Kit and method of infection for efficient infection of aav 3d organoids

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a kit for efficient AAV infection of 3D organoids and an infection method. The present application first provides a kit for efficient AAV infection of organoids in vitro, which comprises a box body and reagents, wherein the reagents comprise a basic culture medium configured for 3D organoid culture, optional nutritional supplements for promoting growth of the 3D organoids, and an AAV infection special culture medium. The AAV infection special culture medium comprises a TLTD infection-promoting system and specific components. The present application can achieve efficient AAV in vitro infection of organoids, and the organoids maintain a relatively high survival rate. In addition, the kit provided by the present application is simple to assemble and operate, the infection system provided is stable and controllable, and is suitable for infection of 3D organoids of various sources.

Description

[0001] Divisional application

[0002] This application is a divisional application of Chinese Patent Application No. 2025107757970, filed on June 11, 2025, entitled "A reagent kit and method for infecting organoids with highly efficient in vitro AAV". Technical Field

[0003] This invention belongs to the field of biomedical technology, specifically relating to a kit and infection method for AAV to efficiently infect 3D organoids. Background Technology

[0004] Adeno-associated virus (AAV) gene therapy is a method of treating diseases by introducing new genes into the human body through AAV viral vectors. However, to date, only a few AAV-based gene therapy drugs have been approved for clinical trials. This is mainly because most current AAV gene therapy drugs have potential risks to subjects using AAV vectors and insufficient transduction efficiency, leading to their discontinuation before clinical trials. Therefore, further optimization of AAV gene therapy methods is needed to achieve better efficacy.

[0005] Organoids are three-dimensional structures derived from tissue-specific cells. They share highly similar structural features and functional properties with autologous tissues. Research on AAVs in 3D organoids is still in its early stages. Currently, there are four methods for AAV infection of 3D organoids: (1) AAVs are directly added to the culture medium after organoid digestion and passage. Since the organoids are encapsulated by matrix gel or hydrogel, AAVs have 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 organoid infection success rate is low. (3) Organoids are digested into 2D cells for AAV infection. This also destroys the 3D morphology of the organoids, resulting in a low organoid infection success rate. (4) Matrigel-supported planar infection (MSPI) method: This method places organoids on a flat Matrigel layer and then adds AAV solution. Although it maintains the normal 3D morphology of organoids, AAV fails to make sufficient contact with 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.

[0006] In summary, existing methods for AAV-infected organoids generally suffer from problems such as complex and time-consuming procedures, low infection efficiency, low success rates, and easy organoid death. Therefore, developing an accurate, easy-to-operate, short-cycle, and highly efficient AAV gene therapy efficacy evaluation model for preclinical and clinical applications is an urgent problem to be solved in the field of translational medicine. Summary of the Invention

[0007] The purpose of this invention is to provide a kit and method for efficiently infecting organoids with AAV, partially solving or alleviating the aforementioned shortcomings of the prior art. This method achieves highly efficient virus delivery while minimizing damage to the three-dimensional structure and physiological polarity of the organoids, significantly improving gene transduction efficiency, and maintaining the activity and survival rate of the organoids. The specific technical solution adopted in this invention is as follows.

[0008] In a first aspect, the present invention provides a kit for efficiently in vitro infecting organoids with AAV.

[0009] A kit for AAV infection of 3D organoids, the kit comprising a housing 100 and reagents, the housing comprising a culture scaffold 110 and blank wells 120; the culture scaffold 110 is detachably suspended on the blank wells 120, the length of the culture scaffold 110 being less than the depth of the blank wells 120; one culture scaffold 110 and one blank well 120 constitute a set of independent culture units;

[0010] The culture scaffold 110 includes an annular scaffold 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 scaffold support portion 130 and the culture cavities 140 are connected by a scaffold cavity 131.

[0011] The scaffold cavity 131 is a porous membrane structure. The pores of the membrane structure allow AAV and nutrients to pass through, but block the entry of 3D organoids.

[0012] The reagents include a basal culture medium for 3D organoid culture, optional nutritional supplements to promote the growth of 3D organoids, and a special culture medium for AAV infection.

[0013] The AAV infection-specific culture medium includes a TLTD infection-promoting system and a specific component; the TLTD infection-promoting system consists of 4-hydroxyethylpiperazine ethanesulfonic acid, TAT-HA2 peptide, LAH4 peptide, THR-FLRFAMIDE peptide, 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; the concentration of sodium dehydroacetate is less than 100 μg / mL;

[0014] The basal culture medium includes DMEM / F12 basal culture medium, a mixture of DMEM / F12 basal culture medium and IMDM culture medium, or DMEM culture medium.

[0015] The THR-FLRFAMIDE polypeptide is a trade name, and its sequence is H-Thr-Phe-Leu-Arg-Phe-NH2.

[0016] The "TLTD" in the TLTD pro-infection system described in this invention is an abbreviation for the components TAT-HA2 polypeptide (T), LAH4 polypeptide (L), THR-FLRFAMIDE polypeptide (T), and teniposide (D).

[0017] In some specific embodiments, the ratio of DMEM / F12 basal medium to IMDM medium is 1:1.

[0018] The term "conical shape" refers to a structure whose overall shape resembles a cone, but with a relatively gentle or even flat bottom, rather than a cone-shaped structure.

[0019] Furthermore, the optional nutritional supplement for promoting 3D organoid growth comprises one or more ingredients selected from the group consisting of β-mercaptoethanol, L-propionamide-L-glutamine, B-27 supplement, N-2 supplement, human insulin, epidermal growth factor, recombinant human fibroblast growth factor 10, putrescine, progesterone, selenite, linoleic acid, penicillin / streptomycin, A83-01, Y-27632 supplement, gastrin I, nicotinamide, N-acetyl-L-cysteine, trichomoniasis, ascorbic acid, rosiglitazone, TEAD-IN-3, triiodo-L-thyroxine, and D,L-α-tocopherol.

[0020] Furthermore, the optional nutritional supplement for promoting 3D organoid growth comprises one or more ingredients selected from the group consisting of serum albumin, laminin, Noggin, fibronectin, elastin, R-Spondin 1 supplement and Wnt-3a supplement.

[0021] Furthermore, each of the support portions 130 extends downward into three independent culture chambers 140; each culture chamber 140 consists 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.

[0022] Furthermore, the support portion 130 is made of polystyrene; the cavity 131 is made of PDMS membrane.

[0023] Preferably, the AAV infection-specific culture medium comprises the following components: 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, 200 µmol / L TAT-HA2 peptide, 200 μmol / L LAH4 peptide, 0.1 mmol / L THR-FLRFAMIDE peptide, 1 μmol / L teniposide, 1% protein component (1% human serum albumin or 1% human low-density lipoprotein), and 50 μg / mL sodium dehydroacetate.

[0024] In another aspect, the present invention provides a method for efficiently infecting organoids with AAV in vitro.

[0025] The method for infecting 3D organoids with AAV using the above-described kit for AAV-infected organoids includes the following steps:

[0026] S01: Use the basal culture medium and optional nutrient supplements in the kit to culture organoids, and select a suitable three-dimensional matrix (such as Matrigel, BME, etc.) for the type of organoid to maintain the 3D morphological stability of the organoids.

[0027] S02: Once the cultured organoids have reached the developmental maturity required for viral infection, the cultured organoids are digested using the matrix gel and then resuspended in organoid culture medium; the diameter of the mature organoids is 100-400 μm.

[0028] S03: The resuspended organoid is vertically added from the culture scaffold 110 of the kit, allowing the organoid to fall naturally into the single culture chamber 140;

[0029] S04: Add AAV to the blank well 120 of the kit and mix it with the AAV infection-specific culture medium in the kit to form a viral solution. Then, place the culture scaffold 110 containing organoids into the scaffold to immerse the organoids in the viral solution. Then, place the kit in a dynamic environment to infect the organoids with AAV. The volume ratio of the organoids to the viral solution is 0.5-0.6:1.

[0030] S05: After organoid infection is completed, the organoids are separated from the viral fluid by removing the culture scaffold 110 from the kit.

[0031] Furthermore, the working dose of the AAV is expressed in terms of the multiplicity of infection (MOI), which ranges from 1E+4 to 1E+10.

[0032] 1E represents 10 raised to the power of 10.

[0033] Furthermore, the kit was placed in a dynamic environment to allow AAV to infect organoids; the shaking parameters were set to 100-300 r / min; the shaking temperature range was set to 32-37℃; and the shaking time was set to 1-2 h.

[0034] Furthermore, the organoids are normal human organoids, including intestinal organoids, liver organoids, lung organoids, kidney organoids, or pancreatic organoids.

[0035] Those skilled in the art can choose a combination of optional nutritional supplements to achieve the goal of cultivating mature organoids, depending on the type of organoid they wish to cultivate.

[0036] In the above method, the cultured organoids reach the developmental maturity required for viral infection, including (but not limited to) the presence of polar structures (e.g., cells arranged in a top-to-bottom direction, obvious cavity structures, and clear localization of polar-related proteins (e.g., ZO-1, E-cadherin), morphology and volume meeting preset standards (e.g., diameter range of 100–400 μm, stable morphology, clear boundaries, dense cell arrangement, and no large amount of debris or necrotic areas), expression of relevant differentiation or maturation markers reaching a set level (e.g., enhanced expression of Albumin, CYP3A4, HNF4α, etc. in liver organoids), and culture time reaching a specific node (e.g., liver organoids are usually cultured for no less than 10 days); that is, the matrix gel of the cultured organoids is digested and then resuspended in organoid culture medium.

[0037] Furthermore, the organoids described in S01 may be derived from animal tissues such as those of humans, primates, mice, pigs, and dogs, or obtained by differentiation from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0038] As a preferred embodiment, the volume ratio of the organoid sample to the AAV virus solution is 1 / 2.

[0039] Furthermore, the type of AAV is not limited, including multiple AAV serotypes (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, etc.).

[0040] Furthermore, mature organoids with polar structures in S02 typically have a diameter of 100-300 μm, with a maximum of no more than 400 μm, and are morphologically stable, clearly defined, with densely arranged cells and no large amount of debris or necrotic areas.

[0041] Furthermore, the presence of polar structures in SO2 refers to the cells being arranged in a top-to-bottom direction, with distinct cavity structures and clearly localized polar-related proteins (such as ZO-1 and E-cadherin).

[0042] Furthermore, the expression of relevant differentiation or maturation markers reaching the set level refers to enhanced expression of markers such as albumin, cytochrome P450 enzyme CYP3A4, and hepatic nuclear factor HNF4α in liver organoids; enhanced expression of markers such as Villin (villi structure-related), MUC2 (goblet cell marker), and Chromogranin A (CHGA, endocrine cell marker) in intestinal organoids; and enhanced expression of markers such as SPC (SFTPC, type II alveolar cell marker), AQP5 (type I alveolar cell marker), and TTF-1 (NKX2.1, lung development-related transcription factor) in lung organoids.

[0043] As a preferred option, the shaking parameter is set to 100-200 r / min.

[0044] Furthermore, the shaking temperature range is set to 32-37℃.

[0045] As a preferred option, the temperature parameter is set to 37℃.

[0046] Furthermore, the AAV virus solution is pre-mixed with the AAV infection-specific culture medium provided in the kit to form the AAV virus solution, and then briefly treated at 4°C for later use (to maintain virus activity).

[0047] Beneficial technical effects:

[0048] (1) This invention provides a specialized culture medium that can promote efficient AAV infection of organoids and maintain organoid viability. This specialized culture medium, through the synergistic effect of the TLTD infection-promoting system and the DHA-S component, achieves efficient in vitro infection of organoids by AAV virus, maintaining the number and size of organoids at a good level, thereby achieving high organoid survival. Experiments of this invention show that although both the TLTD infection-promoting system and DHA-S alone can maintain organoid survival 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 effect of the TLTD infection-promoting system and the DHA-S component achieves efficient AAV infection. Furthermore, this invention screened the concentration of DHA-S and found that only low concentrations achieved a positive effect. DHA-S is known to have antibacterial and antifungal properties and is commonly used as an antifungal and preservative additive in food and feed. This invention innovatively proposes that adding DHA-S to AAV-infected organoids can significantly promote AAV infection efficiency. Under the optimal culture conditions of this invention, the AAV infection efficiency of organoids is above 90%. This represents a significant improvement in AAV infection efficiency compared to traditional methods.

[0049] (2) This invention also provides a kit for highly efficient AAV infection of 3D organoids. This kit uses a detachable culture scaffold to culture organoids, allowing the three-dimensional organoids to suspend in the AAV viral solution while maintaining their 3D morphology. This simulates the in vivo environment, including cell-to-cell interactions and the spatial morphology of the cells and their surroundings. The culture scaffold provided by this invention increases the contact and material exchange space between the viral solution and the organoids, thereby improving AAV infection efficiency to a certain extent. Furthermore, this detachable culture scaffold allows for precise control of the start and end times of viral infection, facilitating streamlined operations.

[0050] (3) The kit for high-efficiency AAV infection of 3D organoids provided by this invention has good adaptability and sensitivity, and is suitable for efficient transduction of trace amounts of AAV viral resources. It is especially suitable for experimental scenarios with limited clinical sample volume, 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, and significantly improve viral utilization.

[0051] (4) Finally, the kit provided by this invention is simple to assemble and operate, and the equipped infection system is stable and controllable. It is suitable for 3D organoids from various sources (including those from normal tissues and tumors) and is compatible with commercially available 96-well plate instruments, exhibiting good reproducibility and adaptability. It is suitable for medium- and high-throughput experimental environments and automated platform applications, providing efficient, stable, and reproducible in vitro techniques for viral vector evaluation, gene function research, and organoid drug screening. It also provides solid experimental support for the development and validation of AAV-related clinical gene therapy strategies. Overall, the method for efficient in vitro AAV virus infection of organoids provided by this invention significantly alleviates the problems of complex operating procedures, low organoid survival rate, low viral transfection efficiency, difficulty in maintaining the 3D morphology of the organoids themselves, and the difficulty in culturing suspended organoids in existing methods. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0053] Figure 1 Bright field and fluorescence images (scale bar 100 μm) of AAV (MOI=1E+5) carrying green fluorescent protein infected with normal human liver organoids under different culture conditions in one embodiment of the present invention.

[0054] Figure 2 In one embodiment of the present invention, the number and size of organoids after infection of normal human liver organoids with AAV (MOI=1E+5) carrying green fluorescent protein were verified under different culture conditions.

[0055] Figure 3 In one embodiment of the present invention, the expression of genes after AAV (MOI=1E+5) carrying green fluorescent protein was infected into normal human liver organoids was verified under different culture conditions.

[0056] Figure 4 This is a statistical chart showing the survival rate of normal human liver organoids under different DHA-S addition conditions in one embodiment of the present invention.

[0057] Figure 5 This is a schematic diagram of the overall kit for highly efficient AAV infection of 3D organoids in one embodiment of the present invention;

[0058] Figure 6 This is a schematic diagram of the culture scaffold of the reagent kit in one embodiment of the present invention;

[0059] Figure 7 This is a schematic diagram of a single culture chamber structure of a culture scaffold in one embodiment of the present invention;

[0060] Figure 8 In one embodiment of the present invention, the survival rate of organoids after infecting normal human liver organoids with AAV carrying green fluorescent protein (MOI=1E+5) at different rotation speeds was verified.

[0061] Figure 9 In one embodiment of the present invention, the expression of the GFP gene was verified after AAV carrying green fluorescent protein (MOI=1E+5) was infected into normal human liver organoids using different rotation speeds.

[0062] Figure 10 This is a flowchart illustrating the use of an AAV-based kit for efficiently infecting 3D organoids in one embodiment of the present invention.

[0063] Figure 11 Bright field and fluorescence images (scale bar 100 μm) of AAV carrying green fluorescent protein infected into normal human liver organoids using a kit for high-efficiency AAV infection of 3D organoids in one embodiment of the present invention.

[0064] Figure 12 Bright field and fluorescence images (scale bar 100 μm) of human lung cancer organoids infected with AAV carrying green fluorescent protein using a kit for high-efficiency infection of 3D organoids by AAV in one embodiment of the present invention.

[0065] Figure 13 Bright field and fluorescence images (scale bar 100 μm) of AAV carrying green fluorescent protein infected with human colon cancer organoids using a kit for high-efficiency AAV infection of 3D organoids in one embodiment of the present invention.

[0066] Figure 14 This is a statistical graph of GFP gene expression after human colon cancer organoids are infected with AAV carrying green fluorescent protein using a kit for efficient AAV infection of 3D organoids in one embodiment of the present invention.

[0067] Figure 15 This is a bright-field image and fluorescence image (scale bar 100 μm) of human liver organoids infected with AAV carrying green fluorescent protein using conventional methods in one embodiment of the present invention.

[0068] Summary of attached labeling and identification:

[0069] Box body 100, culture support 110, blank hole 120, support support part 130, support cavity 131, culture cavity 140, opening part 141, culture cavity cavity 142, bottom 143. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0071] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0072] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0073] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0074] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having 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., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0075] Example 1

[0076] This embodiment provides an example of a kit for the efficient infection of normal human liver organoids by AAV.

[0077] This kit contains basal culture medium, optional nutritional supplements, and AAV infection-specific culture medium.

[0078] The basal culture medium was DMEM / F12 basal culture medium.

[0079] Optional nutritional supplements include: serum-free nutrient culture medium supplements: 3.5 μmol / L β-mercaptoethanol, 2 mmol / L L-propionamide-L-glutamine (GlutaMAX), 1× (v / v) B-27 supplement (50×), 1× (v / v) N-2 supplement (100×), 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, and 1% penicillin / streptomycin (P / S).

[0080] Optional nutritional supplements may also include various protein components, such as: 0.05% serum albumin (bovine), 50 μg / mL laminin (extracellular matrix protein); organoid recombinant proteins: 100-150 ng / mL Wnt-3a supplement, 100 ng / mL Noggin protein.

[0081] Optional nutritional supplements may also include organoid culture small molecules, such as: 1 μmol / L LA 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.

[0082] AAV infection-specific culture medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic 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).

[0083] Experimental results: Adding the TLTD infection-promoting system can enhance the infection efficiency of AAV in normal human liver organoids; adding low concentrations of sodium dehydroacetate can promote the growth and development of normal human liver organoids after AAV infection, while also improving AAV transfection efficiency; see [link to results]. Figures 1-3 .

[0084] Example 2

[0085] This embodiment provides an example of a kit for highly efficient AAV infection of human lung organoids.

[0086] This kit contains basal culture medium, optional nutritional supplements, and AAV infection-specific culture medium.

[0087] The basal culture medium is composed of DMEM / F12 and IMDM culture medium in a 1:1 ratio.

[0088] AAV infection-specific culture medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic 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).

[0089] Optional nutritional supplements include: serum-free nutrient medium supplements: 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).

[0090] Optional nutritional supplements may also include various protein components, such as: 0.05% serum albumin (bovine), 50 μg / mL fibronectin, 50 μg / mL laminin, and 150 ng / mL Noggin protein.

[0091] Optional nutritional supplements may also include organoid culture small molecules, such as: 10 μmol / L Y-27632 supplement (ROCK inhibitor), 1 μmol / L rosiglitazone, and 50 nmol / L TEAD-IN-3 (TEAD transcription factor inhibitor).

[0092] Example 3

[0093] This embodiment provides an example of a kit for highly efficient AAV infection of human intestinal organoids (applicable to intestinal organoids such as small intestine, colon, and large intestine).

[0094] This kit contains basal culture medium, optional nutritional supplements, and AAV infection-specific culture medium.

[0095] The basal culture medium is DMEM.

[0096] AAV infection-specific culture medium: TLTD infection-promoting system: 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic 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.

[0097] Optional nutritional supplements include: serum-free nutrient culture medium supplements: 1× (v / v) B-27 Supplement, 1× (v / v) N-2 Supplement, 2 mmol / L L-propionamide-L-glutamine (GlutaMAX), 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, 0.5 μmol / L Triiodo-L-thyroxine, 100 μmol / L Linoleic Acid, 50 μmol / L L-α-Tocopherol, and 1% Penicillin / Streptomycin (P / S).

[0098] Optional nutritional supplements may also include a variety of protein components, such as: 0.05% serum albumin (bovine), extracellular matrix proteins including a combination of 50 μg / mL fibronectin and 20 μg / mL elastin; organoid recombinant proteins: a combination of 100 ng / mL LR-Spondin 1 supplement, 100 ng / mL Wnt-3a supplement, and 100 ng / mL Noggin protein.

[0099] Optional nutritional supplements may also include organoid culture small molecules, such as: 2 μmol / L LA 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.

[0100] Example 4

[0101] This embodiment provides a screening of the amount of sodium dehydroacetate (DHA-S) added, as shown in Table 1.

[0102] Table 1

[0103]

[0104] The results showed that adding a low concentration (less than 100 μg / mL) of DHA-S significantly enhanced the survival rate of organoids, with a maximum survival rate reaching 126.05%. However, the survival rate gradually decreased as the added amount increased. Therefore, this invention selected a low concentration of DHA-S to formulate a special culture medium for AAV infection. The survival rate results are shown in […]. Figure 4.

[0105] Example 5

[0106] This embodiment provides a kit for highly efficient AAV infection of organoids and an operational example.

[0107] like Figure 5-7 As shown. The kit uses 96-well plates for cell culture as the base blank wells, with independent 3D organoid culture devices inside each well, which are 3D printed, as detailed below.

[0108] The AAV high-efficiency organoid infection kit includes a box 100, reagents, and a cap.

[0109] 1. Basic parameters of 96-well plates (consistent with commercially available products, compliant with ANSI / SLAS specifications)

[0110] External dimensions: 127.76 mm × 85.48 mm × 14.35 mm, blank hole 120 single hole inner diameter (orifice): 6.86 ± 0.15 mm, hole depth: 10.85 mm, single hole maximum volume is 360μL.

[0111] 2. Overall dimensions of the culture scaffold 110

[0112] (1) Bracket support part 130 (PS material)

[0113] Outer diameter: 5.70 mm, inner diameter: 5.50 mm, annular wall thickness: 0.10 mm. A scaffold cavity 131 and multiple culture chambers 140 extend downwards from the scaffold support portion 130. The overall height of the culture scaffold 110 is less than the depth of the blank well, for example, set to: 10.00 ±0.05 mm. The diameter of the top annular ring of the scaffold support portion 130 is larger than the diameter of the blank well.

[0114] (2) The cavity 142 of the culture chamber 140 is made of a 0.4 μm polydimethylsiloxane (PDMS) film.

[0115] The culture chamber 140 has a conical structure that is larger at the top and smaller at the bottom. In some preferred embodiments, there are three culture chambers 140 connected by a support cavity 131.

[0116] The scaffold cavity 131 is a cylindrical cavity with an inner diameter smaller than that of the blank pore; a PDMS film is attached inside the cavity; the membrane pore size is 0.4 ± 0.05 μm.

[0117] In some preferred embodiments, the three culture chambers are arranged in an equilateral triangle.

[0118] 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° at the apex and 60° ± 5° at the angle between the generatrix and the bottom surface.

[0119] In the above structure, the PDMS membranes are sealed together.

[0120] 3. Liquid loading capacity

[0121] (1) Culture chamber 110 (for culturing 3D organoids)

[0122] The recommended volume of liquid added is 70-90 μL, preferably 70 μL.

[0123] (2) Blank well 120 (for placing AAV solution)

[0124] The recommended volume of liquid added is 140-160 μL, preferably 140 μL.

[0125] In some preferred embodiments, the volume of organoids added is half the volume of AAV viral solution added.

[0126] 4. Installation and coordination

[0127] The culture support 110 is detachably suspended on the blank hole 120 (the bottom 143 and the bottom of the blank hole 120 have a gap to allow AAV, nutrients and solutions of the culture medium to pass through). The cover (not shown in the figure) is then placed on top and slightly pressed to fix it, so as to achieve good isolation of the liquid in the upper chamber (internal culture chamber) / lower chamber (external annular space) and prevent leakage.

[0128] Reagents: The culture medium is set as a special medium for AAV infection; the special medium for AAV infection includes the TLTD infection-promoting system and specific components.

[0129] The design of each structural component in the reagent kit ensures that:

[0130] (1) A reasonable distribution of the culture space of organoids and the contact with the virus fluid helps to improve infection efficiency and the degree of material exchange.

[0131] (2) The 140-degree culture chamber design, 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 the operation convenient.

[0132] (3) The pores of the PDMS membrane allow AAV and nutrients to pass through, but block 3D organoids from entering the external culture wells.

[0133] Example 6

[0134] This embodiment provides an example of a method for efficiently infecting organoids with AAV.

[0135] The AAV infection-specific culture medium (basal culture medium + TLTD + DHA-S group) of Example 1 was used, and the specific steps are as follows.

[0136] (1) 3D organoids are cultured using commercially available or common culture media. Those skilled in the art will understand that the specific composition of the culture medium used can be routinely adjusted depending on the organoids being cultured.

[0137] (2) When the cultured organoids reach the developmental maturity required for viral infection, the matrix gel of the cultured organoids is digested and then resuspended in organoid culture medium.

[0138] (3) Digest the matrix gel using digestive solutions (e.g., Organoid Dissociation Solution, MCE, catalog number: HY-K6013; Organoid Digestive Solution, Novartis Medical, catalog number: 01K02100) for 2 minutes, then resuspend in organoid culture medium and adjust the density to 2 × 10⁻⁶. 5 Organoids per mL; use a pipette to vertically add the resuspended organoids from the culture scaffold of the kit, allowing the organoids to fall naturally into individual culture chambers; each chamber should contain approximately 3-4 organoids with a diameter of approximately 150-200 μm.

[0139] (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 organoids into a liquid environment to immerse the organoids in the AAV virus solution. Subsequently, place the kit in a dynamic environment to infect the organoids with AAV virus. The volume ratio of the organoids added to the AAV virus solution is 0.5:1 (the AAV is tagged with GFP). 200 μL of sterile 1×PBS solution can be added to the corresponding blank wells to prevent evaporation.

[0140] (5) Place the reagent kit from the previous step into a cell culture incubator equipped with a fixed-track shaker at 37°C and 5% CO2, and shake for infection, preferably at 200 r / min. Figure 7-8 To ensure cell viability and maintain maximum infection efficiency, the process was repeated for 2 hours. Subsequently, sterile 1×PBS for cell preparation was added to a new 96-well plate, the inner loop device was extracted, immersed in 1×PBS, and washed by shaking for 2 minutes, 3 times.

[0141] (6) After organoid infection is completed, the organoids are separated from the virus solution by removing the culture scaffold from the kit (based on density and growth status of various organs; for example, 5 wells of normal human liver organoids are typically collected), and then re-inoculated into a matrix gel (48 wells, 25 μL of matrix gel / well) for 6-72 hours of culture (for example, 6 hours for normal human liver organoids). See Figure 8-10 .

[0142] The results showed that different shaking rates had a significant impact on the survival rate of organoids and the efficiency of viral infection.

[0143] Example 7

[0144] The method described in Example 6 enables AAV to efficiently infect normal human liver organoids.

[0145] The concentration of AAV virus is MOI=1E+5.

[0146] The infection status of the normal human liver organoids infected in this embodiment was determined by observing GFP under a fluorescence microscope. As shown in the figure, green fluorescence indicates successful AAV infection of the 3D liver organoids. Furthermore, based on fluorescence expression, the infection efficiency was determined to be over 90%, and the 3D morphology of the organoids was preserved. Figure 11 .

[0147] Example 8

[0148] The method described in Example 6 enables AAV to efficiently infect human lung cancer organoids.

[0149] The concentration of AAV virus is MOI=1E+6.

[0150] The infection status of the successfully infected 3D lung cancer organoids in this embodiment was determined by observing GFP using a fluorescence microscope. As shown in the figure, green fluorescence indicates successful AAV infection of the 3D lung organoids. Furthermore, based on fluorescence expression, the infection efficiency was determined to be over 90%, and the 3D morphology of the organoids was preserved. Figure 12 .

[0151] Example 9

[0152] The method described in Example 6 enables AAV to efficiently infect human colon cancer organoids.

[0153] The concentration of AAV virus is MOI=1E+7.

[0154] The infection status of the colon cancer 3D organoids infected in this embodiment was determined by observing GFP using a fluorescence microscope. As shown in the figure, green fluorescence indicates successful AAV infection of the colon cancer 3D organoids. Furthermore, based on the fluorescence intensity, the infection efficiency can be determined to be above 95%. Figure 13.

[0155] Example 10

[0156] This embodiment analyzes the gene expression of human colon cancer 3D organoids after infection in Example 8.

[0157] (1) RNA was extracted from AAV-infected colon cancer 3D organoids (negative control group) and AAV-infected colon cancer 3D organoids (experimental group). The specific steps are as follows:

[0158] 1) Organoid recycling.

[0159] After infection, remove the basal culture medium from the normal human liver organoid tubes and add cell recovery buffer (200 μL / well) to each well in the mixture of matrix gel and organoids. Place on ice for 20 min. After gently resuspending each well by pipetting, transfer each group of organoids to a 15 mL BD tube and centrifuge at 4°C, 1000 rpm, for 5 min.

[0160] 2) Organoid cleaning.

[0161] Discard the supernatant if possible, resuspend in pre-cooled DPBS, centrifuge at 4°C, 1000 rpm, for 5 min. Repeat 2-3 times.

[0162] 3) Lysis and extraction of total RNA from organoids.

[0163] Resuspend the organoids in 1 mL of lysis buffer (Trizol reagent) and transfer to a 1.5 mL RNase-free EP tube. Add 0.2 mL of chloroform to the centrifuge tube containing the lysate, vortex thoroughly for 20 seconds, and incubate at room temperature for 5 minutes. Centrifuge at 12000 rpm at 4°C for 10 minutes, then transfer the upper aqueous phase containing total RNA to a new centrifuge tube. Avoid contact with the organic phase and intermediate layer, which contain DNA and protein. Add an equal volume of isopropanol to the upper aqueous phase, invert several times to mix, and incubate at room temperature for 5 minutes. Centrifuge at 12000 rpm at 4°C for 15 minutes; RNA precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 mL of 75% ethanol per mL of Trizol, and gently invert to mix to wash away the RNA precipitate. Centrifuge at 12000 rpm at 4°C for 2 minutes, discard the liquid, being careful not to discard the RNA precipitate. Invert the tube to air dry at room temperature for 5-10 minutes. Add an appropriate amount of DEPC-treated water to dissolve the RNA precipitate. After measuring the OD value to quantify the RNA concentration, store at -80℃.

[0164] 4) qPCR

[0165] First-strand cDNA was synthesized using M-MLV reverse transcriptase. Real-time quantitative PCR (qPCR) was performed in 20 μL of SYBR Green PCRMaster Mix (Roche, Germany). All reactions were performed in duplicate. See [link to relevant documentation] Figure 14 .

[0166] Comparative Example 1

[0167] This example provides a conventional method for infecting normal human liver 3D organoids with AAV. The method involves digesting and passaged the liver organoids (digesting them into small cell clusters of 5-6 cells and single cells), then directly adding AAV with an MOI of 1E+5 to the culture medium. The organoids are cultured for another 3 days. The infected liver 3D organoids are then observed using a fluorescence microscope to determine the GFP level. As shown in the figure, the absence of green fluorescence indicates that AAV infection of the liver 3D organoids was unsuccessful. See [link to example]. Figure 15 .

[0168] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A kit for AAV in vitro infection of 3D organoids, characterized in that, The kit includes a box (100) and reagents. The box includes a culture scaffold (110) and a blank well (120). The culture scaffold (110) is detachably suspended on the blank well (120). The length of the culture scaffold (110) is less than the depth of the blank well (120). One culture scaffold (110) and one blank well (120) constitute a set of independent culture units. The culture scaffold (110) includes an annular scaffold support (130) and a plurality of conical culture cavities (140) extending downward, which are larger at the top and smaller at the bottom. The scaffold support (130) and the culture cavities (140) are connected by a scaffold cavity (131). The scaffold cavity (131) is a porous membrane structure. The pores of the membrane structure allow AAV and nutrients to pass through, but block the entry of 3D organoids. The reagents include a basal culture medium for 3D organoid culture, optional nutritional supplements to promote the growth of 3D organoids, and a special culture medium for AAV infection. The AAV infection-specific culture medium includes a TLTD infection-promoting system and a specific component; the TLTD infection-promoting system consists of 4-hydroxyethylpiperazine ethanesulfonic acid, TAT-HA2 peptide, LAH4 peptide, THR-FLRFAMIDE peptide, 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; the concentration of sodium dehydroacetate is less than 100 μg / mL; The basal culture medium includes DMEM / F12 basal culture medium, a mixture of DMEM / F12 basal culture medium and IMDM culture medium, or DMEM culture medium.

2. The kit for AAV infection of 3D organoids as described in claim 1, characterized in that, The optional nutritional supplements for promoting 3D organoid growth comprise one or more ingredients selected from the group consisting of β-mercaptoethanol, L-propionamide-L-glutamine, B-27 supplement, N-2 supplement, human insulin, epidermal growth factor, recombinant human fibroblast growth factor 10, putrescine, progesterone, selenite, linoleic acid, penicillin / streptomycin, A83-01, Y-27632 supplement, gastrin I, nicotinamide, N-acetyl-L-cysteine, trichomoniasis, ascorbic acid, rosiglitazone, TEAD-IN-3, triiodo-L-thyroxine, and D,L-α-tocopherol.

3. The kit for AAV infection of 3D organoids as described in claim 1, characterized in that, The optional nutritional supplements for promoting 3D organoid growth comprise one or more ingredients selected from the group consisting of serum albumin, laminin, Noggin, fibronectin, elastin, R-Spondin 1 supplements, and Wnt-3a supplements.

4. The kit for AAV infection of 3D organoids as described in claim 1, characterized in that, Each of the support portions (130) extends downward into three independent culture chambers (140); each culture chamber (140) consists of an opening (141), a culture chamber body (142), and a bottom (143); the opening portion (141) is made of polystyrene, and the culture chamber body (142) and the bottom (143) are made of PDMS membrane.

5. The kit for AAV infection of 3D organoids as described in claim 1, characterized in that, The support portion (130) of the stent is made of polystyrene; the cavity of the stent (131) is made of PDMS membrane.

6. The kit for AAV infection of 3D organoids as described in claim 1, characterized in that, The AAV infection-specific culture medium comprises the following components: 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, 200 µmol / L TAT-HA2 peptide, 200 μmol / L LAH4 peptide, 0.1 mmol / L THR-FLRFAMIDE peptide, 1 μmol / L teniposide, 1% protein component, and 50 μg / mL sodium dehydroacetate.

7. A method for infecting organoids with AAV using the kit for in vitro infection of 3D organoids with AAV according to any one of claims 1-6, characterized in that, Includes the following steps: S01: Use the basal culture medium and optional nutrient supplements in the kit to culture organoids, and select a three-dimensional matrix suitable for the type of organoid to maintain the 3D morphological stability of the organoids; S02: Once the cultured organoids have reached the developmental maturity required for viral infection, the cultured organoids are digested using the matrix gel and then resuspended in organoid culture medium; the diameter of the mature organoids is 100-400 μm. S03: The resuspended organoid is vertically added from the culture scaffold (110) of the kit, allowing the organoid to fall naturally into a single culture chamber (140); S04: Add AAV to the blank well (120) of the kit and mix it with the AAV infection-specific culture medium in the kit to form a viral solution. Then, place the culture scaffold (110) containing organoids into the scaffold to immerse the organoids in the viral solution. Then, place the kit in a dynamic environment to infect the organoids with AAV. The volume ratio of the organoids added to the viral solution is 0.5-0.6:

1. S05: After organoid infection is completed, the organoids are separated from the viral fluid by removing the culture scaffold (110) from the kit.

8. The method as described in claim 7, characterized in that, The working dose of the AAV is expressed in terms of the multiplicity of infection, which ranges from 1E+4 to 1E+10.

9. The method as described in claim 7, characterized in that, Place the kit in a dynamic environment to infect organoids with AAV; set the shaking parameters to 100-300 r / min; set the shaking temperature range to 32-37℃; and set the shaking time to 1-2 h.

10. The method as described in claim 7, characterized in that, The organoids mentioned are normal human organoids, including intestinal organoids, liver organoids, lung organoids, kidney organoids, or pancreatic organoids.

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