Subcutaneous construction method of multi-tissue human organoid-animal chimera and application thereof

Abstract

This invention belongs to the field of biotechnology and relates to in vivo culture technology of human organoids, xenotransplantation models, organoid vascularization technology, and translational research in regenerative medicine. It provides a method for subcutaneous construction of multi-tissue human organoid-animal chimeras and its application. The method involves transplanting in vitro cultured organoids into the subcutaneous space of immunodeficient animals, and using the host microenvironment to achieve rapid vascularization and functional maturation of the organoids. This method solves the problems of existing organoid in vitro culture lacking a vascular network, which easily leads to central necrosis, and the complexity, invasiveness, and difficulty in dynamic observation of traditional in vivo transplantation methods. It is applicable to the in vivo construction of organoids from multiple tissues such as the heart, brain, and lungs, and can be applied in drug screening, human cell-host immune interaction research, and regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to human organoid in vivo culture technology, xenotransplantation models, organoid vascularization technology, and regenerative medicine translational research. Specifically, it relates to the subcutaneous construction method of multi-tissue human organoid-animal chimeras and their applications. Background Technology

[0002] Organoid technology, a significant breakthrough in stem cell and regenerative medicine, can simulate the complex structure and some functions of human organs under three-dimensional in vitro culture conditions, providing a promising research platform for disease modeling, drug development, and personalized medicine. However, traditional in vitro culture systems have fundamental limitations, primarily the lack of a functional vascular network and a complete systemic physiological microenvironment. Relying on passive diffusion for nutrient acquisition, organoids generally face core hypoxia and nutrient depletion after reaching a critical size, leading to central necrosis and thus limiting their long-term survival and further development of macroscopic structures. More critically, the in vitro culture environment isolates neuroendocrine regulation, immune cell interactions, and systemic signaling between organs, making it difficult for organoids to achieve truly long-term functional maturation and fully simulate complex physiological and pathological processes in vivo.

[0003] To overcome the bottlenecks of in vitro culture, current technologies attempt to transplant organoids into living animals, leveraging the host microenvironment to promote vascularization and functional maturation. Common transplantation sites include subcapsular renal, intrahepatic, and intrasplenic sites. Subcapsular renal transplantation is frequently used due to its relatively rich blood supply. However, these existing transplantation strategies still have significant limitations. The procedures typically involve complex microsurgical techniques, resulting in significant trauma and a high risk of complications such as bleeding or host organ damage, posing challenges to reproducibility and animal survival rates. Furthermore, these deep transplantation sites make long-term, non-invasive, in-situ real-time monitoring of the dynamic processes following organoid transplantation (such as survival, vascularization, growth, and functional changes) extremely difficult. Studies often rely on endpoint histological analysis, making it difficult to obtain continuous dynamic data. Simultaneously, due to surgical complexity and limitations of suitable transplantation sites, the parallel construction of multi-tissue-derived organoid chimeras in the same host for comparative studies or systemic toxicity assessments remains challenging. The unique local microenvironment of certain transplantation sites may also have non-specific effects on the maturation of non-originating organoids, limiting the universality of this technology.

[0004] Therefore, developing a novel in vivo organoid construction strategy that is simple to operate, minimally invasive, suitable for long-term dynamic observation, and has broad tissue compatibility has become a key requirement for promoting the development of this technology towards higher biomimetic models and its practical application. Summary of the Invention

[0005] The purpose of this invention is to provide a method for subcutaneous construction of multi-tissue human organoid-animal chimeras and its application. By mixing early organoids pre-cultured in vitro with a biocompatible matrix and transplanting them into the subcutaneous space of immunodeficient animals, the host microenvironment is utilized to achieve rapid vascularization and functional maturation of the organoids. This solves the problems of existing organoid in vitro culture lacking a vascular network, which easily leads to central necrosis, and traditional in vivo transplantation methods being complex, invasive, and difficult to dynamically observe.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A method for subcutaneous construction of a multi-tissue human organoid-animal chimera, comprising the following steps:

[0007] S1. In vitro pre-culture: Human pluripotent stem cells are induced to differentiate in vitro to form organoids derived from specific tissues;

[0008] S2, Matrix Encapsulation: The organoid is mixed with a biocompatible matrix to form an organoid-matrix complex;

[0009] S3. Subcutaneous transplantation: The organoid-matrix complex is transplanted into the subcutaneous space of an immunodeficient animal;

[0010] S4. In vivo maturation: The transplanted organoid undergoes vascularization and functional maturation within the animal, forming a tissue chimera containing human cells and host animal cells.

[0011] Preferably, the specific tissue is selected from one of the heart, brain, or lung tissue, and the developmental stage of the organoid is the precursor cell stage or the early organoid stage.

[0012] Preferably, the biocompatible matrix is ​​Matrigel or a biological scaffold containing pro-angiogenic factors.

[0013] Preferably, the immunodeficient animal is an immunodeficient mouse, and the subcutaneous transplantation is performed by making an incision in the subcutaneous tissue on the back of the mouse and constructing a subcutaneous transplantation space.

[0014] The present invention further provides a vascularized human heart organoid-animal chimera constructed by the above method.

[0015] Preferably, it comprises human TNNT2+ cardiomyocytes and a host animal-derived vascular endothelial cell network coupled to said cardiomyocytes.

[0016] The present invention further provides a long-lasting human brain organoid-animal chimera constructed by the above method.

[0017] Preferably, it has a layered cortical structure and is perfused by a vascular network derived from the host animal, without a central necrotic region.

[0018] The present invention further provides a human lung organoid-animal chimera with a branched structure constructed by the above method.

[0019] Preferably, it has a complex airway epithelial branching structure and a mature basement membrane.

[0020] The present invention further provides the application of the above-mentioned chimera in drug screening or efficacy evaluation.

[0021] The present invention further provides the application of the above-mentioned chimera in the study of the interaction between human cells and the host immune system.

[0022] The present invention further provides the application of the above-mentioned chimera as a source of tissue or organ transplant donors in regenerative medicine.

[0023] The beneficial effects of this invention are:

[0024] 1. Achieved efficient vascularization and functional maturation of organoids, solving the problem of core necrosis.

[0025] This invention transplants organoids into a well-vascularized subcutaneous space, leveraging the host microenvironment to spontaneously induce angiogenesis, forming a chimera coupling a network of vascular endothelial cells derived from the host animal with human functional cells. Compared to traditional in vitro culture, this method effectively overcomes central necrosis caused by the lack of blood vessels in organoids, significantly prolongs the in vivo survival time of organoids, and promotes further functional maturation of their structures (such as cortical stratification in brain organoids and branching structure formation in lung organoids).

[0026] 2. Simple to operate, minimally invasive, and easy to promote.

[0027] Compared to the complex deep microsurgical transplantation surgeries such as subcapsular renal transplantation and intracranial transplantation in existing technologies, the subcutaneous transplantation technique used in this invention does not require sophisticated microsurgical instruments, is simple and quick to operate, causes less trauma to the host animal, has a high postoperative survival rate, and is highly reproducible, making it suitable for widespread application in general biomedical laboratories.

[0028] 3. Facilitates long-term, non-invasive dynamic observation

[0029] The superficiality of subcutaneous transplantation sites allows researchers to perform long-term, non-invasive, in-situ real-time monitoring of the grafts. Combined with pre-labeling with fluorescent dyes such as DiR and small animal in vivo imaging systems, the survival, growth, and degradation processes of organoids can be continuously tracked in the same animal, acquiring dynamic data and significantly reducing the number of experimental animals used, thus conforming to the 3R principle.

[0030] 4. It has broad organizational compatibility and universality.

[0031] The method of this invention is not limited to a single tissue source and is applicable to human organoids of multiple tissue types, such as the heart, brain, and lungs. By constructing organoid chimeras of different tissues at different sites on the body surface of the same host animal, parallel studies of multiple organs can be achieved, providing a new platform for systemic toxicity assessment and multi-organ interaction research.

[0032] 5. The constructed chimeric structure is complete and functionally mature.

[0033] Cardiac organoid (CO) chimeras: These contain human TNNT2+ cardiomyocytes and are coupled to the host vascular network, exhibiting rhythmic pulsation and mature electrophysiological characteristics.

[0034] Brain organoid (BO) chimeras: have a layered cortical structure, are perfused by host blood vessels, have enhanced neuronal activity, and have no central necrotic area.

[0035] Lung branch organoids (LBO) chimeras: possess complex airway epithelial branching structures and mature basement membranes, more closely resembling the characteristics of adult lung tissue.

[0036] 6. Broad application prospects

[0037] The multi-tissue organoid-animal chimera constructed in this invention, based on its vascularization, functional maturity, and in situ human-mouse cell chimerism, can be directly applied to drug screening and efficacy evaluation, human cell-host immune interaction research, and regenerative medicine tissue / organ transplant donor sources, providing a highly biomimetic technical model for related research fields. Attached Figure Description

[0038] Figure 1This invention validates the structure and function of CO transplantation in mice. (A) IVIS images of mice transplanted with COs (- / + DiR staining) and heart organoids extracted 14 days later; (B) Representative bright-field images of COs and PT-COs cultured in vitro on day 14, scale bar: 100 μm; the area of ​​COs was measured using ImageJ, n=9; (C) H&E staining images of PT-CO sections on day 14, outlined in blue dashed lines, scale bar: 100 μm; (D) Immunofluorescence staining images of PT-CO sections on day 14 after transfer: DAPI (blue), CD31 (human, red), WT1 (green), scale bar: 100 μm; (E) Immunofluorescence staining images of PT-CO sections on day 14 after transfer: DAPI (blue), CD31 (mouse, red), scale bar: 20 μm; (F) Representative images of COs and PT-COs cultured in vitro on day 60, scale bar: 100 μm and 1 cm, area of ​​CO measured using ImageJ, n=9; (G) Transmission electron microscopy image of PT-CO, showing sarcomeres (S, yellow arrow) and mitochondria (M); (H) Immunofluorescence staining image on day 60 after PT-CO transfer: DAPI (blue), CD31 (human, red), and WT1 (green), scale bar: 100 μm; (I) VEGF protein concentrations in mice and humans as determined by ELISA, data expressed as mean ± standard deviation, n=3, #: P < 0.001 compared with other groups, ***: P < 0.001, **: P < 0.01; (J) Draft illustration depicting the transplantation process and outcome);

[0039] Figure 2This invention verifies the structure and function of BO transplanted in mice ((A) Gross observation of transplanted BO; (B) Fixation and staining of BO sections after transplantation, Human-CD31 (green) and mouse-CD31 (red); Human-CD31 (green) and Occludin (red); Human-CD31 (green) and ZO-1 (red) co-stained; Scale bar, 1000 μm, inset, 20 μm; (C) After in vitro culture of BO (Day 45), transplantation into mice for 4 weeks, injection of Evans blue and observation of its distribution in BO and surrounding tissues (Figure C: (a) Mouse image 4 weeks after BO transplantation, before Evans blue injection; (b) After Evans blue injection; (c) Gross observation of subcutaneous BO; (d) Enlarged image of Evans blue stained blood vessels, and photographs of subcutaneous blood vessels in surrounding tissues; (ef) In vitro stereomicroscopic photographs of subcutaneous BO in mice after tail vein injection of Evans blue, Scale bar, 50 μm). (μm); (D) After in vitro culture of cardiac organoids (Day 60), they were transplanted into mice and cultured for 8 weeks. Observation of mice after tail vein injection of Evans blue and observation of sections after removal of cardiac organoids).

[0040] Figure 3 This invention provides structural verification of LBO transplantation in mice ((A) IVIS image of a mouse with transplanted LBOs (+DiR staining); (B) IVIS image of cut skin tissue from the back of a mouse; (C) IVIS image of an LBO graft removed from the back skin of a mouse; (D) Gross image of cut skin tissue from the back of a mouse; (E) Image of an LBO graft removed from the back skin of a mouse). Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Example 1: Heart Organoid Chimera

[0045] 1. Method

[0046] 1.1 In vitro induction and pretreatment of human CO (cardiac organoids)

[0047] Cell source: Human embryonic stem cells H9 (Beina Chuanglian Biotechnology Co., Ltd.)

[0048] Induction scheme:

[0049] Stage 1: EB formation period (day 0-1)

[0050] Preparation of reagents and consumables: One day in advance, place the ultra-low adsorption U-bottom 96-well plate (Qingdao Jindian Biochemical Equipment Co., Ltd., #WP96-6CCUSH), 50 mL sample trough, and 200 μL sterile pipette tip box at -20 ℃ for pre-cooling.

[0051] Day 0: hESCs were digested into single cells, and the cells were counted using a plate blue assay, at a density of 3.5 x 10⁶ cells per well (96-well plate). 4 Collect cell suspension from each cell. Mix an appropriate amount of cell suspension with 10 5 A small amount of matrix gel (placed on ice) was pipetted into 21 mL of culture medium (containing CHIR99021 (Selleck, #S1263) at a final concentration of 6 μM, Y-27632 (Sigma-Aldrich, #Y0503) at a final concentration of 7.5 μM, RPMI1640 (Gibco™, #21870075) + 2% B27 (Gibco™, #A1895601) at a final concentration of 7.5 μM). The prepared cell suspension was then transferred to a pre-chilled 50 mL loading well. Using an eight-channel 300 μL pipette, the cell suspension was added to pre-chilled ultra-low adsorption U-bottom 96-well plates (200 μL / well). The 96-well plates were then centrifuged at 2000 r / min for 10 min in a refrigerated centrifuge, and finally placed in a cell culture incubator for incubation.

[0052] Stage 2: Mesodermal induction period (days 1-2)

[0053] Day 1: Discard the culture medium from the 96-well plate at a rate of 180 μL / well, and add culture medium (RPMI 1640 + 10% B27 + CHIR 99021 to a final concentration of 1.5 μM) at a rate of 180 μL / well. After changing the medium, place the 96-well plate in a cell culture incubator and incubate for 24 h.

[0054] Day 2: Discard the culture medium from the 96-well plate at a rate of 180 μL / well, add culture medium (RPMI 1640 + 10% B27) at a rate of 180 μL / well, change the medium, and then place the 96-well plate in a cell culture incubator for 24 h.

[0055] Stage 3: Cardiac mesodermal induction period (days 3-6)

[0056] Day 3: Discard the culture medium from the 96-well plate at a rate of 180 μL / well, and add culture medium (RPMI 1640 + 10% B27 + IWR-1-endo (Selleck, #S7086) at a final concentration of 2.5 μM) at a rate of 180 μL / well. After changing the medium, place the 96-well plate in a cell culture incubator and incubate for 72 h.

[0057] Stage 4: Maturation and Cultivation Period (day 6+)

[0058] Day 6: Discard and add 180 μL of culture medium (RPMI 1640 + 10% B27) to each well of the 96-well plate. After changing the medium, place the 96-well plate in a cell culture incubator for incubation. Change the medium every 2 days thereafter. Around Day 10, regular CO2 pulsation can be observed, indicating successful differentiation.

[0059] 1.2 Construction of the subcutaneous transplant microenvironment and transplantation surgery

[0060] Animal model: 6-8 week old, male NOD-SCID mice weighing 18-22 g were selected and housed in an SPF-grade environment with a temperature controlled at 22-24 ℃, humidity at 40-60%, 12-hour light-dark cycle, and free access to food and water. They were acclimatized for 7 days before the experiment.

[0061] Subcutaneous transplantation: Mice were anesthetized by isoflurane inhalation (induction concentration 3–4%, maintenance concentration 1.5–2%), and the skin on the back was disinfected (75% ethanol + iodine). A small incision of about 1 cm was made subcutaneously on the right back of the mouse using ophthalmic scissors. After COs matured in vitro, 10 COs were transferred from the culture dish to the cannula of the trocar. Subsequently, the trocar was used to puncture the subcutaneous tissue on the right back of the mouse, and the COs in the cannula were pushed into the subcutaneous tissue of the mouse using the needle core. COs stained with 20 μM DiR (Beijing Fluorescence Biotechnology Co., Ltd., #2207) for 30 min were transplanted into the mice and maintained for 30 days. After transplantation, an optical in vivo imaging system (IVIS) was used. ® The Lumia series III (PerkinElmer) was used to image mice to verify the survival of transplanted COs in mice.

[0062] 1.3 In vivo maturation and observation of chimeras

[0063] Observation period: Observations were conducted at fixed locations at weeks 1, 2, 4, 6, and 8 after transplantation, and animals were euthanized at the end of week 8 for analysis.

[0064] Observation method: Before transplantation, COs were co-incubated with DiR dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, excitation / emission wavelength: 748 / 780 nm) (concentration: 20 μM, incubation at 37 ℃ for 30 min). After washing with PBS to remove unbound dye, subcutaneous transplantation was performed. At each observation time point, fluorescence imaging was performed on mice using a small animal in vivo imaging system (excitation filter 745 nm, emission filter 800 nm) to dynamically track the survival, localization, and proliferation trend of COs.

[0065] 2. Results Analysis

[0066] 2.1 Chimera formation and vascularization

[0067] The survival of transplanted COs in mice was detected by in vivo imaging. Fluorescence was still detectable 14 days after transplantation. Figure 1 (A). Furthermore, it was observed that 10 COs began to fuse during transplantation, eventually forming a larger organoid tissue (referred to as: post-transplantation CO, PT-CO) on day 14, with a diameter exceeding 1 mm, three times the size of a single CO cultured in vitro during the same period. Figure 1 (Middle B). H&E staining of PT-CO sections revealed the heart muscle structure ( Figure 1 (C). WT1-positive human epicardial cells and mature human blood vessels were also detected in PT-CO. Figure 1 (D, white arrow). Furthermore, the results showed that the mouse blood vessels penetrated PT-CO, which was evidenced by the cross-sectional structure of the detected mouse blood vessels. Figure 1 (E). Mature blood vessels can be observed on the surface of PT-CO, and their chambers are filled with varying proportions of body fluid or blood. Figure 1 (Middle F). Ultrastructural analysis of a pair of PT-COs identified well-defined cardiac sarcomeres ( Figure 1 (G). The mature blood vessels of PT-CO have been proven to be of human origin (G). Figure 1 (Middle H, white arrow). To investigate the reasons for vascular maturation, this invention further analyzed the concentrations of human and mouse vascular endothelial growth factor (VEGF) in PT-CO and mouse vascular chambers. The results showed that the concentration of human VEGF in PT-CO (fluid extracted from the chamber) and in vitro COs (from the culture medium) was higher than that in mouse serum, while the concentration of mouse VEGF in PT-CO was higher than that in in vitro COs (…). Figure 1(I) This indicates that mouse blood flow permeated into the chambers of the PT-CO. These results suggest that vascular maturation of the PT-CO is induced by mouse VEGF in local blood perfusion (transplantation process and results are as follows). Figure 1 As shown in J).

[0068] H&E staining: First, organoids are collected and fixed with 4% paraformaldehyde. Then, they are pre-embedded with agarose or HistoGel to prevent the loss of small samples in subsequent processing. Next, after dehydration with graded ethanol, clearing with xylene, and paraffin wax impregnation, the samples are oriented and embedded into paraffin blocks and cut into 3-5 micrometer thin slices and mounted on anti-detachment slides. Finally, the samples are dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin for nuclei, differentiated with hydrochloric acid alcohol, blued with bluing solution, stained with eosin for cytoplasm, and then dehydrated and cleared before mounting. This allows the tissue structure, cell polarity, and nucleoplasmic contrast of the organoids to be clearly presented under a microscope.

[0069] Example 2: Brain organoid chimera

[0070] 1. Method

[0071] 1.1 In vitro induction and pretreatment of human BO (brain organoids)

[0072] Cell source: Human embryonic stem cells H9 (Beina Chuanglian Biotechnology Co., Ltd.)

[0073] Induction scheme:

[0074] Brain organoids were differentiated using the STEMdiff™ Brain Organoid Kit (stem cell, #08570).

[0075] 1.2 Construction of the subcutaneous transplant microenvironment and transplantation surgery

[0076] Animal model: 6-8 week old, male NOD-SCID mice weighing 18-22 g were selected and housed in an SPF-grade environment with a temperature controlled at 22-24 ℃, humidity at 40-60%, 12-hour light-dark cycle, and free access to food and water. They were acclimatized for 7 days before the experiment.

[0077] Subcutaneous transplantation: Mice were anesthetized by isoflurane inhalation (induction concentration 3-4%, maintenance concentration 1.5-2%), and the skin on their backs was disinfected (75% ethanol + iodine). A small incision of about 1 cm was made subcutaneously on the right back of the mouse using ophthalmic scissors. After maturation in vitro, 10 boron spores were transferred from the culture dish to the cannula of the trocar. Subsequently, the trocar was used to puncture the subcutaneous tissue on the right back of the mouse, and the boron spores in the cannula were pushed into the subcutaneous tissue of the mouse using the needle core. The boron spores stained with 20 μM DiR (Beijing Fluorescence Biotechnology Co., Ltd., #2207) for 30 min were transplanted into the mice and maintained for 30 days. After transplantation, an optical in vivo imaging system (IVIS) was used. ® The Lumia series III (PerkinElmer) was used to image mice to verify the survival of transplanted BOs in mice.

[0078] 1.3 In vivo maturation and observation of chimeras

[0079] Observation period: Observations were conducted at fixed locations at weeks 1, 2, 4, 6, and 8 after transplantation, and animals were euthanized at the end of week 8 for analysis.

[0080] Observation method: Before transplantation, BOs were co-incubated with DiR dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, excitation / emission wavelength: 748 / 780 nm) (concentration: 20 μM, incubation at 37 ℃ for 30 min). After washing with PBS to remove unbound dye, subcutaneous transplantation was performed. At each observation time point, fluorescence imaging of mice was performed using a small animal in vivo imaging system (excitation filter 745 nm, emission filter 800 nm) to dynamically track the survival, localization, and proliferation trend of BOs.

[0081] 2. Results Analysis

[0082] Mature boron endothelial cells (BO) were successfully transplanted subcutaneously into the right back of SCID mice. In vivo and histological studies confirmed a significant increase in transplanted BO volume. Human-derived (Human-CD31+) endothelial cells successfully integrated into the mouse host circulation and formed a rich microvascular network and tight junctions (ZO-1+). Figure 2 China A- Figure 2 (B). More importantly, tail vein injection of Evans blue confirmed that a functional BBB-like structure that strictly rejects exogenous dyes had been formed in this chimera. Figure 2 C- Figure 2 (D).

[0083] Example 3: Lung Branch Organoid Chimera

[0084] 1. Method

[0085] 1.1 In vitro induction and pretreatment of human LBO (branched organoid lung)

[0086] Cell source: Human embryonic stem cells H9 (Beina Chuanglian Biotechnology Co., Ltd.)

[0087] Induction protocol: Branched lung organoids were differentiated using the STEMdiff™ Lung Organoid Kit with Branched Structures (stem cell, #100-0195).

[0088] 1.2 Construction of the subcutaneous transplant microenvironment and transplantation surgery

[0089] Animal model: 6-8 week old, male NOD-SCID mice weighing 18-22 g were selected and housed in an SPF-grade environment with a temperature controlled at 22-24 ℃, humidity at 40-60%, 12-hour light-dark cycle, and free access to food and water. They were acclimatized for 7 days before the experiment.

[0090] Subcutaneous transplantation: Mice were anesthetized by isoflurane inhalation (induction concentration 3-4%, maintenance concentration 1.5-2%), and the skin on the back was disinfected (75% ethanol + iodine). A small incision of about 1 cm was made subcutaneously on the right back of the mouse using ophthalmic scissors. After maturation in vitro, 10 LBOs were transferred from the culture dish to the cannula of the trocar. Subsequently, the trocar was used to puncture the subcutaneous tissue on the right back of the mouse, and the LBOs in the cannula were pushed into the subcutaneous tissue of the mouse using the needle core. The LBOs stained with 20 μM DiR (Beijing Fluorescence Biotechnology Co., Ltd., #2207) for 30 min were transplanted into the mice and remained there for 30 days. After transplantation, an optical in vivo imaging system (IVIS) was used. ® The Lumia series III (PerkinElmer) was used to image mice to verify the survival of transplanted LBOs in mice.

[0091] 1.3 In vivo maturation and observation of chimeras

[0092] Observation period: Observations were conducted at fixed locations at weeks 1, 2, 4, 6, and 8 after transplantation, and animals were euthanized at the end of week 8 for analysis.

[0093] Observation method: Before transplantation, LBOs were co-incubated with DiR dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, excitation / emission wavelength: 748 / 780 nm) (concentration: 20 μM, incubation at 37 ℃ for 30 min). After washing with PBS to remove unbound dye, subcutaneous transplantation was performed. At each observation time point, fluorescence imaging of mice was performed using a small animal in vivo imaging system (excitation filter 745 nm, emission filter 800 nm) to dynamically track the survival, localization, and proliferation trend of LBOs.

[0094] 2. Results Analysis

[0095] The results showed that IVIS in vivo imaging revealed specific fluorescence signals at the transplantation site in mice transplanted with DiR-labeled LBO, demonstrating that LBO was successfully colonized in mice and maintained its activity. Figure 3 (A); Imaging of the back skin tissue and ex vivo graft confirmed that the fluorescence signal originated from the graft itself rather than diffusion or background signal. Figure 3 China B- Figure 3 (C) The macroscopic photograph visually demonstrates the morphological location of the graft in the host tissue and its structure after complete detachment. Figure 3 D- Figure 3 The results (E) comprehensively demonstrate that LBO can survive stably, be accurately positioned, and maintain structural integrity after in vivo transplantation, laying the foundation for subsequent functional verification.

[0096] In summary, based on the completion of the above three embodiments, the content of this invention can be summarized as follows:

[0097] This invention successfully establishes a method for constructing multi-tissue human organoid-animal chimeras based on subcutaneous transplantation. By transplanting in vitro cultured human organoids derived from the heart, brain, lungs, etc., into the subcutaneous space of immunodeficient mice, the rapid vascularization and long-term functional maturation of the organoids are achieved by leveraging the host's rich vascular network and systemic microenvironment.

[0098] In Example 1, the heart organoids formed a chimera after transplantation, containing human TNNT2+ cardiomyocytes and a host-derived vascular network, maintaining rhythmic pulsation, and the ultrastructure showed typical myosarcoma structure. In Example 2, the brain organoids formed a functional blood-brain barrier structure after transplantation, with human and mouse vascular endothelial cells coupling with each other and no central necrosis area. In Example 3, the lung branch organoids maintained a complex airway epithelial branching structure in vivo, with intact graft morphology, and in vivo imaging and anatomical observation jointly verified its stable colonization and structural integrity.

[0099] The human organoid-animal chimeras for the heart, brain, and lungs constructed in this invention possess core characteristics such as in vivo vascularization, structural integrity, mature function, and in-situ chimerism with human-host cells. These characteristics provide a solid structural and functional foundation for their applications in drug screening and efficacy evaluation, research on human cell-host immune system interactions, and the source of tissue / organ transplant donors in regenerative medicine. Using conventional techniques for cardiovascular / nervous / respiratory system drug screening, chimeras of corresponding tissue types can be directly used for efficacy evaluation, target validation, and tissue penetration testing of relevant drugs. Based on conventional research methods in this field, such as immune cell labeling and co-localization analysis, the in-situ chimerism of human-mouse cells in chimeras can be used to study the interaction mechanisms between human functional cells and host immune cells. Furthermore, using conventional tissue transplantation procedures, vascularized and functionally mature chimeras can serve as potential functional donor precursors for repairing defects in heart, brain, and lung tissues, promoting clinical translational research in regenerative medicine. The chimeras constructed in this invention provide research models and technical carriers that more closely resemble human physiological characteristics in the aforementioned fields.

[0100] The three embodiments collectively demonstrate that this subcutaneous construction method has advantages such as simple operation, minimal trauma, ease of dynamic observation, and broad tissue compatibility. It effectively overcomes the shortcomings of traditional organoid in vitro culture, such as lack of vascularization and susceptibility to central necrosis, as well as the complexity and difficulty in real-time monitoring of existing in vivo transplantation methods. The chimeras constructed in this invention show broad application prospects in drug screening, immune interaction research, and regenerative medicine donor sources, providing a reliable technical platform for the in vivo functional maturation and translational application of human organoids from multiple tissue sources.

[0101] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for subcutaneous construction of multi-tissue human organoid-animal chimeras, characterized in that, Includes the following steps: S1. In vitro pre-culture: Human pluripotent stem cells are induced to differentiate in vitro to form organoids derived from specific tissues; S2, Matrix Encapsulation: The organoid is mixed with a biocompatible matrix to form an organoid-matrix complex; S3. Subcutaneous transplantation: The organoid-matrix complex is transplanted into the subcutaneous space of an immunodeficient animal; S4. In vivo maturation: The transplanted organoid undergoes vascularization and functional maturation within the animal, forming a tissue chimera containing human cells and host animal cells.

2. The method according to claim 1, characterized in that, The specific tissue is selected from one of the heart, brain, or lung tissues, and the developmental stage of the organoid is the precursor cell stage or the early organoid stage.

3. The method according to claim 1, characterized in that, The biocompatible matrix is ​​Matrigel or a biological scaffold containing pro-angiogenic factors.

4. The method according to claim 1, characterized in that, The immunodeficient animal is an immunodeficient mouse, and the subcutaneous transplantation is performed by making an incision in the subcutaneous tissue on the back of the mouse and constructing a subcutaneous transplantation space.

5. A vascularized human heart organoid-animal chimera, characterized in that, It is constructed by the method according to any one of claims 1-4, comprising human TNNT2+ cardiomyocytes and a host animal-derived vascular endothelial cell network coupled to said cardiomyocytes.

6. A long-lasting human brain organoid-animal chimera, characterized in that, It is constructed by the method described in any one of claims 1-4, has a layered cortical structure, is perfused by a vascular network derived from the host animal, and has no central necrotic region.

7. A human lung organoid-animal chimera with a branched structure, characterized in that, It is constructed by the method described in any one of claims 1-4 and has a complex airway epithelial branching structure and a mature basement membrane.

8. The use of the chimera according to any one of claims 5-7 in drug screening or efficacy evaluation.

9. The use of the chimera according to any one of claims 5-7 in the study of the interaction between human cells and the host immune system.

10. The use of the chimera according to any one of claims 5-7 as a source of tissue or organ transplant donor in regenerative medicine.

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