Human placenta organoid gene intervention method based on adeno-associated virus transfection
By screening for AAV-DJ serotypes and optimizing the cell-to-virus volume ratio and transfection time, the problem of low transfection efficiency in human placental organoids was solved, achieving efficient and stable gene intervention and providing a reliable platform for studying placental development and pregnancy-related diseases.
Patent Information
- Application Number
- CN202511720595.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-02-06
AI Technical Summary
In the existing technology, adeno-associated virus (AAV) has low transfection efficiency and high cytotoxicity in human placental organoids, making it difficult to achieve efficient and controllable gene intervention, and there is a lack of efficient transfection methods suitable for this type of three-dimensional structure.
AAV-DJ was identified as a highly efficient serotype through screening, and parameters such as a cell-to-virus volume ratio of 5:1 and a transfection time of 10 min were optimized to achieve efficient and stable gene intervention.
It achieved a high transfection efficiency of nearly 80% for human placental organoids, maintained the viability and morphology of the organoids, provided a reliable research platform, broke through the limitations of traditional models, and provided a reliable experimental platform for the study of placental development and pregnancy-related diseases.
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Figure CN121472330A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biomedical technology, in particular to a human placenta organoid gene intervention method based on adeno-associated virus transfection. BACKGROUND
[0002] In the study of placental development and pregnancy-related diseases, traditional methods often rely on animal models or immortalized trophoblast cell lines as experimental platforms. Although animal models have certain advantages in terms of overall physiological environment, their species differences prevent them from accurately simulating the unique structural and functional characteristics of human placenta, especially in terms of gene function and signal pathway research. Commonly used in vitro cell models, such as human choriocarcinoma cell lines (e.g., BeWo, JEG-3, etc.), are convenient to culture and operate, but they have lost many characteristics of primary cells after long-term passage, and cannot reproduce the three-dimensional structure of placental villi and cell heterogeneity in vivo, making it difficult to truly reflect the cellular behavior and molecular mechanisms during placental development.
[0003] In recent years, organoid technology has shown good application prospects in the study of multiple tissues and organs as an emerging in vitro model. Human placental organoids can highly simulate the organizational structure, cell types, and partial functional characteristics of human placenta, providing an ideal platform for studying placental development and related diseases. However, current gene function research on placental organoids is still in its infancy, and there is a lack of efficient, stable, and suitable gene intervention methods for this type of three-dimensional structure. Although there have been attempts to use lentivirus, adenovirus, and other vectors for gene transfection in existing technologies, these methods generally have low transfection efficiency, high cytotoxicity, or difficulty in achieving cell type-specific expression in placental organoids, which severely restricts their application in functional genomics research.
[0004] Adeno-associated virus (AAV) is a non-pathogenic viral vector with low immunogenicity, high transfection efficiency, and the ability to infect non-dividing cells, and has been widely used in gene therapy and functional research of various tissues and cell types. However, there are many AAV serotypes, and their tissue tropism differs significantly. Currently, there is no systematic research to determine the AAV serotype suitable for efficient transfection of human placental organoids and its optimal transfection conditions. In addition, the dense structure of placental organoids and the abundance of extracellular matrix also pose technical challenges for effective viral vector delivery. Therefore, developing an AAV-based transfection method for human placental organoids that can achieve efficient and controllable gene intervention has become a key technical problem that needs to be solved in the field. SUMMARY
[0005] The application aims to provide an adeno-associated virus transfection-based human placenta organoid gene intervention method to solve the problems in the prior art.
[0006] To achieve the above-mentioned purpose, the application provides the following solutions.
[0007] The application provides an adeno-associated virus transfection-based human placenta organoid gene intervention method, comprising the following steps.
[0008] (1) Collecting early pregnancy placenta villus cells and constructing primary placenta organoids;
[0009] (2) After digesting the primary placenta organoids into single cells, mixing the single cell suspension of the primary placenta organoids with adeno-associated viruses capable of interfering with target genes for transfection;
[0010] The serum type of the adeno-associated viruses is selected from AAV-6 or AAV-DJ; the mixed volume ratio of the single cell suspension of the primary placenta organoids to the adeno-associated viruses capable of interfering with target genes is 5:1 or 10:1; and the transfection time is 5-10 min.
[0011] Further, the serum type of the adeno-associated viruses is AAV-DJ.
[0012] Further, the mixed volume ratio of the single cell suspension of the primary placenta organoids to the adeno-associated viruses capable of interfering with target genes is 5:1.
[0013] Further, the transfection time is 10 min.
[0014] Further, the transfection temperature is 37℃.
[0015] Further, in step (2), the primary placenta organoids with a diameter of 100-200 μm are digested into single cells after being cultured for 7-10 days.
[0016] Further, the single cells of the primary placenta organoids after transfection are mixed with Matrigel, reseeded and cultured to obtain the placenta organoids after gene intervention.
[0017] The application further provides a gene-intervened human placenta organoid obtained by the human placenta organoid gene intervention method.
[0018] The application also provides application of the genetically intervened human placenta organoid in preparation of a model for researching functions of placenta development related genes or mechanisms of pregnancy related diseases.
[0019] The application also provides application of the genetically intervened human placenta organoid in screening or evaluating intrauterine gene therapy strategies.
[0020] The application discloses the following technical effects:
[0021] The application successfully provides a high-efficiency, stable and high-specificity human placenta organoid gene intervention method, and solves the core problems of low transfection efficiency and difficulty in effective delivery in a three-dimensional structure in the prior art.
[0022] The application first realizes controllable gene operation in an organoid model highly simulating the structure and function of human placenta, breaks through the limitations of traditional two-dimensional cell models and animal models, and provides a reliable and physiological experimental platform for in-depth research on placenta development mechanisms, molecular foundations of pregnancy related diseases and screening of intrauterine gene therapy strategies. BRIEF DESCRIPTION OF DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.
[0024] Figure 1 A schematic diagram of transfecting organoids by different serotypes of adeno-associated viruses;
[0025] Figure 2 Comparison of results of transfecting organoids by different serotypes of adeno-associated viruses; wherein A is a fluorescence microscope graph of transfecting organoids by different serotypes of adeno-associated viruses; B is transfection efficiency of transfecting organoids by different serotypes of adeno-associated viruses; and C is the number of organoids after transfecting organoids by different serotypes of adeno-associated viruses.
[0026] Figure 3Transfection ratio and transfection time comparison of AAV-DJ serotype adeno-associated virus transfected placenta organoids; Wherein, A is the fluorescence microscope graph of the organoid transfected by the AAV-DJ serotype adeno-associated virus; B is the transfection efficiency of the organoid transfected by the AAV-DJ serotype adeno-associated virus; C is the number of organoids after the organoid is transfected by the AAV-DJ serotype adeno-associated virus;
[0027] Figure 4 The growth of the placental organoid after the target gene CLOCK in the placental organoid is knocked out by using the adeno-associated virus transfection method of the application. DETAILED DESCRIPTION
[0028] The detailed description of the various exemplary embodiments of the application is not to be considered as limiting the application, but rather as a description of certain aspects, features, and embodiments of the application.
[0029] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range of values, and any other stated value or stated range of values within the stated range is also included within the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the content of this specification and that of any document incorporated by reference, the content of this specification controls.
[0031] Various modifications and changes can be made to the specific implementation of the present application described in this specification without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.
[0032] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including, but not limited to.
[0033] Abbreviations involved in the present application:
[0034] DMEM: Dulbecco's Modified Eagle Medium, a commonly used cell culture medium.
[0035] F12: Ham's F-12 Nutrient Mixture, another commonly used cell culture medium, often used in combination with DMEM.
[0036] DMEM / F12: A 1:1 mixture of DMEM and F12.
[0037] FBS: Fetal Bovine Serum, fetal bovine serum.
[0038] EDTA: Ethylenediaminetetraacetic acid, a metal ion chelator commonly used in cell digestion solution.
[0039] GFP: Green Fluorescent Protein, a marker protein used to report successful transfection.
[0040] Y-27632: A ROCK (Rho-associated coiled-coil containing protein kinase) inhibitor commonly used to prevent apoptosis in primary cells.
[0041] N-2 (N2 Supplement): A serum replacement additive used for neuronal cell culture, also used in some organoid culture systems.
[0042] B-27 (B27 Supplement): A serum-free culture additive widely used in neuronal and organoid culture.
[0043] Key terms related to the invention:
[0044] Human Placental Organoids: Refers to the micro-tissue formed by human placental cells under three-dimensional (3D) in vitro culture conditions, which can simulate the structure and function of placenta in vivo.
[0045] Transduction: In this invention, it specifically refers to the process of introducing exogenous genes into placental organoid cells using lentivirus as a vector.
[0046] Genetic Intervention: refers to the process of changing the function of a specific gene in a cell through techniques such as gene knockout, gene overexpression, or gene editing.
[0047] Transfection Efficiency: refers to the percentage of cells that successfully introduced exogenous genes and expressed (such as GFP positive) in the total number of cells.
[0048] Example 1 Construction of human placental organoids
[0049] 1. Tissue collection and pretreatment
[0050] Collect placental villus tissue from 8-week-old placental villus tissue derived from social factors induced abortion (belonging to medical waste), and immediately place it in pre-cooled tissue preservation medium. The composition of the tissue preservation medium is: 90wt% DMEM / F12 base medium, 1wt% N-2 additive, 2wt% B-27 additive, 100 μg / mL primary cell antibiotic, and 0.5 μg / mL Y-27632. The tissue is transported to the laboratory at 4°C.
[0051] 2. Enzymatic step digestion
[0052] a. Scrape the placental villus tissue and transfer it to 10 mL of 0.25% Trypsin-0.02% EDTA solution, and digest it in a 37°C metal bath at a speed of 810 rpm for 5-8 min.
[0053] b. Filter the above digestion material using a 100 μm cell filter, collect the filtrate, and immediately terminate the digestion with an equal volume of DMEM / F12 medium containing 20% FBS.
[0054] c. Centrifuge the filtrate at room temperature at a centrifugal force of 600 ×g for 5 min, collect the precipitate, and obtain a primary mixture of placental villus cells and blood cells.
[0055] d. Transfer the incompletely digested tissue remaining on the 100 μm filter to 10 mL of 1.0 mg / mL collagenase V solution prepared from 10% FBS-DMEM / F12, and continue to digest it in a 37°C metal bath at a speed of 810 rpm for 8 min.
[0056] e. Filter this digestion solution again using a 100 μm cell filter, and centrifuge the filtrate at room temperature at a centrifugal force of 600 ×g for 5 min, collect the precipitate, and obtain a secondary mixture of placental villus cells and blood cells.
[0057] 3. Cell purification
[0058] a. Combine the cell pellets obtained in steps c and e above, resuspend with sufficient DMEM / F12 medium and wash once, centrifuge at 600 x g for 5 min at room temperature.
[0059] b. Discard the supernatant, add 5 times volume of red blood cell lysis buffer to the cell pellet, mix gently by pipetting, stand at room temperature for 2 min for lysis, centrifuge at 600 x g for 5 min.
[0060] c. Discard the supernatant after lysis centrifugation, immediately dilute with DMEM / F12 medium, and centrifuge at 600 x g for 5 min at room temperature.
[0061] d. Discard the supernatant, resuspend the cell pellet with DMEM / F12 medium again and wash once, obtain the purified placental villus cells after centrifugation.
[0062] 4. Organoid culture and formation
[0063] a. Count the purified placental villus cells, resuspend the cells with organoid culture medium.
[0064] The specific components of the organoid culture medium include (all percentages are volume percentages): DMEM / F12 medium, 90%; N-2 supplement, 1%; B-27 supplement, 2%; primary cell antibiotic, 100 μg / mL; N-acetyl-L-cysteine, 204 μg / mL; L-glutamine, 292 μg / mL; recombinant human epidermal growth factor, 50 ng / mL; CHIR99021, 698 μg / mL; recombinant human R-spondin 1, 200 ng / mL; recombinant human fibroblast growth factor, 100 ng / mL; recombinant human hepatocyte growth factor, 50 ng / mL; prostaglandin E2, 882 ng / mL; Y-27632, 2.5 μg / mL; bovine serum albumin, 1 μg / mL.
[0065] b. Mix the cell suspension with Matrigel at a volume ratio of 1:3 on ice, avoiding the generation of air bubbles.
[0066] c. Take 30 μL of the mixture and drop it in the center of the well of a 48-well cell culture plate to form a gel droplet. Place the culture plate in a 37°C incubator, invert and culture for 3 min, then upright and continue to stand for 10-15 min to allow the Matrigel to completely solidify.
[0067] d. After gelation is complete, carefully add 300 pL organoid culture medium per well. Place the culture plates in an incubator at 37 °C, 5% CO2, and change the fresh medium every 2-3 days.
[0068] e. After about 8 days of culture, three-dimensional placental organoids with a diameter of about 100-200 pm and compact structure can be observed under a microscope.
[0069] Example 2 Optimization of AAV viral transfection of human placental organoids
[0070] 1. Digestion of organoids into single cells
[0071] a. Select primary placental organoids that have been cultured for 7-10 days, have good morphology, and have a diameter of 100-200 pm for transfection. Discard the old culture medium, and add 400 pL pre-cooled DMEM / F12 to each well, and gently blow to disperse the droplets. Collect the organoid-containing suspension into a 1.5 mL centrifuge tube. Repeat this operation once to ensure complete collection.
[0072] b. Centrifuge the collected organoid suspension at 600 x g for 5 min at 4 °C, and discard the supernatant.
[0073] c. To completely digest the organoids, add 400 pL of 0.25% Trypsin-0.02% EDTA solution preheated to 37 °C to the cell pellet, and gently and thoroughly resuspend using a 1 mL pipette.
[0074] d. Place the centrifuge tube in a 37 °C incubator for 5 min.
[0075] e. After incubation, immediately add 1 mL of DMEM / F12 to stop the digestion, and centrifuge at 600 x g for 5 min at 4 °C to obtain a single cell pellet.
[0076] f. Resuspend the cells with an appropriate amount of DMEM / F12 and count them, adjust the cell concentration, and prepare a cell suspension with a concentration of 2 x 10 5 -6 x 10 5 cells / mL for standby use.
[0077] 2. AAV transfection and parameter optimization
[0078] a. Serum type screening: Take equal amounts of the above cell suspension, and mix with different serum types of recombinant adeno-associated viruses (including AAV-2, AAV-5, AAV-6, AAV-8, AAV-9, and AAV-DJ). Set the volume ratio of cell suspension to virus to be 10:1, and mix and incubate at 37 °C for 10 min. After incubation, remove the unbound virus by centrifugation, and resuspend with culture medium.
[0079] b. Transfection time and ratio optimization: AAV-DJ serotype was selected, and different transfection conditions were set. The volume ratio of cell suspension to virus was tested at 5:1, 10:1 and 20:1, respectively, and at each ratio, three transfection incubation times of 5 min, 10 min and 20 min were set (all at 37°C).
[0080] 3. Post-transfection culture and efficiency evaluation
[0081] a. After completing virus incubation, centrifuge all cell-virus mixtures at 500 x g for 5 min, and carefully remove the supernatant.
[0082] b. Resuspend the cell pellet with 1 mL of fresh DMEM / F12 medium, and centrifuge at 600 x g for 5 min to obtain the transfected cells.
[0083] c. Count the cells and adjust the concentration to 2 x 10 5 -6 x 10 5 / mL to obtain a cell suspension.
[0084] d. Mix the cell suspension with Matrigel at a mass ratio of 1:3 on ice, take 30 μL and inoculate in a 48-well plate, and follow the operation of Part 4 of Example 1 to solidify and culture.
[0085] e. After 48-72 h of transfection, observe and take photos of the bright field and green fluorescence (GFP) images of each group of organoids using a fluorescence inverted microscope. Calculate the transfection efficiency by counting the ratio of the number of GFP-expressing organoids to the total number of organoids.
[0086] 4. Results
[0087] In order to explore the effect of specific genes on placental function and uterine function, gene intervention on organoids is an important step to realize the research of gene function. The present application completes the gene intervention of human placental organoids. First, green fluorescent gene is carried by adeno-associated virus to transfect human placental organoids, and the expression of green fluorescent gene is observed to determine the success of gene transfer ( Figure 1 ). As shown in Figure 2 , it can be seen from the figure that among the different serotypes of adeno-associated virus AAV-2, AAV-5, AAV-6, AAV-8, AAV-9 and AAV-DJ for transfection of placental organoids, the green fluorescence expression of AAV-DJ transfected organoids is the most, indicating the highest transfection efficiency ( Figure 2 ); in addition, different serotypes have a certain effect on the growth of organoids, such as Figure 2As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. Figure 2 As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest.
[0088] The transfection time of the adeno-associated virus was 5 min, 10 min and 20 min, and the transfection of different cell-virus volume ratios was compared. As shown in FIG. 8A, Figure 3 Figure 3 As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. Figure 3 As shown in FIG. 9B, the transfection efficiency of the cell-virus volume ratio 5:1 was the highest, reaching nearly 80%, and the transfection efficiency of the cell-virus volume ratio 20:1 was the lowest, being about 50%. In terms of the transfection time, the transfection efficiency of the cell-virus volume ratio 5:1 was the highest when the transfection time was 20 min, but the transfection efficiency of the cell-virus volume ratio 5:1 was the lowest when the transfection time was 20 min. Figure 3 As shown in FIG. 9C, the transfection time of 20 min led to a decrease in the cell viability of most cells, which significantly affected the number of spheroids of the organoids. Therefore, the cell-virus volume ratio 5:1 and the transfection time of 10 min can achieve a better transfection effect.
[0089] As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. Figure 4 As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. Figure 4 As shown in FIG. 9A, the number of spheroids of the AAV-9 group was the largest, followed by the AAV-5, AAV-6 and AAV-9 groups, and the number of spheroids of the AAV-2 and AAV-8 groups was the smallest. However, in terms of morphology, the number of spheroids of the AAV-9 group was the largest, but in terms of the number of cells, the number of spheroids of the AAV-2 and AAV-8 groups was the smallest.
[0090] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art should fall within the protection scope of the present application.
Claims
1. A method for gene intervention in human placental organoids based on adeno-associated virus transfection, characterized in that, Includes the following steps: (1) Collect placental villus cells from early pregnancy and construct primary placental organoids; (2) After digesting the primary placental organoids into single cells, the single-cell suspension of the primary placental organoids is mixed with an adeno-associated virus that can interfere with the target gene for transfection. The adeno-associated virus serotype is selected from AAV-6 or AAV-DJ; the mixing volume ratio of the primary placental organoid single-cell suspension to the adeno-associated virus capable of intervening in the target gene is 5:1 or 10:1; and the transfection time is 5-10 min.
2. The method for gene intervention in human placental organoids according to claim 1, characterized in that, The serotype of the adeno-associated virus is AAV-DJ.
3. The method for gene intervention in human placental organoids according to claim 1, characterized in that, The volume ratio of the single-cell suspension of the primary placental organoid to the adeno-associated virus capable of intervening in the target gene is 5:
1.
4. The method for gene intervention in human placental organoids according to claim 1, characterized in that, The transfection time was 10 minutes.
5. The method for gene intervention in human placental organoids according to claim 1, characterized in that, The transfection temperature was 37°C.
6. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (2), primary placental organoids cultured for 7-10 days and with a diameter between 100-200 μm are digested into single cells.
7. The method for gene intervention in human placental organoids according to claim 1, characterized in that, Transfected primary placental organoids were mixed with matrix gel, re-inoculated, and cultured to obtain genetically modified placental organoids.
8. A gene-interventional human placental organoid obtained by the gene intervention method for human placental organoids as described in any one of claims 1-7.
9. The application of the gene-interventional human placental organoid as described in claim 8 in the preparation of models for studying the function of placental development-related genes or the mechanisms of pregnancy-related diseases.
10. The use of a genetically modified human placental organoid as described in claim 8 in screening or evaluating intrauterine gene therapy strategies.