Human placenta organoid gene intervention method based on lentivirus transfection

Abstract

The invention discloses a human placenta organoid gene intervention method based on lentivirus transfection, and belongs to the technical field of biological medicine. According to the method, key parameters of lentivirus transfection, namely the volume ratio of cell suspension to lentivirus to DMEM (Dulbecco Modified Eagle Medium) of 10: 2: 40 and the optimal transfection time of 4 hours, are optimized and determined, so that high transfection efficiency is ensured, the adverse effect of the virus on the activity of the organoid is remarkably reduced, and good organoid balling capacity and normal growth state are maintained. According to the invention, stable and controllable gene intervention is realized in a three-dimensional organoid model which highly simulates the structure and function of a human placenta for the first time, and the obtained placenta organoid subjected to gene intervention has a great significance for deeply researching the molecular mechanism of placenta development in a highly bionic in-vitro environment and exploring the pathogenesis of pregnancy-related diseases such as preeclampsia and the like. And an ideal research platform which is reliable and close to the physiological state is provided by screening or evaluating intrauterine gene therapy strategies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for gene intervention of human placental organoids based on lentivirus transfection. Background Technology

[0002] The human placenta, a key organ at the maternal-fetal interface during pregnancy, plays a crucial role in maintaining pregnancy and fetal health through normal development. Abnormal placental function is closely associated with various pregnancy complications such as preeclampsia and fetal growth restriction. Currently, the main models for studying placental development and related diseases include animal models and in vitro cultured trophoblast cell lines. However, animal models exhibit physiological differences between species, making it difficult to fully simulate the structure and function of the human placenta; while traditional trophoblast cell lines are prone to phenotypic drift during long-term passage and lack the three-dimensional structure and cellular heterogeneity of in vivo placental tissue, limiting their application value in mechanistic studies.

[0003] In recent years, human placental organoids have emerged as a novel in vitro model, effectively mimicking the three-dimensional structure and cellular composition of placental tissue, providing a platform closer to physiological conditions for studying placental development and disease mechanisms. However, current methods for genetic manipulation of placental organoids are still immature, particularly in achieving efficient and stable gene knockout or overexpression. Commonly used transfection methods, such as liposome transfection and electrotransfection, are inefficient in organoids and easily damage cell viability, making it difficult to achieve effective gene intervention while maintaining the three-dimensional structure of the organoid.

[0004] Lentiviral transfection, a technique capable of infecting non-dividing cells and achieving stable integration of exogenous genes, is theoretically applicable to gene modification of organoids. However, in practice, lentiviral transfection conditions (such as viral titer, infection time, and cell state) significantly affect transfection efficiency and organoid survival. Currently, there are no reports on lentiviral transfection systems for human placental organoids, and a systematic approach to optimizing transfection parameters and evaluating the subsequent growth status of organoids is lacking. Therefore, developing an efficient and low-toxicity lentiviral transfection method suitable for human placental organoids has become a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a gene intervention method for human placental organoids based on lentivirus transfection, thereby addressing the problems existing in the prior art. This invention provides an efficient, stable, and low-toxicity lentivirus transfection method by optimizing the volume ratio of cell suspension, lentivirus, and culture medium, as well as the transfection time. This method achieves efficient gene intervention while maintaining the three-dimensional structure and viability of human placental organoids, providing a reliable experimental platform for studying placental development mechanisms and pregnancy-related diseases.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for gene intervention in human placental organoids based on lentivirus transfection, comprising the following steps:

[0008] (1) Collect placental villus cells from early pregnancy and construct primary placental organoids;

[0009] (2) Lentiviral packaging and concentration were carried out using packaging cell lines to obtain high-titer lentiviruses;

[0010] (3) After digesting the primary placental organoids obtained in step (1) into single cells, a single cell suspension is obtained and transfected using the high-titer lentivirus obtained in step (2) to achieve gene intervention.

[0011] In step (3), the volume ratio of the single-cell suspension, the high-titer lentivirus, and DMEM during transfection is 10:1:40 or 10:2:40; the incubation time for transfection is 2-6 hours.

[0012] Furthermore, in step (3), the volume ratio of the single-cell suspension, the high-titer lentivirus, and DMEM during transfection is 10:2:40.

[0013] Furthermore, in step (3), the incubation time for transfection is 4 hours.

[0014] Furthermore, in step (3), the incubation temperature for transfection is 37°C.

[0015] Furthermore, in step (2), the packaging cell line is 293T cells, and the viral plasmids used include plasmids containing Gag / Pol function, plasmids containing Rev function, plasmids containing VSV-G envelope protein, and target gene transfer plasmids. The mass ratio of the Gag / Pol function plasmid, the Rev function plasmid, the VSV-G envelope protein plasmid, and the target gene transfer plasmid is 1:1:1:4.

[0016] Furthermore, after the lentivirus is packaged, it is transfected with a transfection reagent for 20 minutes, and the viral supernatant is collected at 24 h and 48 h after transfection. The high-titer lentivirus is obtained by ultracentrifugation and concentration.

[0017] Furthermore, in step (3), the primary placental organoids cultured for 7-10 days and with a diameter between 100-200 μm are digested into single cells.

[0018] The present invention also provides a human placental organoid obtained by the aforementioned human placental organoid gene intervention method after gene intervention.

[0019] The present invention also provides an application of the gene-interventional human placental organoids described above in the preparation of models for studying the function of placental development-related genes or the mechanisms of pregnancy-related diseases.

[0020] The present invention also provides the application of the aforementioned gene-interventional human placental organoids in screening or evaluating intrauterine gene therapy strategies.

[0021] The present invention discloses the following technical effects:

[0022] This invention successfully provides a highly efficient, stable method for gene intervention in human placental organoids with minimal impact on cell viability. It systematically solves the core problem in existing technologies—low efficiency and difficulty in achieving effective gene manipulation while maintaining three-dimensional structure—due to the lack of targeted transfection systems. By optimizing and establishing key parameters for lentiviral transfection—a volume ratio of cell suspension, lentivirus, and DMEM of 10:2:40, and an optimal transfection time of 4 hours—high transfection efficiency is ensured while significantly reducing the adverse effects of the virus on organoid viability, maintaining good organoid spheroidization ability and normal growth.

[0023] This invention marks the first time that stable and controllable gene intervention has been achieved in a three-dimensional organoid model that highly simulates the structure and function of the human placenta, overcoming the limitations of traditional two-dimensional trophoblast cell lines and animal models with significant species differences. The resulting gene-interventional placental organoids provide a reliable and physiologically close research platform for in-depth research into the molecular mechanisms of placental development, exploring the pathogenesis of pregnancy-related diseases such as preeclampsia, and screening or evaluating intrauterine gene therapy strategies in a highly biomimetic in vitro environment. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A schematic diagram illustrating lentivirus packaging and transfection of placental organoids;

[0026] Figure 2 The cell survival rate after lentivirus transfection at different times with different virus ratios;

[0027] Figure 3The images show the organoid transfection results at different time points with different viral ratios; where A is a fluorescence micrograph of organoids transfected with lentivirus at different time points with different viral ratios; B is the spheroidization rate of organoids transfected with lentivirus at different time points with different viral ratios; and C is the transfection efficiency of organoids transfected with lentivirus at different time points with different viral ratios.

[0028] Figure 4 To investigate the growth of placental organoids after knocking out a target gene using lentiviral transfection, the following data were collected: A shows single-cell sequencing analysis of placental cell differentiation potential (CytoTRACE), cell phenotype, and target gene (GNAS) expression; B shows a dimensionality reduction map based on UMAP (Uniform Manifold Approximation and Projection) illustrating the expression distribution of the CLOCK gene in placental organoid cells; C shows a morphological observation of placental organoids, comparing the differences in organoid growth and fluorescence expression between CLOCK gene knockout (CLOCK-KO) and control (CON) groups using bright-field and fluorescence (GFP) imaging. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Abbreviations used in this invention:

[0035] DMEM: Dulbecco's Modified Eagle Medium, a commonly used cell culture medium.

[0036] F12: Ham's F-12 Nutrient Mixture, another commonly used cell culture medium, often used in combination with DMEM.

[0037] DMEM / F12: A 1:1 mixture of DMEM and F12 in culture medium.

[0038] FBS: Fetal Bovine Serum.

[0039] PBS: Phosphate-Buffered Saline.

[0040] EDTA: Ethylenediaminetetraacetic acid, a metal ion chelating agent commonly used in cell digestion solutions.

[0041] GFP: Green Fluorescent Protein, a marker protein used to report successful transfection.

[0042] scRNA-seq: Single-Cell RNA Sequencing.

[0043] CTBs: Cytotrophoblasts.

[0044] Y-27632: A ROCK (Rho-associated coiled-coil containing proteinkinase) inhibitor, commonly used to prevent primary cell apoptosis.

[0045] N2 Supplement: A serum replacement additive used in neuronal cell culture, and also used in some organoid culture systems.

[0046] B27 Supplement: A serum-free culture additive widely used in neuron and organoid culture.

[0047] Polybrene: a cationic polymer commonly used to improve lentivirus transfection efficiency.

[0048] Puromycin: An antibiotic used to screen cells that have been successfully transfected with a virus carrying a resistance gene (such as the puromycin resistance gene).

[0049] Key terms used in this invention:

[0050] Human placental organoids: These are miniature tissues formed by in vitro three-dimensional (3D) culture of cells derived from the human placenta, which can mimic the structure and function of the placenta in vivo.

[0051] Lentivirus: A type of retroviral virus that, after genetic engineering, can be used as a vector to introduce foreign genes into cells. It can infect dividing and non-dividing cells and achieve long-term stable gene expression.

[0052] Transduction: In this invention, it specifically refers to the process of introducing exogenous genes into placental organoid cells using lentiviruses as vectors.

[0053] Genetic intervention refers to the act of altering the function of specific genes in cells through techniques such as gene knockout, gene overexpression, or gene editing.

[0054] Virus Packaging: refers to the process of co-transfecting lentiviral vector plasmids and packaging plasmids (such as pMD2.G, psPAX2, etc.) into packaging cells such as 293T to produce infectious viral particles.

[0055] Virus concentration refers to the process of enriching virus particles in a viral stock solution using methods such as ultracentrifugation to obtain a viral suspension with a higher titer.

[0056] Transfection efficiency: refers to the percentage of cells that successfully introduce and express foreign genes (such as GFP positive) out of the total number of cells.

[0057] Sphere Formation Rate (Efficiency): This refers to the percentage of digested single cells that can successfully form organoid spheres in three-dimensional culture. It is an important indicator for measuring the survival and proliferation capacity of organoids.

[0058] Pseudotemporal Trajectory Analysis: A computational method based on scRNA-seq data used to infer dynamic changes in cells along developmental processes or differentiation pathways.

[0059] Example 1: Construction of human placental organoids

[0060] 1. Tissue collection and preprocessing

[0061] Placental villus tissue from 8-week-old fetuses was collected from placental villus tissue derived from miscarriages due to social factors (classified as medical waste) and immediately placed in pre-cooled tissue preservation medium. The tissue preservation medium consisted of 90 wt% DMEM / F12 basal medium, 1 wt% N-2 additive, 2 wt% B-27 additive, 100 μg / mL primary cell antibiotic, and 0.5 μg / mL Y-27632. The tissues were transported to the laboratory at 4°C.

[0062] 2. Stepwise enzyme digestion

[0063] a. Scrape the placental villi tissue and transfer it to 10 mL of a solution containing 0.25% Trypsin and 0.02% EDTA. Digest the solution in a metal bath at 37°C at 810 rpm for 5-8 min.

[0064] b. Filter the digested 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.

[0065] c. Centrifuge the filtrate at 600 × g for 5 min at room temperature, collect the precipitate, and obtain a primary mixture of placental villus cells and blood cells.

[0066] d. Transfer the incompletely digested tissue remaining on the 100 μm filter to 10 mL of a 1.0 mg / mL collagenase V solution prepared with 10% FBS-DMEM / F12, and continue digestion in a 37°C metal bath at 810 rpm for 8 min.

[0067] e. Filter the digestion solution again using a 100 μm cell filter. Centrifuge the filtrate at 600 × g for 5 min at room temperature, collect the precipitate, and obtain a mixture of secondary placental villus cells and blood cells.

[0068] 3. Cell purification

[0069] a. Combine the cell pellets obtained in steps c and e above, resuspend them in sufficient DMEM / F12 medium and wash once, then centrifuge at 600 × g for 5 min at room temperature.

[0070] b. Discard the supernatant, add 5 times the volume of red blood cell lysis buffer to the cell pellet, gently pipette to mix, incubate at room temperature for 2 min for lysis, and centrifuge at 600 ×g for 5 min.

[0071] c. After lysis and centrifugation, discard the supernatant, immediately add DMEM / F12 medium for dilution, and centrifuge at 600 ×g for 5 min at room temperature.

[0072] d. Discard the supernatant, resuspend the cells in DMEM / F12 medium, wash the cell pellet once, and centrifuge to obtain purified placental villus cells.

[0073] 4. Organoid Culture and Formation

[0074] a. Count the purified placental villus cells and resuspend the cells in organoid culture medium.

[0075] The specific components of the culture medium (all percentages are by volume) are as follows: DMEM / F12 medium, 90%; with N-2 additive, 1%; B-27 additive, 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-vertebral protein 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; fetal bovine serum albumin, 1 μg / mL.

[0076] b. Mix the cell suspension and matrix gel at a volume ratio of 1:3 on ice until homogeneous, avoiding the formation of air bubbles.

[0077] c. Take 30 μL of the mixture and drop it into the center of each well in a 48-well cell culture plate to form a gel droplet. Place the culture plate in a 37°C incubator and incubate upside down for 3 min, then invert it and let it stand for another 15 min to allow the matrix gel to completely solidify.

[0078] d. After solidification, carefully add 300 μL of organoid culture medium to each well. Incubate the culture plate in a 37°C, 5% CO2 incubator, replacing the medium with fresh medium every 2-3 days.

[0079] e. After about 8 days of culture, three-dimensional placental organoids with a diameter of about 100-200 μm and a dense structure can be observed under a microscope.

[0080] Example 2 Lentiviral Packaging and Concentration

[0081] 1. Preparation of packaging cells

[0082] 293T cells were cultured in DMEM medium containing 10% FBS. Transfection was performed when the cells reached 60-70% confluence in 10 cm culture dishes.

[0083] 2. Plasmid transfection

[0084] Prepare a viral packaging plasmid mixture by mixing plasmids containing Gag / Pol function, Rev function, VSV-G envelope protein, and target gene transfer plasmid at a mass ratio of 1:1:1:4. Mix the total plasmid mixture with an appropriate amount of transfection reagent LIPO3.0 in serum-free DMEM and incubate at room temperature for 20 min to form the transfection complex.

[0085] 3. Virus production

[0086] The transfection complex was added dropwise to the 293T cells that had been replaced with serum-free medium. After incubation at 37°C for 8 h, the medium containing the transfection complex was discarded and replaced with fresh DMEM medium containing 10% FBS.

[0087] 4. Virus collection and concentration

[0088] Cell supernatants containing lentiviral particles were collected at 24 h and 48 h post-transfection. The collected supernatants were filtered through a 0.45 μm filter to remove cell debris. The filtrate was transferred to 50 mL ultracentrifuge tubes and centrifuged at 4500 × g for 10 min at 4 °C. Most of the supernatant was discarded, and the bottom 200 μL of concentrated virus solution was retained, aliquoted, and stored at -80 °C for later use.

[0089] Example 3 Optimization of Lentiviral Transfection

[0090] 1. Organoids are digested into single cells.

[0091] a. Select primary placental organoids cultured for 7-10 days with good morphology and a diameter between 100-200 μm for transfection. Discard the old culture medium, add 400 μL of pre-cooled DMEM / F12 to each well, gently pipette to disperse the droplets, and collect the suspension containing the organoids into a 1.5 mL centrifuge tube. Repeat this operation once to ensure complete collection.

[0092] b. Centrifuge the collected organoid suspension at 600 ×g for 5 min at 4°C and discard the supernatant.

[0093] c. To thoroughly digest the organoids, add 400 μL of 0.25% Trypsin-0.02% EDTA solution preheated to 37°C to the cell pellet and gently and thoroughly resuspend by pipetting with a 1 mL pipette.

[0094] d. Incubate the centrifuge tubes in a 37°C incubator for 5 min.

[0095] e. After incubation, immediately add 1 mL of DMEM / F12 to terminate digestion, and centrifuge at 600 ×g for 5 min at 4°C to obtain a single-cell pellet.

[0096] f. Resuspend cells in an appropriate amount of DMEM / F12 and count them. Adjust the cell concentration to prepare a cell concentration of 1×10⁻⁶. 6 -2×10 6 Prepare a cell suspension of cells / mL for later use.

[0097] 2. Optimization of transfection conditions

[0098] Different transfection conditions were set to determine the optimal parameters.

[0099] a. Virus ratio: Set two virus addition ratios:

[0100] (I) Cell suspension:lentivirus:DMEM volume ratio = 10:1:40;

[0101] (II) Cell suspension: Lentiviral: DMEM volume ratio = 10:2:40.

[0102] b. Transfection time: For each virus proportion group, four transfection time points were set: 2 h, 4 h, 6 h and 12 h.

[0103] c. Transfection process: Polybrene was added to each transfection system to a final concentration of 5 μg / mL to improve infection efficiency. The cell-virus mixture was incubated at 37°C for the corresponding set times (2 h, 4 h, 6 h, and 12 h).

[0104] 3. Post-transfection treatment and culture

[0105] a. After incubation, add 1 mL of DMEM / F12 to each transfection system, mix by pipetting, centrifuge at 600 ×g for 5 min, and discard the supernatant containing the virus.

[0106] b. Resuspend the cell pellet in DMEM / F12 and wash again by centrifugation.

[0107] c. Count the cells and adjust the cell density to 2 × 10⁻⁶. 5 -6×10 5 Cells / mL were used to obtain a cell suspension.

[0108] d. Mix the cell suspension and matrix gel at a volume ratio of 1:3 on ice and resuspend the cells.

[0109] e. Seed 30 μL of cell-Matrix gel mixture in each well of a 48-well plate and fix the gel as described in Part 4 of Example 1.

[0110] f. After solidification, add 250-300 μL of fresh organoid culture medium to each well and incubate at 37°C.

[0111] 4. Results

[0112] Figure 1 This is a schematic diagram of lentivirus packaging and organoid transfection, illustrating the lentivirus packaging system and the specific process of organoid transfection used in this invention.

[0113] Figure 2 Cell survival was assessed at different lentiviral transfection times (2 h, 4 h, 6 h, and 12 h). Trypan blue staining results showed that the cell death rate was close to 100% after 12 h of treatment, indicating that lentiviral intervention time exceeding 12 h had a significant adverse effect on cell survival.

[0114] Figure 3 The effects of different lentivirus transfection times (2 h, 4 h and 6 h) on organoid transfection efficiency were demonstrated. Figure 3 A represents the morphology of placental organoids observed under a bright field microscope. It is evident that the organoid has a spherical structure and that the green fluorescent protein (GFP) carried by the transfection plasmid is clearly expressed. Figure 3 B statistically analyzed the number of organoids formed under different treatment times and different virus ratios. The results showed that the organoid formation rate was highest after 2 hours of treatment and lowest after 6 hours of treatment, indicating that the virus treatment time was negatively correlated with the organoid formation ability. Figure 3C compared organoid transfection efficiencies under different ratios and treatment times. It was found that the transfection efficiency was highest when the transfection treatment time was 4 h and the volume ratio of cell suspension:lentivirus:DMEM was 10:2:40.

[0115] Figure 4 Figure A shows the cell clustering and pseudo-time sequence analysis results of placental single-cell sequencing, indicating that cytotroph cells are the starting point of development. Figure 4 The B result indicates that the CLOCK gene is highly expressed during the initiation stage of cytotrophoblast development. Figure 4 The results showed that after knocking out the CLOCK gene, the spheroidization rate and growth status of placental organoids decreased significantly, further verifying the feasibility of the lentivirus knockout method used in this invention in functional studies.

[0116] The method provided by this invention enables those skilled in the art to successfully construct human placental organoids and utilize lentiviruses to achieve efficient and low-toxicity gene intervention, providing a reliable technical platform for subsequent research on placental development and related disease mechanisms.

[0117] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for gene intervention in human placental organoids based on lentivirus transfection, characterized in that, Includes the following steps: (1) Collect placental villus cells from early pregnancy and construct primary placental organoids; (2) Lentiviral packaging and concentration were carried out using packaging cell lines to obtain high-titer lentiviruses; (3) After digesting the primary placental organoids obtained in step (1) into single cells, a single cell suspension is obtained and transfected using the high-titer lentivirus obtained in step (2) to achieve gene intervention. In step (3), during transfection, the volume ratio of the single-cell suspension, the high-titer lentivirus, and DMEM is 10:1:40 or 10:2:40; the incubation time for transfection is 2-6 hours.

2. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (3), during transfection, the volume ratio of the single-cell suspension, the high-titer lentivirus, and DMEM is 10:2:

40.

3. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (3), the incubation time for transfection is 4 hours.

4. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (3), the incubation temperature for transfection is 37°C.

5. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (2), the packaging cell line is 293T cells, and the viral plasmids used include plasmids containing Gag / Pol function, plasmids containing Rev function, plasmids containing VSV-G envelope protein, and target gene transfer plasmids. The mass ratio of the Gag / Pol function plasmid, the Rev function plasmid, the VSV-G envelope protein plasmid, and the target gene transfer plasmid is 1:1:1:

4.

6. The method for gene intervention in human placental organoids according to claim 1, characterized in that, After packaging, the lentivirus was transfected with a transfection reagent for 20 minutes, and the viral supernatant was collected at 24 h and 48 h after transfection. The high-titer lentivirus was obtained by ultracentrifugation and concentration.

7. The method for gene intervention in human placental organoids according to claim 1, characterized in that, In step (3), the primary placental organoids cultured for 7-10 days and with a diameter between 100-200 μm are digested into single cells.

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.

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