Organ-like infection method based on microinjection
By using microinjection to precisely inject fluorescently labeled pathogens into organoids, the problem of matrix gel barrier limitation is solved, enabling efficient and targeted infection while preserving the three-dimensional structure and physiological characteristics of organoids, thus improving infection efficiency and result reliability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-03
- Publication Date
- 2026-04-03
AI Technical Summary
Existing organoid infection methods are limited by the physical barrier of matrix gel, resulting in low infection efficiency, poor reproducibility, and large deviations between infection results and in vivo conditions. It is difficult to achieve precise and effective interaction between pathogens and organoid epithelial cells, and it is also difficult to preserve the three-dimensional structure and physiological characteristics of organoids.
The microinjection method is used to precisely inject fluorescently labeled pathogens into organoids through a microinjection needle. The fluorescent labeling supports real-time observation of the infection site, enabling targeted infection of pathogens and organoids, avoiding digestion of matrix gel, and preserving the physiological structure and function of organoids to the greatest extent.
It significantly improves infection efficiency and specificity, the infection model is closer to the real physiological state in vivo, the infection process is visualized, and it is convenient to track the distribution and interaction of pathogens in organoids. It is applicable to the study of infection of various types of organoids and pathogens.
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Figure CN121780348A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell infection technology, specifically relating to an organoid infection method based on microinjection. Background Technology
[0002] Organoids, as micro-tissues formed in vitro in three dimensions and possessing organ-specific structures and functions, can highly mimic the cellular composition and physiological characteristics of organs in vivo, thus showing great potential in disease modeling, drug development, and regenerative medicine research.
[0003] However, in infectious disease research, traditional two-dimensional cell models, due to their lack of spatial structure and cellular heterogeneity, struggle to accurately reflect the natural infection process of pathogens. While animal models can provide a more comprehensive in vivo environment, they suffer from drawbacks such as long processing times, high costs, and ethical restrictions.
[0004] To overcome these limitations, researchers have attempted to co-culture pathogens with organoids. However, in existing organoid culture systems, matrix gel, as a key material mimicking the basement membrane of cells in vivo, forms a high-density protein network structure, providing a natural physical barrier against microorganisms such as bacteria and viruses. Existing research has confirmed that matrix gel encapsulating organoids significantly hinders effective contact between pathogens and organoid epithelial cells, leading to low infection efficiency and difficulty in control.
[0005] To overcome the matrix gel barrier and enable pathogens to access organoid cells, current technologies have attempted to remove matrix gel through protease digestion or enzyme-free digestive solutions. However, while mild digestive agents such as neutral proteases can reduce cell damage to some extent, the digestion process inevitably disrupts the original self-assembly spatial structure of the organoids, leading to loss of epithelial cell polarity and disintegration of intercellular connections, thus negating the advantage of mimicking the in vivo physiological microenvironment. For example, after digestion, the unique villous-crypt structure of intestinal organoids is destroyed, making it impossible to reproduce the natural pathways by which pathogens cross the epithelial barrier, resulting in a significant discrepancy between the infection mechanism research results and the actual in vivo situation.
[0006] Therefore, while matrix gel provides structural support for organoids, its physical barrier hinders the effective interaction between pathogens such as bacteria and viruses and organoid epithelial cells. In other words, existing organoid infection methods are limited by the physical barrier of matrix gel, resulting in low infection efficiency, poor reproducibility, and significant deviations between infection outcomes and in vivo conditions. Furthermore, it is difficult to achieve precise and effective interaction between pathogens and organoid epithelial cells, thus failing to fully preserve the three-dimensional structure and physiological characteristics of organoids. Summary of the Invention
[0007] To address the problems of existing technologies, this invention proposes a microinjection-based organoid infection method. The aim is to overcome the physical barrier of matrix gel by precisely injecting fluorescently labeled pathogens into the organoid, thereby achieving efficient and targeted infection while preserving the physiological structure and function of the organoid to the greatest extent possible. This solves the problems of low efficiency, poor reproducibility, and large deviation between infection results and in vivo status in current organoid infection methods.
[0008] This invention discloses an organoid infection method based on microinjection, specifically comprising:
[0009] A glass capillary tube is drawn into a microinjection needle with a tip diameter of 1-5 µm using a needle puller. The microinjection needle is connected to a pressure-type microinjection pump via a flexible tubing, and the volume of pathogen suspension injected per injection is precisely controlled by pressure adjustment. The pathogen suspension is labeled with a lipophilic fluorescent dye, collected by centrifugation, and resuspended in sterile PBS buffer. The labeled pathogen suspension is then transferred into the microinjection needle using a pipette. The organoid is placed under a stereomicroscope, and the target area is located using a fluorescence microscope. The microinjection needle is inserted into the target area, and the labeled pathogen suspension is injected to complete the infection.
[0010] Preferably, the glass capillary is a borosilicate glass capillary with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm.
[0011] Preferably, the injection volume of the pathogen suspension is calculated based on the multiple of infection (MOI), where MOI = pathogen quantity : organoid cell quantity; the pathogen suspension is a bacterial suspension or a viral suspension, specifically, the MOI of the bacterial suspension is 1~100, and the MOI of the viral suspension is 0.01~10; the injection volume of the pathogen suspension is 2µL~50µL, wherein the injection volume is adaptively adjusted according to the size of the organoid; the concentration of the pathogen suspension is dynamically adjusted based on the MOI and the injection volume.
[0012] Preferably, when the pathogen is bacteria, the bacterial suspension to be infected is co-incubated with the lipophilic fluorescent dye to allow the bacterial membrane to bind the fluorescent label.
[0013] When the pathogen is a virus, the viral suspension is labeled using a gene-edited fluorescent virus or a lipophilic fluorescent dye.
[0014] Preferably, the lipophilic fluorescent dye is at least one of DiI dye, DiO dye, and DiR dye, and the dye concentration is 1~10µM; the labeling conditions for pathogen suspension are: room temperature incubation for 10-30 min.
[0015] Preferably, the organoid is one of the following: human iPS whole lung organoid, intestinal organoid, lung organoid, and liver organoid.
[0016] Preferably, the tip of the microinjection needle is inserted obliquely into the organoid, penetrating the surface and entering the organoid cavity to a depth of 1 / 3 to 1 / 2 of its diameter.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0018] This invention utilizes microinjection to achieve targeted infection of pathogens within organoids. Pathogens can effectively bypass the matrix gel barrier, achieving targeted infection of specific regions within the organoids, significantly improving infection efficiency and specificity. Simultaneously, this invention eliminates the need for matrix gel digestion, maximizing the preservation of the organoid's complete three-dimensional structure, cell polarity, and intercellular connections, making the infection model closer to the actual physiological state in vivo, thereby improving the reliability of the results.
[0019] Secondly, fluorescent labeling supports real-time observation and subsequent analysis of infection sites, making the infection process visible and facilitating precise tracking of pathogen distribution and interactions within organoids. Furthermore, microinjection is applicable to various types of organoids and a wide range of bacterial and viral pathogen infections, demonstrating good versatility. Attached Figure Description
[0020] Figure 1 This is an image of Mycobacterium abscessus infecting organoids according to Example 1 of the present invention;
[0021] Figure 2 Images of sections of organoids infected with different types of diseases in Example 1 of this invention, stained with HE and TUNEL.
[0022] Figure 3 This is an image of RSV virus infecting organoids according to Embodiment 2 of the present invention. Detailed Implementation
[0023] To better understand the content of this invention, specific embodiments will be used to further illustrate the invention below. The following embodiments are based on the technology of this invention and provide detailed implementation methods and operating steps. However, the scope of protection of this invention is not limited to the following embodiments; that is, all other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0024] This invention discloses a microinjection-based organoid infection method. Targeting pathogenic microorganism infections (such as enteropathogenic bacteria, respiratory bacteria, RSV virus, and influenza virus), it precisely introduces fluorescently labeled bacteria into specific regions (such as bronchial epithelial tubular structures and alveolar-like vesicle structures) through microinjection, simulating the process of pathogens invading host cells in vivo. This method solves the problem of pathogenic microorganisms being unable to penetrate the matrix gel barrier in traditional co-culture.
[0025] Specifically, it includes:
[0026] Preparation of microinjection needles: Glass capillaries are drawn into microinjection needles with a tip diameter of 1-5µm using a needle puller. The tip is fine enough to penetrate the surface of organoids while avoiding excessive tissue damage.
[0027] Injection system connection and control: The microinjection needle is connected to a pressure-type microinjection pump via a flexible tubing. The volume of pathogen suspension injected in a single injection is precisely controlled by adjusting the pressure. The pathogen suspension is a bacterial suspension or a viral suspension. The injection volume is adjusted according to the size of the organoid space.
[0028] As one embodiment of the present invention, regarding the preparation method of pathogen suspension, activated pathogens are inoculated into MH medium and cultured at 37°C with shaking until the logarithmic growth phase; the bacterial cells are collected by centrifugation and resuspended after washing with sterile PBS; the turbidity of the bacterial suspension is measured using a turbidimetric tube or spectrophotometer, and the bacterial concentration is calculated according to a standard curve; the required bacterial quantity is calculated based on MOI=100 and the amount of organoid cells, and the bacterial suspension is adjusted to the corresponding concentration according to the single injection volume, thus obtaining the pathogen suspension to be injected. The culture conditions of pathogenic microorganisms and organoids are safe and mature existing technologies, and will not be described in detail here.
[0029] Pathogen labeling and transfer: The pathogen suspension was labeled with a lipophilic fluorescent dye and then transferred to a microinjection needle using a pipette to obtain a labeled pathogen suspension.
[0030] As one embodiment of the present invention, when the pathogen is bacteria: the bacterial suspension to be infected is co-incubated with a lipophilic fluorescent dye to allow the bacterial membrane to bind the fluorescent label so as to track the infection site under a fluorescence microscope;
[0031] As one embodiment of the present invention, when the pathogen is a virus: a gene-edited fluorescent virus or a lipophilic fluorescent dye is used to label the viral suspension so as to track the infection site under a fluorescence microscope;
[0032] For bacteria, their OD 600The conversion relationship between OD value and CFU / mL varies depending on the bacterial species. The method involves determining the corresponding CFU of bacteria with different OD values under logarithmic growth conditions (OD value range 0.4~1.0) through plate colony counting, thus establishing an OD value conversion relationship. 600 Linear relationship between value and CFU / mL;
[0033] The viral titer was determined using the plaque assay, a classic virological method, with a titer range of 1E6 PFU / mL to 1E9 PFU / mL.
[0034] In one embodiment of the present invention, the lipophilic fluorescent dye is at least one of DiI dye-orange-red fluorescence (1,1'-tetratetraimino-3,3,3',3'-tetramethylindole carbapenem iodide), DiO dye-green fluorescence (3,3'-tetratetraimino-3,3,3',3'-tetramethylindole carbapenem perchlorate), and DiR dye-near-infrared fluorescence (near-infrared derivatives of DiI).
[0035] Organoid localization and injection: The organoid is placed under a stereomicroscope, the target area is located using a fluorescence microscope, the microinjection needle is precisely inserted into the target area, and the pathogen suspension is slowly injected to complete the infection.
[0036] In this invention, the pathogen is a bacterium or a virus, specifically Mycobacterium abscessus, Acinetobacter baumannii, RSV virus, influenza virus, etc.; the organoids are human iPS whole lung organoids, intestinal organoids, lung organoids, liver organoids, etc.
[0037] The present invention can be further understood through the following embodiments;
[0038] Example 1
[0039] This embodiment utilizes microinjection to precisely introduce Mycobacterium abscessus into the entire lung organoid of human iPS and tracks the infection process.
[0040] 1. Material preparation
[0041] Human iPS whole lung organoids: After one month of in vitro culture, organoids with complex three-dimensional structures (such as tubular structures of bronchial epithelium and alveolar-like vesicle structures) are formed.
[0042] Mycobacterium abscessus: Laboratory standard strain (i.e., international standard strain ATCC 19977) TM ).
[0043] DIO dye: final concentration is 1µM.
[0044] Needle puller: Model SutterP-2000, used for preparing microinjection needles.
[0045] Microinjection system: includes pressure-type microinjection pump, flexible tubing and stereomicroscope.
[0046] Sterile PBS buffer.
[0047] 2. Operating Procedures
[0048] A glass capillary tube was drawn into a microinjection needle with a tip diameter of 2µm using a needle puller; ensuring that the needle tip was fine enough to penetrate the surface of the organoid without causing excessive damage, and that the inner diameter was sufficient for bacteria to pass through; the prepared microinjection needle was then connected to a pressure-type microinjection pump via a flexible tubing.
[0049] A negative pressure test was conducted to ensure the system was well sealed, and the injection volume of the bacterial suspension was precisely controlled by adjusting the pressure of the syringe pump. Here, the injection volume was 50µL.
[0050] Mycobacterium abscessis was co-incubated with DIO dye under sterile conditions for 30 minutes to allow the DIO dye to bind to the bacterial membrane, thus fluorescently labeling the bacteria. After co-incubation, the labeled bacteria were collected by centrifugation and resuspended in sterile PBS buffer to prepare a 1×10⁻⁶ solution. 8 A CFU / mL labeled bacterial suspension was prepared; the labeled bacterial suspension was transferred into a microinjection needle using a sterile pipette.
[0051] The whole lung organoid was placed on an organoid culture chamber and placed under an inverted fluorescence microscope; the alveolar cavities were located under the microscope; a microinjection needle was precisely inserted into the alveolar cavity, and a suspension of labeled bacteria with a 10 MOI (multiple of infection) was slowly injected.
[0052] After injection, the organoids were cultured under suitable conditions for 2 hours. Subsequently, the green fluorescent signal (DIO labeling) inside the organoids was observed using a fluorescence microscope to confirm whether the bacteria had successfully entered and localized to the target area.
[0053] Please refer to the details. Figure 1-2 ;
[0054] Figure 1 This is an image of Mycobacterium abscessus infection within organoids, as shown in Example 1.
[0055] Control group (uninfected): Organoids that have not undergone any treatment, show no fluorescent signal, and are morphologically intact.
[0056] Co-incubation infection group: The labeled bacteria were directly co-incubated with the organoids, but no microinjection was performed; the results showed that the fluorescence signal inside the organoids was weak or even absent, indicating that the bacteria could not effectively penetrate the matrix gel barrier to enter the organoids, and the infection efficiency was extremely low.
[0057] Injection infection group: Labeled bacteria were directly injected into the organoids using microinjection. The results showed that obvious green fluorescent signals appeared inside the organoids, and the fluorescent signals were concentrated in the injection area, indicating that the bacteria successfully and accurately entered the organoids and caused infection.
[0058] Figure 2 Illustrations of histopathological changes (HE staining) and apoptosis (TUNEL staining) of organoids under different infection modes.
[0059] Organoid HE staining:
[0060] Control group (uninfected): Organoid tissue structure is intact, and cells are arranged in a regular manner.
[0061] Co-incubation infection group: No obvious changes were observed in tissue morphology, and the inflammatory response was not obvious.
[0062] Injection infection group: Organoid sections after injection infection showed a significant increase in inflammasomes and disordered cell structure, indicating that injection infection can effectively induce inflammatory responses in lung organoids.
[0063] Organoid TUNEL staining:
[0064] TUNEL staining is a commonly used experimental technique for detecting DNA breaks during apoptosis. Its core principle is that when cells undergo apoptosis, endogenous nucleases are activated, leading to DNA double-strand breaks and the generation of numerous 3'-OH ends. Catalyzed by terminal deoxynucleotidyl transferase (TdT), fluorescein- or biotin-labeled dUTPs are attached to these DNA break ends, allowing for the specific identification of apoptotic cells through fluorescence microscopy or chemical staining.
[0065] The results showed that after TUNEL staining, the intensity of DNA fragmentation and the proportion of positive cells in the organoid sections of the injection-infected group were much higher than those in the uninfected group and the co-incubation-infected group, indicating that injection infection can significantly induce apoptosis in organoid cells, further confirming its ability to effectively infect organoids.
[0066] In summary, after HE and TUNEL staining of co-incubated infected organoids, it was found that compared with co-incubated infected organoids, microinjection infected organoids showed a significant increase in inflammasomes and more pronounced apoptosis. This clearly demonstrates that the microinjection infection method provided by this invention can effectively and accurately infect lung organoids and induce a significant host response.
[0067] Example 2
[0068] Compared with Example 1, the main difference is that: in this example, the pathogen Mycobacterium abscessus is replaced with gene-edited RSV virus with a reporter gene; the viral titer is 2E8pfu / mL; the injection is at a titer of 3MOI, the number of organoid cells is 5E5, and a total of 1.5E6 viral particles are required; each organoid is injected with 7.5µL of viral suspension.
[0069] Figure 3 Images of RSV virus infection within organoids in this embodiment are shown. In the RSV-infected group, a clear RSV-N fluorescent signal is visible, overlapping with the nuclear signal of DAPI, indicating that the RSV virus successfully invaded the organoid cells, achieving effective infection. In the Mock group, almost no RSV-N fluorescent signal is visible, only the nuclear signal of DAPI is present; this indicates that the Mock group was indeed not infected by RSV, further verifying that the fluorescent signal in the RSV-infected group is caused by the virus itself.
[0070] Because pre-labeled viruses are used, the virus suspension can be directly observed and transferred into a microinjection needle.
[0071] This invention can have other embodiments based on the above preparation method, which will not be listed one by one. Therefore, any simple modifications, equivalent changes and alterations made by any person skilled in the art to the above embodiments without departing from the scope of the technical solution of this invention shall still fall within the scope of the technical solution of this invention.
Claims
1. A method for organoid infection based on microinjection, characterized in that: A glass capillary tube is drawn into a microinjection needle with a tip diameter of 1-5µm using a needle puller; the microinjection needle is connected to a pressure-type microinjection pump via a flexible tubing, and the volume of pathogen suspension injected in a single injection is controlled by pressure regulation. Pathogen suspensions were labeled with lipophilic fluorescent dyes, collected by centrifugation, and resuspended in sterile PBS buffer. A labeled pathogen suspension is obtained and transferred to the microinjection needle using a pipette. The organoid is placed under a stereomicroscope, and the target area is located using a fluorescence microscope. The microinjection needle is inserted into the target area, and the labeled pathogen suspension is injected to complete the infection.
2. The organoid infection method based on microinjection as described in claim 1, characterized in that, The glass capillary is a borosilicate glass capillary with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm.
3. The organoid infection method based on microinjection as described in claim 1, characterized in that, The pathogen suspension is a bacterial suspension or a viral suspension; the MOI of the bacterial suspension is 1~100, and the MOI of the viral suspension is 0.01~10; the injection volume of the pathogen suspension is 2µL~50µL.
4. The organoid infection method based on microinjection as described in claim 3, characterized in that, When the pathogen is bacteria, the bacterial suspension to be infected is co-incubated with the lipophilic fluorescent dye to allow the bacterial membrane to bind the fluorescent label. When the pathogen is a virus, the viral suspension is labeled using a gene-edited fluorescent virus or a lipophilic fluorescent dye.
5. The organoid infection method based on microinjection as described in claim 1, characterized in that, The lipophilic fluorescent dye is at least one of DiI dye, DiO dye, and DiR dye.
6. The organoid infection method based on microinjection as described in claim 1, characterized in that, The organoid is one of the following: human iPS whole lung organoid, intestinal organoid, lung organoid, and liver organoid.
7. The organoid infection method based on microinjection as described in claim 1, characterized in that, The tip of the microinjection needle is inserted obliquely into the organoid, penetrating the surface and entering the organoid cavity to a depth of 1 / 3 to 1 / 2 of its diameter.