tight junctions between cells and the original tissue polarity, leading to biological distortion of the organoid model. Furthermore, the recombination process is lengthy, and cell survival rates are low.

[0005] Pain Point 3: The "uncontrollability" of traditional physical transfection (referring to traditional electroporation / microinjection);

[0006] Microinjection: Although precise, it has extremely low throughput, which cannot meet the needs of large-scale screening.

[0007] Traditional suspension electroporation: Due to the random distribution of organoid clumps in the suspension, the electric field distribution is uneven. To penetrate multiple cell layers, high voltages are often required, leading to severe Joule thermal damage and massive cell death.

[0008] In summary, existing technologies have fallen into a technical paradox where "structural integrity" and "transfection efficiency" are mutually exclusive. There is an urgent need in this field for a new method that can overcome the physical barrier of matrix gel, achieving active and efficient gene delivery without damaging the precise three-dimensional structure of organoids.

[0009] To address this, a membrane-mediated organoid transfection method is proposed. Summary of the Invention

[0010] This invention aims to solve the problems of low transfection efficiency, matrix gel barrier obstruction, and structural damage of organoid viruses in the prior art. It proposes a filter membrane-mediated organoid virus transfection method, which improves infection efficiency by uniformly spreading organoids on the surface of a microporous filter membrane and using electroporation technology to precisely drive virus particles to penetrate the residual matrix gel barrier.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A filter membrane-mediated organoid virus transfection method, the method comprising the following steps:

[0013] (1) Provides a mildly digested organoid suspension in which the organoids retain residual matrix gum for maintaining their three-dimensional structure;

[0014] (2) The organoid suspension is placed in a container with a microporous filter membrane and centrifuged. Centrifugal force is used to trap the organoids and spread them on the surface of the microporous filter membrane to form an organoid film.

[0015] (3) Transfer the microporous filter membrane carrying the organoid membrane to the electroporation device so that the side of the microporous filter membrane away from the organoid comes into contact with the medium containing the virus vector without air bubbles.

[0016] (4) Apply an electric field by passing an electric current, and use the electrophoretic force generated by the electric field to drive the negatively charged virus vector through the microporous filter membrane and the residual matrix gel, and enter the cells in the organoid membrane.

[0017] Preferably, in step (1), the volume percentage of the residual matrix gel is controlled at 10%-20% so that the organoids maintain their clump shape while detaching from the culture medium substrate.

[0018] By limiting the specific volume percentage of the residual matrix gel, the highest transfection throughput is achieved while maintaining the 3D microenvironment.

[0019] Preferably, in step (1), the mild proteolytic enzyme is TrypLE™ Express or Dispase II, and the treatment includes incubation at 37±1°C for 5 to 8 minutes until the organoid edges are blurred but not disintegrated into single cells as observed under a microscope;

[0020] By limiting mild digestion parameters, the reproducibility of the process and the activity of organoids are ensured, preventing over-digestion (leading to apoptosis, anokikis) or under-digestion (leading to excessively large clumps and severe accumulation).

[0021] Preferably, in step (2), the pore size of the microporous filter membrane is 50 μm ± 5 μm, which is configured to retain organoid clumps while allowing viral vectors and current to pass through; the surface of the microporous filter membrane is hydrophilically treated to reduce non-specific adsorption of viral vectors;

[0022] Using a pore size of 50 μm, smaller than the diameter of the organoid, ensures that the organoid remains on the membrane; at the same time, it is much larger than the diameter of the virus and matrix gel fragments to prevent the filter membrane from clogging and causing abnormal centrifugation pressure. This is also the physical basis for forming a uniform "organoid membrane".

[0023] Preferably, in step (2), the organoids are centrifuged at a relative centrifugal force of 700g to 900g for 4 to 6 minutes to overcome the viscous resistance of the residual matrix adhesive and form a single-layer distributed film on the filter membrane surface.

[0024] Preferably, in step (3), the electroporation device has a bayonet structure for horizontally fixing the microporous filter membrane; the viral vector has a titer of ≥1×10⁻⁶ for lentivirus or adeno-associated virus. 8 TU / mL or ≥1×10¹²vg / mL.

[0025] Preferably, the parameters of the electric field include: a voltage of 20V to 40V, a pulse duration of 20 seconds to 40 seconds, the electric field being applied as a square wave pulse, and maintaining a constant voltage within a single pulse cycle.

[0026] Preferably, the electroporation parameters are selected from one of the following based on the type of the viral vector:

[0027] When the viral vector is a lentivirus, the voltage is 25V±2V and the pulse duration is 30 seconds±5 seconds.

[0028] When the viral vector is adeno-associated virus, the voltage is 20V±2V and the pulse time is 20 seconds±5 seconds.

[0029] When the viral vector is adenovirus, the voltage is 30V±2V and the pulse duration is 30 seconds±5 seconds.

[0030] Preferably, the procedure also includes step (5) subsequent culture and recovery steps: after electroporation, the organoids are eluted from the microporous filter membrane by low-speed centrifugation and resuspended in a culture medium containing 3%-5% matrix gel for recovery culture to prevent anodic apoptosis; the low-speed centrifugation is 100g±20g for 3-5 minutes.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. By spreading organoids into a single-layer film and applying electrophoretic force, this invention overcomes the steric hindrance effect of matrix gel. Experimental data show that the virus penetration index (core fluorescence intensity / peripheral fluorescence intensity) of this method is greater than 0.8, which is significantly better than the <0.2 of the traditional immersion method, thus eliminating the "eggshell effect".

[0033] 2. A low voltage window of 20V-40V was used to avoid thermal damage from high-voltage electrophoresis; combined with the protection of 10%-20% residual matrix gel, the cell survival rate of the transfected organoids remained above 90%, and the proliferation capacity was not significantly different from that of the untreated group.

[0034] 3. It is compatible with multiple viral vectors such as lentivirus, adenovirus, and adeno-associated virus, and is suitable for mature organoids from different sources (such as intestine, liver, lung, etc.). The operation process is standardized and can be scaled up to high throughput.

[0035] 4. The hydrophilic PC filter membrane material and the centrifugation window of 700-900g were clearly defined to ensure the uniform spreading of organoids on the filter membrane and reduce the coefficient of variation (CV) of transfection efficiency between batches to less than 5%. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the confocal microscopy image taken after organoid infection with lentivirus according to the present invention;

[0037] Figure 2 This diagram illustrates a comparison of the transfection efficiency of the present invention, the conventional method, and WTcontrol. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The present invention can be further understood through the following embodiments;

[0040] Example 1

[0041] In this embodiment, human liver organoids are processed and transfected using a lentiviral vector via centrifugation and electroporation to drive viral expression.

[0042] 1. Material preparation:

[0043] Organoid model: Human normal liver organoids, cultured in a Matrigel microenvironment containing factors such as R-Spondin1, Noggin, and EGF.

[0044] Extracellular matrix (ECM): cellular matrix gel, reduced growth factor (GFR), protein concentration is approximately 8-10 mg / mL.

[0045] Microporous membrane: Hydrophilic polycarbonate track-etched membrane, 50μm pore size, 13mm diameter, 10μm thickness, porosity approximately 15%. (PC membrane was chosen to avoid the non-specific adsorption of viruses by nylon membrane and to reduce mechanical damage to cells due to its smooth surface.)

[0046] Gentle digestive enzyme: TrypLE™ ExpressEnzyme, free of phenol red and animal-derived ingredients.

[0047] Viral vector:

[0048] Lentiviral: titer determined to be 2.5 x 10⁻⁶ 8 TU / mL (measured by HEK293T cell flow cytometry).

[0049] Adeno-associated virus: titer 1.2 x 10⁻⁶ 13 vg / mL.

[0050] Adenovirus: titer 5 x 10 10 PFU / mL.

[0051] Electroporation device: Improved horizontal plate electrode electroporation tank, with a fixed electrode spacing (Gap) of 4mm, connected to BTX ECM830 square wave electroporator.

[0052] Statistical analysis: All quantitative data were obtained from at least three independent biological replicates (n≥3), and results are expressed as mean ± standard deviation (Mean±SD). Differences between groups were assessed using one-way ANOVA combined with Tukey's post-hoc test, and p < 0.05 was considered statistically significant.

[0053] 2. Experimental procedure:

[0054] Step 1: Organoid pretreatment (gentle digestion and detachment from the substrate);

[0055] ① Collect human liver organoids cultured to day 7 with a diameter of approximately 200-300 μm, and wash once with cold PBS to remove the culture medium.

[0056] ② Add TrypLE™ Express digestion solution preheated to 37°C, with a volume 5 times that of the organoid precipitate.

[0057] ③ Incubate at a constant temperature of 37℃ for 6 minutes. Gently blow on the container every 2 minutes during this time.

[0058] ④ Termination of digestion: Under a microscope, when the organoid macrospheres dissociate into dense cell clusters of 20-50 cells, and the edges are slightly blurred but have not dispersed into single cells, immediately add 3 times the volume of cold culture medium containing 10% fetal bovine serum (FBS) to terminate digestion.

[0059] ⑤ Control of residual matrix gel: At this point, matrix gel has not been completely removed from the suspension. Based on trypan blue staining and microscopic examination, the volume percentage of residual matrix gel is estimated to be about 15%. This residual gel is crucial for maintaining the structural stability of cell clusters during subsequent centrifugation and electroporation.

[0060] Step 2: Organoid collection and centrifugation;

[0061] ① Transfer the above organoid suspension to a specially designed centrifuge tube with a 50μm PC filter membrane at the bottom (the waste collection chamber is located below the filter membrane).

[0062] ② Place it in a horizontal rotor centrifuge, set the relative centrifugal force to 800g, centrifuge time to 5 minutes, and temperature to 4℃.

[0063] ③ After centrifugation, the liquid medium was removed through the filter membrane, while the organoid clusters were retained and densely spread on the filter membrane surface, forming a uniformly thick, moist "cell-matrix gel film". Microscopic examination showed no multilayer stacking.

[0064] Step 3: Filter membrane transfer and virus droplet loading;

[0065] ① Add 50 μL of lentivirus concentrate (2.5 x 10⁻⁶) to the negative electrode plate of the electroporation cell. 8 TU / mL).

[0066] ② Carefully remove the PC filter membrane carrying the organoid membrane with tweezers, and place the side of the filter membrane away from the cell (i.e., the bottom) flat on the virus droplet, ensuring that no air bubbles are generated. At this time, the negatively charged virus is located on the negative electrode side.

[0067] ③ Add 50 μL of conductive buffer solution to the surface of the organoid membrane (positive electrode side) and cover it with the positive electrode plate.

[0068] Step 4: Electroporation-driven virus transfection;

[0069] Connect the power supply to the power source and apply a square wave pulse.

[0070] Parameter settings: voltage 25V (field strength approximately 62.5V / cm), single pulse duration 30 seconds (s), pulse interval 1 second, pulse count 1. This method utilizes the electrophoretic force generated by low-voltage long pulses to drive viral particles directionally through the pores of the PC filter membrane and residual matrix gel, enriching them and allowing them to enter the cells, rather than simply relying on high-voltage breakdown.

[0071] Step 5: Subsequent cultivation and screening;

[0072] ① After the electroporation is complete, immediately remove the filter membrane and place it upside down at the opening of a new centrifuge tube.

[0073] ② Rinse the filter membrane surface with 500 μL of cold organoid culture medium containing 5% Matrigel, and centrifuge at 100g for 3 minutes to elute the organoids to the bottom of the tube.

[0074] ③ The recovered organoids were resuspended in Matrigel at normal concentration, seeded into 24-well plates, and incubated at 37°C.

[0075] Results detection: EGFP fluorescence expression was observed by confocal microscopy 48 hours after transfection.

[0076] 1. Transfection efficiency: Flow cytometry analysis showed that the proportion of EGFP-positive cells was 88.5% ± 3.2%.

[0077] 2. Cell viability: ATP luminescence assay showed that the cell viability was 94.2% ± 1.5% compared with the untreated group, with no significant cytotoxicity.

[0078] 3. Morphology: On day 5 of culture, the organoids reassembled into typical hollow spherical structures, and the fluorescence signal was evenly distributed in the core and periphery of the spheres, with no "eggshell effect" observed.

[0079] Example 2: Parameter adaptation verification for different viral vectors.

[0080] To verify the broad applicability of the method of this invention, parameter fine-tuning tests were performed on AAV and adenovirus. The operation steps are the same as in Example 1, except that the electroporation parameters are adjusted as follows:

[0081] Virus type Virus characteristics Optimize voltage (V) Pulse duration (ms) Transfection efficiency (Mean ± SD) Remark Adeno-associated virus Small particles (20nm), uncoated, and negatively charged 20V 20ms 72.1%±4.5% Traditional soaking methods have an efficiency of less than 15%. adenovirus Large particles (90nm), no envelope, and strong immunogenicity 30V 30ms 65.4%±5.1% 30V helps overcome the steric hindrance of large particles. Lentiviral The particles (100 nm) have a coating. 25V 30ms 88.5%±3.2% Balanced coating stability and driving force

[0082] As shown in the table above, this method is effective for all three mainstream viral vectors. Adeno-associated virus (AAV) requires the lowest driving voltage (20V) due to its small size, while adenovirus, due to its large size and lack of lipid envelope buffer, requires a slightly higher voltage (30V) to overcome the matrix gel pore size resistance. The survival rate of all groups remained above 90%.

[0083] Example 3: Window confirmation of key process parameters (comparative experiment);

[0084] To demonstrate the scientific validity and necessity of the parameter range in the claims, this embodiment includes multiple sets of comparative experiments deviating from the preferred range.

[0085] The effect of voltage parameters: With a fixed centrifugal force of 800g, using lentivirus, the efficiency and virulence under different voltages were tested.

[0086] Experimental group Voltage (V) Transfection efficiency (%) Cell viability (%) Results Analysis Control group A 10V 8.3±1.2 98.5±0.5 Voltage too low: The electrophoretic driving force is insufficient to overcome the resistance of the matrix gel. Example 1 25V 88.5±3.2 94.2±1.5 Best window: efficient and virus-free. Control group B 60V 90.1±2.1 65.4±4.8 Mild toxicity: Efficiency reaches a plateau, but thermal damage begins to appear. Control group C 100V 91.5±1.8 15.4±3.2 Excessive voltage: generates a large amount of Joule heat, leading to cell disintegration and death.

[0087] As shown in the table above, the data supports strictly limiting the voltage range in the claims to 20V-40V. Voltages below 20V are invalid, and voltages above 60V are unacceptably toxic.

[0088] Effect of Centrifugal Force (Plate Morphology Analysis): With a fixed voltage of 25V, the effects of different centrifugal forces on the plateau state and final results of organoids were tested.

[0089] Experimental group Centrifugal force (g) Filter membrane coverage morphology (microscopic examination) Transfection uniformity (CV value) Results Analysis Control group D 300g Organoids are stacked in multiple layers 45.6% (Poor) Insufficient centrifugal force: Stacking leads to uneven distribution of electric field and virus. Example 1 800g Single-layer dense flat tiling 4.2% (Excellent) Optimal window: forming an ideal quasi-two-dimensional plane. Control group E 1500g Single layer, but some cells are broken. 12.1% (China) Excessive centrifugal force: Mechanical pressure leads to physical damage (nuclear fragmentation).

[0090] As can be seen from the table above, the data supports limiting the centrifugal force range in the claims to 700g-900g.

[0091] Example 4: Control experiment.

[0092] Traditional method control group;

[0093] 1. Material preparation;

[0094] Human liver organoids: organoids from the same source and cultured in the same batch as in Example 1; unlike in Example 1, the organoids in this group retained the complete Matrigel coating state and were not subjected to digestion and thinning treatment.

[0095] Lentiviral vector: A lentivirus carrying the EGFP reporter gene identical to that in Example 1, with a titer of 2.5 × 10⁻⁶. 8 U / mL.

[0096] Culture system: standard 24-well cell culture plate.

[0097] 2. Operating procedures;

[0098] Step 1: Virus loading;

[0099] Organoids containing intact matrix gel were placed in culture wells, and the original culture medium was aspirated; the same titer (2.5 × 10⁻⁶) as in Example 1 was added directly to the wells. 8 Lentiviral suspension (U / mL); the amount of virus solution is sufficient to cover organoid matrix droplets (e.g., 500 μL).

[0100] Step 2: Incubate statically;

[0101] Place the culture plate in a 37°C constant temperature incubator; keep it in a static state for co-incubation for 4 hours; during this process, there is no active driving (i.e. no centrifugal force is applied and no electroporation is performed), and the virus only relies on passive diffusion to penetrate the matrix gel barrier.

[0102] Step 3: Change the medium and incubate;

[0103] After 4 hours of incubation, the virus-containing culture medium was aspirated, and the cells were washed once with sterile PBS to remove free viruses that had not entered the cells; fresh organoid culture medium was added and cultured for another time.

[0104] Step 4: Result Detection;

[0105] After culturing for another 48 hours (synchronized with Example 1), fluorescence microscopy was used for detection. The results showed that due to the physical barrier of the matrix gel and the lack of active driving force, only sparse and weak fluorescence was observed at the edge of the organoids, and no fluorescence was observed in the internal core region. Quantitative analysis showed that its fluorescence integral density (IntDen) was about 1 / 3 of that of Example 1, proving that the transfection efficiency of the conventional method is significantly lower than that of the method of the present invention when the matrix gel is retained.

[0106] WTControl control group: designed to assess the endogenous autofluorescence level and natural growth status of organoids, providing a negative reference for evaluating the transfection efficiency and structural integrity of Example 1.

[0107] 1. Material Preparation: Human liver organoids: Mature organoids from the same source and batch as in Example 1, maintained in Matrigel, possessing a complete three-dimensional structure. Control medium: Sterile PBS buffer or blank organoid culture medium (free of any lentivirus, adenovirus, or AAV vector). Culture system: Conventional 24-well or 48-well cell culture plates. Detection equipment: Inverted fluorescence microscope with EGFP filter channel, parameters set consistent with Example 1.

[0108] 2. Operational Procedures: Organoid Preparation: Select organoids that are in good growth condition and have uniform morphology; Unlike Example 1, this group does not perform digestion pretreatment, centrifugation, or electroporation to maintain their most original growth microenvironment.

[0109] Simulated loading: In order to control variables, organoids can be "simulated" by adding an equal volume of sterile PBS buffer instead of viral droplets, but without applying an electric field; or they can be kept in the original culture medium directly.

[0110] Simultaneous culture: The organoids were placed in a 37°C constant temperature incubator and cultured in parallel with the experimental group of Example 1; during the process, the culture medium was replaced with fresh medium according to the standard procedure.

[0111] Results: After 48 hours of culture, fluorescence microscopy was performed with the same exposure time, gain, and other parameters as in Example 1. The results showed that only a very weak background signal was detected in the EGFP channel (the IntDen value was about 1 / 20 of that in Example 1), and the organoids in the field of view showed a typical spherical structure with smooth edges and dense structure, without dissociation or fragmentation. This confirmed that the high-intensity fluorescence signal observed in Example 1 was indeed caused by viral transfection mediated by the method of this invention.

[0112] In summary, this embodiment demonstrates that the coupling technology of "centrifugation-microporous membrane-electro-driven" can achieve efficient and uniform introduction of lentiviruses into the human liver organoid model without damaging the three-dimensional structure and activity of the organoids, overcoming the technical problem of low transfection efficiency caused by the matrix gel barrier in the prior art.

[0113] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of filter membrane-mediated organoid viral transfection, comprising, The method comprises the following steps: (1) providing a suspension of organoids treated with mild protease digestion, the organoids in the suspension retaining residual matrigel for maintaining their three-dimensional structure; (2) placing the suspension of organoids in a container with a microporous filter membrane and centrifuging to make the organoids retained and spread on the surface of the microporous filter membrane to form an organoid film; (3) transferring the microporous filter membrane carrying the organoid film to an electroporation device, and contacting the side of the microporous filter membrane away from the organoids with a medium containing viral vectors without air bubbles; (4) applying an electric field by power on, and driving the negatively charged viral vectors to pass through the microporous filter membrane and the residual matrigel into the cells in the organoid film by electrophoretic force generated by the electric field.

2. The method according to claim 1, wherein, In step (1), the volume fraction of the residual matrigel is controlled at 10%-20% to make the organoids maintain a mass shape while being separated from the culture medium.

3. The method according to claim 1, wherein the method is a filter membrane-mediated organoid viral transfection method. In step (1), the mild protease is TrypLE™ Express or Dispase II, and the treatment includes incubation at 37±1℃ for 5-8 minutes until the organoids are observed under a microscope to be blurred at the edges but not dispersed into single cells.

4. The method according to claim 1, wherein the method is based on filter membrane-mediated organoid viral transfection. In step (2), the pore size of the microporous filter membrane is 50μm±5μm, which is configured to retain the organoid mass while allowing the viral vectors and electric current to pass through; the surface of the microporous filter membrane is treated with hydrophilic treatment to reduce non-specific adsorption of viral vectors.

5. The method for organoid virus transfection based on a filter membrane as described in claim 1, characterized in that, In step (2), the relative centrifugal force is 700g-900g for 4-6 minutes to overcome the viscous resistance of the residual matrigel and make the organoids form a monolayer distributed film on the surface of the filter membrane.

6. The method according to claim 4, wherein the method is based on filter membrane-mediated organoid viral transfection. In step (3), the electrical transfer device has a bayonet structure for horizontally fixing the microfiltration membrane; the titer of the viral vector against lentivirus or adeno-associated virus is ≥1 x 10 8 TU / mL or ≥1 x 10¹²vg / mL, respectively.

7. The method according to claim 1, wherein the method is based on filter membrane-mediated organoid viral transfection. In step (4), the parameters of the electric field include: voltage 20V-40V, pulse time 20s-40s, the electric field is applied in the form of square wave pulse, and the voltage is kept constant in a single pulse cycle.

8. The method according to claim 7, wherein the method is a filter membrane mediated organoid viral transfection method. The electroporation parameters are selected as follows according to the type of the viral vector: When the viral vector is a lentivirus, the voltage is 25V±2V and the pulse time is 30s±5s; When the viral vector is an adeno-associated virus, the voltage is 20V±2V and the pulse time is 20s±5s; When the viral vector is an adenovirus, the voltage is 30V±2V and the pulse time is 30s±5s.

9. The method according to claim 1, wherein the method is based on filter membrane-mediated organoid viral transfection. Step (5) is further included for subsequent culture and recovery: after electroporation, the organoids are eluted from the microporous filter membrane by low-speed centrifugation and resuspended in a culture medium containing 3%-5% matrigel by volume concentration for recovery culture to prevent anoikis; the low-speed centrifugation is 100g±20g for 3-5 minutes.

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