A kit for efficiently infecting organoids with AAV in vitro and a method for infecting organoids

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a kit for efficient in vitro AAV infection of organoids and a method for infecting organoids. The present invention first provides a kit for efficient in vitro AAV infection of organoids, the kit comprising a box body and reagents, the box body comprising a culture support and a blank well; the culture support is detachably suspended on the blank well, and one culture support and one blank well constitute a group of independent culture units. The reagent is a special culture medium for AAV infection; it includes a TLTD infection-promoting system and specific components. The present invention can achieve efficient in vitro infection of organoids by AAV virus, and the organoids maintain a high survival rate.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a kit for efficiently infecting organoids with AAV in vitro and a method for infecting organoids. Background Art

[0002] Continuous advances in biomedical technology and genetic engineering have enabled the development of gene therapy approaches for adeno-associated virus (AAV)-related diseases. AAV gene therapy involves introducing new genes into the human body via viral vectors to treat disease. However, to date, only a few AAV-based gene therapy drugs have been approved for clinical trials. This is primarily due to the potential risks to AAV recipients and insufficient transduction efficiency, leading to the discontinuation of most AAV-based gene therapy approaches before clinical trials. Therefore, further optimization of AAV gene therapy approaches is needed to achieve greater efficacy.

[0003] Organoids are three-dimensional structures derived from tissue-specific cells. Organoids have highly similar structural features, cell structures, and functional properties to autologous tissues. Research on AAV in 3D organoids is still in its infancy. Currently, there are four ways to infect 3D organoids with AAV: (1) Directly adding AAV to the culture medium after digestion and passage of the organoids. Since the organoids are wrapped in matrix gel or hydrogel, AAV has difficulty entering the gel structure, resulting in low infection efficiency and low infection success rate. (2) After mechanically destroying the formed organoids, they are prepared into 2D cells for AAV resuspension infection. Since the use of mechanical force destroys the 3D morphology of the organoids and even causes damage to the organoids, the cell death rate after infection is high and the success rate of organoid infection is low. (3) Digesting the organoids into 2D cells for AAV infection also destroys the 3D morphology of the organoids, resulting in a low success rate of organoid infection. (4) Matrigel-supported planar infection (MSPI) method: This method places organoids on a flat Matrigel layer and then adds AAV solution. Although the normal 3D morphology of the organoids is maintained, AAV fails to fully contact the organoids, resulting in unstable infection efficiency. In addition, this method is time-consuming and cumbersome to prepare, and requires the preparation of a Matrigel platform. Therefore, this method is not suitable for large-scale preparation and use.

[0004] In summary, existing AAV organoid infection methods are generally complex and time-consuming, with low infection efficiency and success rate, as well as organoids that are prone to death. Therefore, developing accurate and easy-to-use AAV gene therapy efficacy evaluation models for preclinical and clinical use, with short culture cycles and efficient delivery, is an urgent challenge in the field of translational medicine. Summary of the Invention

[0005] The present invention aims to provide a kit for efficiently in vitro AAV infection of organoids and a method for efficiently infecting organoids with AAV based on this kit, partially resolving or alleviating the aforementioned deficiencies in the prior art. This kit enables highly efficient viral delivery without disrupting the three-dimensional structure and physiological polarity of organoids, significantly improving gene transduction efficiency while maintaining the activity and survival rate of organoids. The present invention specifically employs the following technical solutions.

[0006] In a first aspect, the present invention provides a kit for efficiently infecting organoids with AAV in vitro.

[0007] A kit for efficiently infecting organoids with AAV in vitro, the kit comprising a box body 100 and reagents, the box body comprising a culture support 110 and a blank well 120; the culture support 110 is detachably suspended on the blank well 120, the length of the culture support 110 being less than the depth of the blank well 120; one culture support 110 and one blank well 120 forming a group of independent culture units;

[0008] The culture support 110 includes a circular support support portion 130 and a plurality of conical culture cavities 140 extending downward, which are larger at the top and smaller at the bottom. The support support portion 130 and the culture cavities 140 are connected by a support cavity 131.

[0009] The reagent is set as a special culture medium for AAV infection; the special culture medium for AAV infection includes a TLTD infection-promoting system and specific components;

[0010] The TLTD infection-promoting system consists of 4-hydroxyethylpiperazineethanesulfonic acid, TAT-HA2 polypeptide, LAH4 polypeptide, THR-FLRFAMIDE polypeptide, teniposide and at least one protein component; the specific component is a low concentration of sodium dehydroacetate; the protein component is human serum albumin or human low-density lipoprotein; and the concentration of the sodium dehydroacetate is less than 100 μg / mL.

[0011] The quasi-cone shape refers to a structure whose overall shape is similar to a cone, but the bottom is relatively flat or even flat, rather than a conical structure.

[0012] Furthermore, each of the bracket support portions 130 extends downwardly into three independent culture chambers 140; each of the culture chambers 140 is composed of an opening 141, a culture chamber body 142 and a bottom 143; the opening 141 is made of polystyrene, and the culture chamber body 142 and the bottom 143 are made of PDMS membrane.

[0013] Furthermore, the material of the bracket support portion 130 is polystyrene; the material of the bracket cavity 131 is PDMS film.

[0014] As a preferred embodiment, the AAV infection-specific culture medium specifically comprises the following components: 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid, 200 µmol / L TAT-HA2 polypeptide, 200 μmol / L LAH4 polypeptide, 0.1 mmol / L THR-FLRFAMIDE polypeptide, 1 μmol / L teniposide, 1% protein component (human serum albumin with a mass concentration of 1% or human low-density lipoprotein with a mass concentration of 1%) and 50 μg / mL sodium dehydroacetate.

[0015] Another aspect of the present invention provides a method for efficiently infecting organoids with AAV in vitro.

[0016] The method for efficiently infecting organoids with AAV virus using the above kit comprises the following steps:

[0017] S01: Cultivating 3D organoids;

[0018] S02: When the cultured organoids reach the developmental maturity required for viral infection, the Matrigel in which the organoids are cultured is digested and then resuspended in organoid culture medium;

[0019] The cultured organoids reach the developmental maturity required for viral infection, including (but not limited to) the appearance of polar structures (e.g., cells arranged in an apical-basal direction, distinct lumen structures, and clear localization of polarity-related proteins (e.g., ZO-1, E-cadherin)), meet preset morphology and volume standards (e.g., a diameter range of 100–400 μm, stable morphology, clear boundaries, dense cell arrangement, and the absence of large amounts of debris or necrotic areas), and reach set expression levels of relevant differentiation or maturation markers (e.g., enhanced expression of albumin, CYP3A4, and HNF4α in liver organoids), and reach a specific culturing time (e.g., liver organoids are typically cultured for at least 10 days); i.e., the Matrigel in which the organoids are cultured is digested and then resuspended in organoid culture medium;

[0020] S03: vertically adding the resuspended organoids from the culture support 110 of the kit, allowing the organoids to naturally fall into the single culture chamber 140;

[0021] S04: AAV is added to the blank well 120 of the kit and mixed with the AAV infection-specific culture medium provided in the kit to form an AAV virus liquid. The culture support 110 containing the organoid is then placed therein so that the AAV virus liquid submerges the organoid. The kit is then placed in a dynamic environment so that the AAV virus infects the organoid. The volume ratio of the added amount of the organoid to the AAV virus liquid is 05-06:1.

[0022] Furthermore, the organoids described in S01 can be derived from human, primate, mouse, pig, dog and other animal tissues, or differentiated from induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC);

[0023] Furthermore, the organoid types described in S01 include but are not limited to intestinal organoids, liver organoids, lung organoids, kidney organoids, pancreatic organoids, etc.;

[0024] Furthermore, the culture conditions in S01 use corresponding three-dimensional matrices (such as Matrigel, BME, etc.) and conventional organoid culture medium formulas according to the organoid type to maintain the stability of organoid morphology.

[0025] Preferably, the volume ratio of the organoid sample volume to the AAV virus solution is 1 / 2.

[0026] Furthermore, the AAV type is not limited, including multiple AAV serotypes (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, etc.).

[0027] Furthermore, mature organoids with polar structures in S02 are usually 100-300 μm in diameter, with a maximum diameter of no more than 400 μm, and have stable morphology, clear boundaries, dense cell arrangement, and no large amounts of debris or necrotic areas.

[0028] Furthermore, the polar structure in S02 means that the cells are arranged in a top-bottom direction, the lumen structure is obvious, and the polarity-related proteins (such as ZO-1 and E-cadherin) are clearly positioned.

[0029] Furthermore, the expression of relevant differentiation or maturation markers reaches the set level, which means that, for example, the expression of albumin, cytochrome P450 enzyme CYP3A4, and hepatic nuclear factor HNF4α in liver organoids is enhanced; for example, the expression of Villin (related to villus structure), MUC2 (goblet cell marker), and Chromogranin A (CHGA, an endocrine cell marker) in intestinal organoids is enhanced; for example, the expression of SPC (SFTPC, a type II alveolar cell marker), AQP5 (a type I alveolar cell marker), and TTF-1 (NKX2.1, a lung development-related transcription factor) in lung organoids is enhanced.

[0030] Furthermore, the working dose of the AAV is expressed as a multiplicity of infection (MOI), and the MOI of the AAV is in the range of 1E+4-1E+10.

[0031] 1E represents a power of 10.

[0032] Furthermore, the kit is placed in a dynamic environment to allow AAV to infect the organoids; the shaking parameters are set to 100-300 r / min; and the shaking time range is set to 1-2 hours.

[0033] As a preference, the shaking parameter is set to 100-200 r / min.

[0034] Furthermore, the shaking temperature range was set to 32-37°C.

[0035] As a preference, the temperature parameter is set to 37°C.

[0036] Furthermore, the AAV virus liquid is mixed with the AAV infection-specific culture medium provided in the kit in advance to form the AAV virus liquid, and is briefly treated at 4° C. for later use (to maintain viral activity).

[0037] Furthermore, after the organoid infection is completed, the organoid is separated from the virus solution by removing the culture scaffold 110 from the kit.

[0038] Beneficial technical effects:

[0039] (1) The present invention provides a novel culture medium for AAV-infected organoids. The culture medium achieves efficient in vitro infection of organoids by AAV virus through the synergistic combination of the TLTD infection-promoting system and the DHA-S component, and the number and size of the organoids are maintained at a good level, thereby proving that the survival rate of the organoids is high. The experiments of the present invention show that although the use of the TLTD infection-promoting system and DHA-S alone can maintain the survival of the organoids at a high level, only the synergistic use of the TLTD infection-promoting system and DHA-S can significantly increase the number of AAV viruses entering the organoids, that is, the synergistic combination of the TLTD infection-promoting system and the DHA-S component achieves efficient infection of AAV. In addition, the present invention also screened the concentration of DHA-S and found that only a low concentration of addition can achieve a positive effect. DHA-S is known to have antibacterial and antifungal properties and is commonly used as an antifungal and preservative additive for food and feed. The present invention innovatively proposes that the addition of DHA-S components to AAV-infected organoids can significantly promote the efficiency of AAV infection. Under the most preferred culture conditions of the present invention, the AAV infection efficiency of the organoids is above 90%. Compared with traditional methods, the efficiency of AAV infection of organoids is significantly improved.

[0040] (2) The present invention also provides a kit for efficiently infecting 3D organoids with AAV. The kit uses a detachable culture scaffold device to culture organoids, so that the three-dimensional structure of the organoids can be suspended in the AAV virus liquid, while maintaining the 3D morphology of the organoids themselves, thereby simulating the in vivo environment, including the interaction between cells, the influence between cells and the surrounding environment, and other spatial morphological issues. The culture scaffold device provided by the present invention increases the contact and material exchange space between the virus liquid and the organoids, thereby improving the efficiency of AAV infection to a certain extent. In addition, this detachable culture scaffold device can also accurately control the start and end time of viral infection, facilitating process-based operations.

[0041] (3) The kit for efficient AAV infection of 3D organoids provided by the present invention has good adaptability and sensitivity, and is suitable for efficient transduction of trace amounts of AAV viral resources. It is particularly suitable for experimental scenarios with limited clinical sample size, scarce viral resources, or small-scale organoid culture. It can achieve high infection efficiency even under conditions of limited viral dose or small infection volume, significantly improving virus utilization.

[0042] (4) Finally, the kit provided by the present invention is simple to assemble and operate, and the infection system it is equipped with is stable and controllable. It is applicable to 3D organoids from a variety of sources (including normal tissue sources and tumor sources), and is compatible with the relevant instruments of 96-well plates available on the market, with good repeatability and adaptability. It is suitable for medium- and high-throughput experimental environments and automated platform applications, providing efficient, stable, and reproducible in vitro technical means for viral vector evaluation, gene function research, and organoid drug screening, and also provides solid experimental support for the development and verification of AAV-related clinical gene therapy strategies. Overall, the method of efficient in vitro AAV virus infection of organoids provided by the present invention significantly alleviates the problems of the existing methods, such as the complexity of the operating procedures, low organoid survival rate, low viral transfection efficiency, difficulty in maintaining the 3D morphology of the organoid itself, and difficulty in culturing suspended organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0044] Figure 1 Bright field and fluorescence images (scale bar, 100 μm) of human normal liver organoids infected with AAV carrying green fluorescent protein (MOI = 1E+5) under different culture medium conditions in one of the embodiments of the present invention;

[0045] Figure 2 In one embodiment of the present invention, the number and size of human normal liver organoids were verified after infection with AAV carrying green fluorescent protein (MOI = 1E+5) under different culture medium conditions;

[0046] Figure 3 In one embodiment of the present invention, the expression of genes after AAV carrying green fluorescent protein (MOI = 1E+5) was infected into normal human liver organoids under different culture medium conditions was verified;

[0047] Figure 4 This is a statistical graph showing the survival rate of normal human liver organoids screened under conditions of adding different amounts of DHA-S in one embodiment of the present invention;

[0048] Figure 5 This is an overall schematic diagram of a kit for efficiently infecting 3D organoids with AAV in one embodiment of the present invention;

[0049] Figure 6 This is a schematic diagram of the structure of a culture support of a kit in one embodiment of the present invention;

[0050] Figure 7 This is a schematic diagram of the structure of a single culture chamber of a culture support in one embodiment of the present invention;

[0051] Figure 8 In one embodiment of the present invention, the survival rate of human normal liver organoids was verified after AAV carrying green fluorescent protein (MOI = 1E+5) was infected into the organoids using different rotation speeds;

[0052] Figure 9 In one embodiment of the present invention, the expression of GFP gene was verified after AAV carrying green fluorescent protein (MOI = 1E+5) was infected into normal human liver organoids at different rotation speeds;

[0053] Figure 10 This is a flow chart of infecting organoids using a kit for efficiently infecting 3D organoids using AAV in one embodiment of the present invention;

[0054] Figure 11 Bright field and fluorescence images of normal human liver organoids infected with AAV carrying green fluorescent protein using a kit for efficiently infecting 3D organoids with AAV in one embodiment of the present invention (scale bar 100 μm);

[0055] Figure 12 Bright field and fluorescence images of human lung cancer organoids infected with AAV carrying green fluorescent protein using a kit for efficiently infecting 3D organoids with AAV in one embodiment of the present invention (scale bar 100 μm);

[0056] Figure 13 Bright field and fluorescence images of human colon cancer organoids infected with AAV carrying green fluorescent protein using a kit for efficiently infecting 3D organoids with AAV in one embodiment of the present invention (scale bar: 100 μm);

[0057] Figure 14 This is a statistical graph of GFP gene expression after AAV carrying green fluorescent protein was infected into human colon cancer organoids using a kit for efficiently infecting 3D organoids with AAV in one embodiment of the present invention;

[0058] Figure 15 Bright field and fluorescence images of human liver organoids infected with AAV carrying green fluorescent protein using conventional methods in one embodiment of the present invention (scale bar: 100 μm).

[0059] Summary of reference numerals:

[0060] The box body 100 , the culture rack 110 , the blank well 120 , the rack support portion 130 , the rack cavity 131 , the culture chamber 140 , the opening 141 , the culture chamber body 142 , and the bottom 143 . DETAILED DESCRIPTION

[0061] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0063] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0064] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0065] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0066] Example 1

[0067] This example provides an example of a kit for efficiently infecting normal human liver organoids with AAV.

[0068] The kit contains basal culture medium, optional nutrient supplements, and specialized medium for AAV infection.

[0069] The basal culture medium is DMEM / F12 basal culture medium.

[0070] Optional nutrient supplements include: serum-free nutrient medium supplement: 3.5 μmol / L β-mercaptoethanol (β-mercaptoethanol), 2 mmol / L L-propionamide-L-glutamine (GlutaMAX), 1× (v / v) B-27 Supplement (B-27 Supplement, 50×), 1× (v / v) N-2 Supplement (N-2 Supplement, 100×), 5 μg / mL human insulin (Human Insulin), 50 ng / mL epidermal growth factor (EGF), 100 ng / mL recombinant human fibroblast growth factor 10 (FGF-10), 16.11 μg / mL putrescine (Putrescine), 6.3 ng / mL progesterone (Progesterone), 5.2 ng / mL selenite (Selenite), and 1% penicillin / streptomycin (P / S).

[0071] Optional nutritional supplements can also include a variety of protein components, such as: 0.05% serum albumin (bovine), 50μg / mL laminin (extracellular matrix protein); organoid recombinant proteins: 100-150ng / mL Wnt-3a supplement, 100ng / mL Noggin protein.

[0072] Optional nutritional supplements can also include organoid culture small molecules, such as: 1 μmol / L 83-01 (ALK5 / TGF-β receptor I kinase inhibitor), 10 μmol / L Y-27632 supplement (ROCK inhibitor), 10 nmol / L gastrin I, 15 nmol / L nicotinamide, 1.25 mmol / L N-acetyl-L-cysteine, and 10 mmol / L forskolin.

[0073] AAV infection-specific medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 200 µmol / L TAT-HA2 peptide, 200 µmol / L LAH4 peptide, 0.1 mmol / L THR-FLRFAMIDE peptide, 1 µmol / L teniposide, and a protein component (e.g., 1% human serum albumin (HSA), and a low-concentration specific component: 50 µg / mL sodium dehydroacetate (DHA-S).

[0074] Experimental results: Adding the TLTD infection-promoting system can promote the infection efficiency of AAV in normal human liver organoids. Adding low-concentration sodium dehydroacetate can promote the growth and development of normal human liver organoids after AAV infection, and at the same time improve the transfection efficiency of AAV. Figure 1-Figure 3 .

[0075] Example 2

[0076] This example provides an example of a kit for efficiently infecting human lung organoids with AAV.

[0077] The kit contains basal culture medium, optional nutrient supplements, and specialized medium for AAV infection.

[0078] The basal culture medium is composed of DMEM / F12 and IMDM culture medium in a 1:1 ratio.

[0079] AAV infection-specific medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 200 μmol / L TAT-HA2 peptide, 200 μmol / L LAH4 peptide, 0.1 mmol / L THR-FLRFAMIDE peptide, 1 μmol / L teniposide, and a protein component (e.g., 1% human low-density lipoprotein (LDL)), and a low-concentration specific component: 50 μg / mL DHA-S.

[0080] Optional nutrient supplements include: Serum-free nutrient medium supplement: 1× (v / v) B-27 supplement, 1× (v / v) N-2 supplement, 3.5 μmol / L β-mercaptoethanol, 2 mmol / L L-propionamide-L-glutamine (GlutaMAX), 200 μmol / L ascorbic acid, 5 μg / mL human insulin, 50 ng / mL epidermal growth factor (EGF), 100 ng / mL recombinant human fibroblast growth factor 10 (FGF-10), 16.11 μg / mL putrescine, 6.3 ng / mL progesterone, 5.2 ng / mL selenite, 100 μmol / L linoleic acid, and 1% penicillin / streptomycin (P / S).

[0081] Optional nutritional supplements can also include various protein components, such as: 0.05% serum albumin (bovine), 50μg / mL fibronectin, 50μg / mL laminin, 150ng / mL Noggin protein.

[0082] Optional nutritional supplements can also include organoid culture small molecules, such as: 10 μmol / L Y-27632 supplement (ROCK inhibitor), 1 μmol / L rosiglitazone, 50 nmol / L TEAD-IN-3 (TEAD transcription factor inhibitor).

[0083] Example 3

[0084] This example provides an example of a kit for efficiently infecting human intestinal organoids (applicable to intestinal organoids such as the small intestine, colon, and large intestine) with AAV.

[0085] The kit contains basal culture medium, optional nutrient supplements, and specialized medium for AAV infection.

[0086] The basal culture medium was DMEM.

[0087] AAV infection-specific culture medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 200 μmol / L TAT-HA2 peptide, 200 μmol / L LAH4 peptide, 0.1 mmol / L THR-FLRFAMIDE peptide, 1 μm / L teniposide, and a protein component (e.g., 1% human serum albumin (HSA)); and a low-concentration specific component: 50 μg / mL DHA-S.

[0088] Optional nutrient supplements include: Serum-free nutrient medium supplement: 1× (v / v) B-27 supplement (B-27Supplement), 1× (v / v) N-2 supplement (N-2 Supplement), 2 mmol / L L-propionamide-L-glutamine (GlutaMAX), 5 μg / mL human insulin (Human Insulin), 50 ng / mL epidermal growth factor (EGF), 100 ng / mL recombinant human fibroblast growth factor 10 (FGF-10), 16.11 μg / mL putrescine (Putrescine), 6.3 ng / mL progesterone (Progesterone), 5.2 ng / mL selenious acid (Selenite), 0.5 μmol / L triiodo-L-thyronine, 100 μmol / L linoleic acid, 50 μmol / LD, L-α-tocopherol, and 1% penicillin / streptomycin (P / S).

[0089] Optional nutritional supplements can also include a variety of protein components, such as: 0.05% serum albumin (bovine), extracellular matrix proteins including 50 μg / mL fibronectin and 20 μg / mL elastin; organoid recombinant proteins: 100 ng / mL R-Spondin 1 supplement, 100 ng / mL Wnt-3a supplement, and 100 ng / mL Noggin protein.

[0090] Optional nutritional supplements can also include organoid culture small molecules, such as: 2 μmol / L 83-01 (ALK5 / TGF-β receptor I kinase inhibitor), 10 μmol / L Y-27632 supplement (ROCK inhibitor), 15 nmol / L gastrin I, and 15 nmol / L nicotinamide.

[0091] Example 4

[0092] This example provides a screening of the addition amount of sodium dehydroacetate (DHA-S), see Table 1.

[0093] Table 1

[0094]

[0095] The results showed that after 24 hours of normal culture of organoids and addition of different concentrations of DHA-S, the addition of low concentrations (less than 100 μg / mL) of DHA-S significantly enhanced the survival rate of organoids, with the highest survival rate reaching 126.05%. However, as the amount of addition gradually increased, the survival rate gradually decreased. Therefore, the present invention selected low concentrations of DHA-S to compound the AAV infection-specific culture medium, and the survival rate results are shown in Figure 2. Figure 4 .

[0096] Example 5

[0097] This example provides a kit and operation example for efficiently infecting organoids with AAV.

[0098] like Figure 5-7 The kit uses a 96-well cell culture plate as a base, with blank wells containing independent 3D organoid culture devices, which are 3D printed and detailed as follows.

[0099] The kit for efficiently infecting organoids with AAV includes a box body 100, reagents, and a cover.

[0100] 1. Basic parameters of 96-well plates (consistent with commercially available ones and in compliance with ANSI / SLAS standards)

[0101] External dimensions: 127.76 mm × 85.48 mm × 14.35 mm, 120 blank wells, single well inner diameter (well mouth): 6.86 ± 0.15 mm, well depth: 10.85 mm, maximum volume of a single well is 360 μL.

[0102] 2. Overall dimensions of the culture scaffold 110

[0103] (1) Bracket support portion 130 (PS material)

[0104] Outer diameter: 5.70 mm, inner diameter: 5.50 mm, ring wall thickness: 0.10 mm. A support cavity 131 and multiple culture chambers 140 extend downward from the support support portion 130. The entire culture support 110 is lower than the depth of the blank wells, for example, set to 10.00 ± 0.05 mm. The diameter of the ring at the top of the support support portion 130 is larger than the diameter of the blank wells.

[0105] (2) The cavity 142 of the culture chamber 140 is made of a 0.4 μm polydimethylsiloxane (PDMS) film.

[0106] The culture chamber 140 is a cone-like structure that is larger at the top and smaller at the bottom. In some preferred embodiments, the culture chamber 140 is provided in three numbers, and the three culture chambers are connected by the support cavity 131 .

[0107] The stent cavity 131 is a cylindrical cavity with an inner diameter smaller than that of the blank hole. A PDMS film is attached to the cavity. The pore size of the film is 0.4 ± 0.05 μm.

[0108] In some preferred embodiments, the three culture chambers are arranged in an equilateral triangle.

[0109] The dimensions of each culture chamber 140 are as follows: the diameter of the opening 141 is approximately 2.5 mm ± 0.05 μm, the diameter of the bottom 143 is approximately 0.8 mm ± 0.05 μm, and the height of the chamber 142 is approximately 4.50 ± 0.05 mm. The cone angle of the culture chamber 140 is 60° ± 5°, with the angle between the main line and the bottom surface being 60° ± 5°.

[0110] In the above structure, the PDMS films are sealed.

[0111] 3. Liquid loading volume

[0112] (1) Culture chamber 110 (for culturing 3D organoids)

[0113] The recommended liquid addition volume is: 70-90μL, preferably: 70μL.

[0114] (2) Blank well 120 (for placing AAV solution)

[0115] The recommended liquid volume is: 140-160 μL, preferably: 140 μL.

[0116] In some optimal embodiments, the amount of organoids loaded is 1 / 2 of the amount of AAV virus solution loaded.

[0117] 4. Installation and coordination

[0118] The culture support 110 is detachably suspended on the blank hole 120 (a gap exists between the bottom 143 and the bottom of the blank hole 120 to allow AAV, nutrients of the culture medium and the solution to pass through), and is covered with a lid (not shown in the figure) and fixed with slight pressure to achieve good isolation of the liquid in the upper chamber (internal culture cavity) / lower chamber (external annular space) without leakage.

[0119] Reagents: The medium is set to be a special medium for AAV infection; the special medium for AAV infection includes a TLTD infection-promoting system and specific components.

[0120] The design of each structural component in the kit can ensure:

[0121] (1) The reasonable distribution of contact between the culture space of the organoid and the viral fluid helps to improve the infection efficiency and the degree of material exchange.

[0122] (2) The design of the culture chamber 140, which is larger at the top and smaller at the bottom, not only enhances the contact between AAV and 3D organoids, but also facilitates the insertion of a 200μL pipette tip and the rapid removal of organoids, making operation convenient.

[0123] (3) The pores of the PDMS membrane allow AAV and nutrients to pass through, but block the 3D organoids from entering the external culture wells.

[0124] Example 6

[0125] This example provides an example of a method for efficiently infecting organoids with AAV.

[0126] The AAV infection-specific culture medium (basal culture medium + TLTD + DHA-S group) of Example 1 was used, and the specific steps were as follows.

[0127] (1) Cultivating 3D organoids using commercially available or common culture media. Those skilled in the art will appreciate that the specific culture media components used can be routinely adjusted depending on the organoid being cultured.

[0128] (2) When the cultured organoids reach the developmental maturity required for viral infection, the Matrigel used to culture the organoids is digested and then resuspended in organoid culture medium.

[0129] (3) Use digestion solution (e.g., Organoid Dissociation Solution, MCE, Catalog No.: HY-K6013; Organoid Digestion Solution, Novomed Medical, Catalog No.: 01K02100) to digest the matrix gel for 2 minutes, resuspend in organoid culture medium, and adjust the density to 2×10 5 / mL; use a pipette to vertically add the resuspended organoids from the culture support of the kit, allowing the organoids to fall naturally into a single culture chamber; each internal chamber contains approximately 3-4 organoids with a diameter of approximately 150-200μm.

[0130] (4) Add AAV virus solution and the AAV infection-specific culture medium provided with the kit to the blank wells of the kit. Then, place the culture scaffold containing the organoids in a liquid environment so that the AAV virus solution immerses the organoids. Then, place the kit in a dynamic environment so that the AAV virus infects the organoids. The volume ratio of the organoid sample to the AAV virus solution is 0.5:1 (the AAV carries a GFP tag). 200 μL of sterile 1× PBS solution can be added to the corresponding blank side wells to prevent evaporation.

[0131] (5) Place the kit from the previous step in a cell culture incubator with an orbital shaker at 37°C and 5% CO2, and shake for infection, preferably at 200 r / min ( Figure 7-8 , while ensuring cell survival and maintaining maximum infection efficiency), for 2 h. Subsequently, cells were added to a new 96-well plate with sterile 1× PBS, the inner ring device was extracted, and immersed in 1× PBS, shaking and washing for 2 min, three times.

[0132] (6) After the organoid infection is completed, the organoids are separated from the virus solution by removing the culture scaffold from the kit (based on the density and growth status of each organ, taking human normal liver organoids as an example, generally 5 wells are collected), and re-inoculated into the matrix gel (48 wells, 25 μL matrix gel / well) for 6-72 hours (taking human normal liver organoids as an example, generally 6 hours). Figure 8-10 .

[0133] The results showed that different shaking rates had a significant impact on the survival rate of organoids and the efficiency of viral infection.

[0134] Example 7

[0135] Referring to the method of Example 6, AAV was efficiently infected with normal human liver organoids.

[0136] The AAV virus concentration was MOI=1E+5.

[0137] The human normal liver organoids infected in this example were observed for GFP using a fluorescence microscope to determine the infection status. As shown in the figure, green fluorescence indicates that AAV infection of the liver 3D organoids was successful. In addition, based on the fluorescence expression, it can be determined that the infection efficiency reached more than 90%, and the 3D morphology of the organoids was preserved. Figure 11 .

[0138] Example 8

[0139] Referring to the method of Example 6, AAV was efficiently infected with human lung cancer organoids.

[0140] The AAV virus concentration was MOI=1E+6.

[0141] The infected lung cancer 3D organoids in this example were observed for GFP using a fluorescence microscope to determine the infection status. As shown in the figure, green fluorescence indicates that AAV infection of the lung 3D organoids was successful. In addition, based on the fluorescence expression, it can be determined that the infection efficiency reached more than 90%, and the 3D morphology of the organoids was preserved. Figure 12 .

[0142] Example 9

[0143] Referring to the method of Example 6, AAV was efficiently infected with human colon cancer organoids.

[0144] The AAV virus concentration was MOI=1E+7.

[0145] The colon cancer 3D organoids infected in this example were observed for GFP using a fluorescence microscope to determine the infection status. As shown in the figure, green fluorescence indicates that AAV infection of the colon cancer 3D organoids is successful. In addition, based on the fluorescence intensity, it can be determined that the infection efficiency is above 95%. Figure 13 .

[0146] Example 10

[0147] This example performs gene expression analysis on human colon cancer 3D organoids infected in Example 8.

[0148] (1) RNA was extracted from 3D colon cancer organoids that were not infected with AAV (negative control group) and 3D colon cancer organoids that were infected with AAV (experimental group). The specific steps are as follows:

[0149] 1) Organoid recovery.

[0150] For the infected normal human liver organoids, aspirate the basal medium from the wells and add 200 μL of cell recovery buffer to each well of the Matrigel-organoid mixture. Place on ice for 20 minutes. Gently pipette each well to resuspend. Transfer each set of organoids to a 15 mL BD tube and centrifuge at 1000 rpm for 5 minutes at 4°C.

[0151] 2) Organoid cleaning.

[0152] Discard the supernatant as much as possible, resuspend in pre-chilled DPBS, and centrifuge at 4°C, 1000 rpm, for 5 min. Repeat 2-3 times.

[0153] 3) Lyse and extract total RNA from organoids.

[0154] Add 1 mL of lysis buffer (Trizol reagent) to the organoids to resuspend them. Transfer the tube to a 1.5 mL RNase-free EP tube. Add 0.2 mL of chloroform to the tube containing the lysate and mix thoroughly on a shaker for 20 seconds. Incubate at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Then, aspirate the upper aqueous phase containing total RNA into a new tube. Avoid touching the organic phase and the intermediate layer, which contain DNA and proteins. Next, add an equal volume of isopropanol to the upper aqueous phase, invert several times to mix, and allow to settle at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. An RNA pellet will be visible at the bottom of the tube. Discard the supernatant and wash the RNA pellet by adding 1 mL of 75% ethanol per mL of Trizol and gently invert to mix. Centrifuge at 12,000 rpm at 4°C for 2 minutes. Discard the liquid, being careful not to discard the RNA pellet. Allow to air dry inverted at room temperature for 5-10 minutes. Add an appropriate amount of DEPC-treated water to dissolve the RNA precipitate. Measure the OD value to quantify the RNA concentration and store at -80°C.

[0155] 4) qPCR

[0156] First-strand cDNA was synthesized using M-MLV reverse transcriptase. Real-time quantitative PCR (qPCR) was performed in a total volume of 20 μL of SYBR Green PCR Master Mix (Roche, Germany). All reactions were performed in duplicate. Figure 14 .

[0157] Comparative Example 1

[0158] This example provides a comparative method for infecting normal human liver 3D organoids with AAV. After the liver organoids are digested and passaged (digested into small cell clusters and single cells consisting of 5-6 cells), AAV at an MOI of 1E+5 is directly added to the culture medium. The organoids are cultured for 3 days. The infected liver 3D organoids in this example are observed for GFP using a fluorescence microscope to determine the infection status. As shown in the figure, no green fluorescence expression indicates that AAV infection of the liver 3D organoids was unsuccessful. Figure 15 .

[0159] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A kit for efficient in vitro AAV infection of organoids, characterized in that: The kit comprises a box body (100) and reagents, wherein the box body comprises a culture support (110) and a blank well (120); the culture support (110) is detachably suspended on the blank well (120), and the length of the culture support (110) is less than the depth of the blank well (120); one culture support (110) and one blank well (120) constitute a group of independent culture units; The culture support (110) comprises a ring-shaped support portion (130) and a plurality of cone-shaped culture cavities (140) extending downward, which are larger at the top and smaller at the bottom. The support portion (130) and the culture cavities (140) are connected via a support cavity (131). The material of the stent support portion (130) is polystyrene; the material of the stent cavity (131) is a PDMS membrane; the pores of the PDMS membrane allow AAV and nutrients to pass through, but block the entry of 3D organoids; The reagent is a special culture medium for AAV infection; the special culture medium for AAV infection includes a TLTD infection-promoting system and specific components; The TLTD infection-promoting system consists of 4-hydroxyethylpiperazineethanesulfonic acid, TAT-HA2 polypeptide, LAH4 polypeptide, THR-FLRFAMIDE polypeptide, teniposide and at least one protein component; the specific component is a low concentration of sodium dehydroacetate; the protein component is human serum albumin or human low-density lipoprotein; and the concentration of the sodium dehydroacetate is less than 100 μg / mL.

2. The kit for efficient in vitro AAV infection of organoids according to claim 1, characterized in that Each of the support brackets (130) extends downwardly to form three independent culture chambers (140); each of the culture chambers (140) is composed of an opening (141), a culture chamber body (142), and a bottom (143); the opening (141) is made of polystyrene, and the culture chamber body (142) and the bottom (143) are made of a PDMS membrane.

3. The kit for efficient in vitro AAV infection of organoids according to claim 1, wherein: The AAV infection-specific culture medium specifically includes the following components: 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid, 200 µmol / L TAT-HA2 polypeptide, 200 μmol / L LAH4 polypeptide, 0.1 mmol / L THR-FLRFAMIDE polypeptide, 1 μmol / L teniposide, 1% protein component and 50 μg / mL sodium dehydroacetate.

4. A method for efficiently infecting organoids with AAV virus using the kit for efficiently infecting organoids with AAV in vitro according to any one of claims 1 to 3, characterized in that: The following steps are involved: S01: Cultivating 3D organoids; S02: When the cultured organoids reach the developmental maturity required for viral infection, the Matrigel in which the organoids are cultured is digested and the organoids are then resuspended in organoid culture medium; S03: vertically adding the resuspended organoids from the culture support (110) of the kit, and allowing the organoids to naturally fall into a single culture chamber (140); S04: AAV virus is added to the blank well (120) of the kit and mixed with the AAV infection-specific culture medium provided in the kit to form an AAV virus liquid. The culture scaffold (110) containing the organoid is then placed therein so that the AAV virus liquid submerges the organoid. The kit is then placed in a dynamic environment so that the AAV virus infects the organoid. The volume ratio of the added amount of the organoid to the AAV virus liquid is 0.5-0.6:

1.

5. The method according to claim 4, wherein The working dose of the AAV is expressed in terms of multiplicity of infection (MOI), and the MOI of the AAV is in the range of 1E+4-1E+10.

6. The method according to claim 4, wherein The kit was placed in a dynamic environment to allow AAV to infect organoids; the shaking parameters were set to 100-300 r / min; and the shaking time range was set to 1-2 h.

7. The method according to claim 6, wherein Set the shaking temperature range to 32-37°C.

8. The method according to claim 4, wherein The AAV and the AAV infection-specific culture medium provided in the kit are mixed in advance to form an AAV virus liquid, and are briefly treated at 4° C. for later use.

9. The method according to claim 4, wherein After the organoid infection is completed, the organoids are separated from the AAV virus solution by removing the culture scaffold (110) from the kit.

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