A vascularized organoid and its preparation method

Abstract

This invention belongs to the field of organoid preparation technology, specifically relating to a vascularized organoid and its preparation method. Its beneficial effects are that, through subcutaneous transplantation in mice, it was found that the blood vessels of the resulting organoids can be perfused with blood flow; the cultured organoids can grow larger and differentiate into corresponding functional structures.

Description

Technical Field

[0001] This invention belongs to the field of organoid preparation technology, specifically relating to a vascularized organoid and its preparation method. Background Technology

[0002] Organoid technology was named the 2018 Technology of the Year by *Nature Methods* magazine, and it has developed rapidly in recent years. Compared to monolayer cells cultured on plates, organoids possess the main cell types of organs in vivo and can reproduce the structure of real organs. Therefore, organoids have advantages in studying organ development, drug screening, and disease model construction.

[0003] The circulatory system is the first organ to develop in the human body, distributed throughout the entire body. It provides nutrients to all organs, removes waste, and maintains adequate gas exchange. Currently, the main technological barrier facing organoids is the lack of a vascular system. For organoids that have already been constructed, although they have reproduced some organ structures and functions, the main limitation to achieving fully functional organoids in vitro is the lack of a suitable tissue size. The main reason for growth arrest or cell death in all organoids during tissue engineering is the lack of sufficient oxygen and nutrient supply. Therefore, vascularization is a crucial technological limitation that urgently needs to be addressed for all types of organoids. Furthermore, the lack of a suitable vascular network also limits their application in transplantation and repair.

[0004] In recent years, most reports on organoid vascularization have focused on using single endothelial cells (ECs), or even more specifically, using a mixture of ECs and vascular smooth muscle cells (VSMCs) to construct blood vessels. However, these methods fail to form well-developed vascular networks and cannot fully mimic the structure of real blood vessels. Methods using microvascular fragments extracted from adipose tissue for organoid vascularization have been shown to form well-developed vascular structures, but their widespread application is limited by tissue heterogeneity, complex extraction methods, and limited quantities. Recent studies have found that endothelial cells can be found in organoids by regulating signaling pathways such as VEGF, FGF, and BMP during organoid formation, but these cells are mostly found randomly. Overall, numerous research groups worldwide have invested heavily in organoid vascularization, but the formation of perfusion-enabled vascular networks in organoids has not yet been achieved.

[0005] Therefore, the vascularization of organoids is still a relatively open research area, and different methods need to be tried to realize organoids with perfusion and functional vascular network structures.

[0006] Numerous studies have demonstrated that blood vessels are crucial for organ development and the maturation of its cellular function, structure, and metabolism. Therefore, constructing more sophisticated vascularized organoids is of great significance in exploring organ development, drug screening, pathogenic mechanisms, and tissue regeneration, and holds immense application potential. Summary of the Invention

[0007] The purpose of this invention is to construct a vascularized organoid, characterized by the ability to form a hollow, functional, and perfusion-compatible vascular network within the organoid.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a vascularized organoid includes the following steps:

[0010] Step 1: Obtain vascular cell spheres;

[0011] Step 2: Obtain other somatic cell spheres;

[0012] Step 3: Fuse one or more vascular cell spheres prepared in Steps 1 and 2 with one or more other somatic cell spheres, and then culture them to prepare vascularized organoids.

[0013] Furthermore, cell spheres can differentiate from pluripotent stem cells, adult stem cells, or be derived from tissue extracts.

[0014] Furthermore, pluripotent stem cells can be induced pluripotent stem cells for humans or animals.

[0015] Furthermore, the preparation process of vascular cell spheres in step 1 involves culturing stem cells to form pseudo-spheres and then differentiating them into vascular cell spheres; or first inducing stem cells to differentiate into vascular cells under 2D conditions and then aggregating them into spheres.

[0016] Furthermore, the preparation process of other somatic cell spheres in step 2 involves culturing stem cell B to form pseudo-spheres and then differentiating them into somatic cell spheres; or first inducing stem cell B to differentiate into somatic cells under 2D conditions and then aggregating them into spheres; or preparing cell clusters from corresponding cell clusters or cell suspensions extracted from animals.

[0017] Preferably, when the two cell clusters are derived from pluripotent stem cells, the stem cells can be cultured into pseudospheres and then divided into two portions, which are then induced to differentiate into vascular cell spheres and other somatic cell spheres, respectively.

[0018] Furthermore, the fusion process described in step 3 can be carried out using conventional methods in this field.

[0019] Preferably, vascular cell spheres and somatic cell spheres are fused under normal culture conditions, suspension culture conditions, or embedded in a hydrogel material.

[0020] Preferably, the fusion step is performed under suspension culture conditions.

[0021] Preferably, in step 3, the number of somatic cell spheres is 1 to 4.

[0022] Preferably, in step 3, the number of vascular cell spheres is 1 to 10.

[0023] Preferably, in step 3, the number of somatic cell spheres is 1; the number of vascular cell spheres is 2.

[0024] Furthermore, in step 3, the presence of CD31-positive vascular structures in the organoid is considered the completion of this step; preferably, the culture time is 3 to 5 days.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. Subcutaneous transplantation in mice revealed that the blood vessels of the resulting organoids could be perfused with blood flow;

[0027] 2. The cultured organoids can grow larger and differentiate into corresponding functional structures. Attached Figure Description

[0028] Figure 1 Bright-field photograph of the hiPSC cell clone in Example 1;

[0029] Figure 2 EBs formed from hiPSC single-cell suspension in Example 1;

[0030] Figure 3 Bright-field photograph of hiPSC-induced vascular cell spheres from Example 1;

[0031] Figure 4 This is an immunofluorescence image of CD31 in hiPSC-induced vascular cells from Example 1;

[0032] Figure 5 The blood vessels in the vascular cell sphere of Example 1 have a hollow structure;

[0033] Figure 6 Bright-field image of hiPSC-induced cardiomyocyte spheroids from Example 1;

[0034] Figure 7 This is an immunofluorescence image of hiPSC-induced cardiomyocyte spheroids cTNTs from Example 1;

[0035] Figure 8 Immunofluorescence images of vascularized cardiac organoids from Example 1;

[0036] Figure 9An immunofluorescence image of the vascular sprouting tip in the vascularized cardiac organoid of Experiment Example 1;

[0037] Figure 10 This is an immunofluorescence image of CD31 in the vascular cell spheroid graft of Experiment Example 1;

[0038] Figure 11 Immunofluorescence images of cTNT in cardiomyocyte spheroids and cardiac organoids from Experiment Example 1;

[0039] Figure 12 Immunofluorescence images of F-actin in cardiomyocyte spheroids and cardiac organoids from Experiment Example 1;

[0040] Figure 13 This is a statistical graph of the beating frequency of the vascularized cardiac organoid in Experiment Example 1;

[0041] Figure 14 This is an immunofluorescence image of the vascularized pancreatic islet organoids from Experiment Example 2.

[0042] Figure 15 This is a bright-field photograph of the vascularized pancreatic islet organoid from Experiment Example 3.

[0043] Figure 16 Immunofluorescence images of the neuronal spheres in Experiment Example 4.

[0044] Figure 17 This is an immunofluorescence image of the vascularized neural organoid from Experiment Example 4. Detailed Implementation

[0045] The specific embodiments of the present invention will be further described below with reference to the examples. The following examples are only used to illustrate the technical embodiments of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0046] Example 1

[0047] This invention also provides a method for preparing vascularized cardiac organoids, comprising the following steps:

[0048] (1) hiPSCs were digested into a single-cell suspension and passaged into ultra-low adsorption plates at a ratio of 1:3. The cells were cultured in stem cell culture medium for 1 day to induce pluripotent stem cells to form embryoid bodies (EBs) (e.g. Figure 2 ), and then the EBs were divided into two parts;

[0049] (2) One of the EBs was induced to form vascular spheroids using a well-defined vascular spheroid induction system for 10 days after 2 days. Fluorescent staining revealed the expression of the vascular-specific marker CD31 (e.g. Figure 3 and 4 ), and has a cavity structure (such as Figure 5);

[0050] (3) Another batch of EBs began 8 days later using a well-defined cardiomyocyte spheroid induction system to induce cardiomyocyte spheroids (e.g. Figure 6 and 7 ), the spontaneous beating of the myocardium;

[0051] (4) After differentiation, one cardiomyocyte sphere and two vascular cell spheres were seeded into one well of a 96-well plate with ultra-low adsorption.

[0052] (5) Add culture medium with clearly defined vascularized heart organoids and 20% fetal bovine serum (FBS) to the first 3 days after inoculation to promote cell survival and cell spheroid fusion;

[0053] (6) Starting on day 4, reduce the FBS content in the culture medium for vascularized cardiac organoids to 2.5%;

[0054] (7) Vascularized cardiac organoids can be obtained on day 5 after inoculation and can be continuously cultured (e.g. Figure 8 ).

[0055] (8) Immunofluorescence results showed that the vascular tip had a budding structure, indicating a tendency to continue growing into the myocardial tissue (e.g. Figure 9 Furthermore, the transplanted blood vessels can perfuse with the host's blood vessels, demonstrating that the blood vessels are a functional, perfusion-enabled vascular network (e.g., Figure 10 );

[0056] (9) Immunofluorescence results showed that blood vessels can promote the orderly arrangement of myocardial structures (e.g. Figure 11 and 12 );

[0057] (10) The obtained vascularized cardiac organoids have a higher beating frequency than simple myocardial bulbs (e.g. Figure 13 ).

[0058] Preferably, hiPSC can be any pluripotent induced stem cell line.

[0059] Preferably, hiPSCs are cultured at a density of 80% before passage using stem cell culture medium.

[0060] Preferably, the stem cell culture medium is Essential 8 or mTeSR.

[0061] Preferably, hiPSCs are digested into a single-cell suspension using 0.5 mM EDTA or Accutase at room temperature or 37°C for 6-8 minutes.

[0062] Preferred, well-defined vascular cell spheroid induction medium is CDM3-V (RIPA1640 as the basal medium, supplemented with 1 mg / ml BSA, 150...). g / ml LAA). Add 6 g / ml LAA for the first two days of differentiation. M CHIR99021, then change the culture medium and add 100 ng / ml VEGF and 50 ng / ml FGF2 to induce until day 10, changing the medium every two days.

[0063] Preferred, well-defined cardiomyocyte spheroid induction medium is CDM3-C (RPMI 1640 as the basal medium, supplemented with 500... g / ml BSA, 200 g / ml LAA). Add 4 g / ml LAA for the first two days of differentiation. M CHIR99021, immediately change the culture medium and add 3 Induction with MIWR for two days, followed by culture with CDM3-C medium until day 8, with medium changed every two days.

[0064] Preferably, a well-defined induction system is constructed to induce EBs into vascular cell spheres and cardiomyocyte spheres, respectively.

[0065] Preferably, the induction start time is differentiated to obtain vascular cell spheres and cardiomyocyte spheres of different sizes, with cardiomyocyte induction occurring later than vascular cell induction, mimicking the sequence of organ development.

[0066] Preferably, the differentiation of the two types of cell spheres ends on the same day, which facilitates the subsequent fusion of cell spheres to form organoids.

[0067] Preferably, this operation is simple and highly repeatable, thus enabling the batch generation of the required number of vascularized cardiac organoids.

[0068] The preferred, well-defined vascularized cardiac organoid culture medium is CDM3-C with 50 ng / ml VEGF and 25 ng / ml FGF2 added, and 20% FBS is specified to promote cell fusion and cell survival.

[0069] Preferably, the vascularized cardiac organoid culture medium with a well-defined composition has an FBS content of 2.5% to ensure the impact of uncertain components in serum on the research in subsequent applications.

[0070] Preferably, the vascularized cardiac organoids obtained can beat autonomously and can be cultured for a long time.

[0071] Example 2

[0072] This invention also provides a method for preparing vascularized islet organoids, wherein the vascular spheres are derived from EB-induced vasculature and the islet spheres are extracted from mouse islets, comprising the following steps:

[0073] (1) The hiPSCs were digested into a single-cell suspension and passaged into an ultra-low adsorption plate at a ratio of 1:3. The cells were cultured in stem cell culture medium for 1 day to induce pluripotent stem cells to form embryoid bodies (EBs).

[0074] (2) One of the EBs was induced to form vascular spheroids for 10 days after 2 days using a well-defined vascular spheroid induction system;

[0075] (3) Mouse islets were extracted from the C57 / BL6 strain and cultured in low-adsorption well plates;

[0076] (4) Five pancreatic islet spheres and five vascular cell spheres were seeded into one well of a 96-well plate with ultra-low adsorption;

[0077] (5) Add pancreatic organoid culture medium and supplement with VEGF, FGF2 and 10% FBS;

[0078] (6) Do not move the culture plate for the first 3 days after inoculation to facilitate cell fusion;

[0079] (7) Starting on day 4 after inoculation, reduce the FBS content in the culture medium for vascularized pancreatic islet organoids to 2.5%;

[0080] (8) Vascularized pancreatic islet organoids (e.g., on day 5 after inoculation) can be obtained. Figure 14 ), and can be continuously cultivated.

[0081] Preferably, hiPSC can be any pluripotent induced stem cell line.

[0082] Preferably, hiPSCs are cultured at a density of 80% before passage using stem cell culture medium.

[0083] Preferably, the stem cell culture medium is Essential 8 or mTeSR.

[0084] Preferably, hiPSCs are digested into a single-cell suspension using 0.5 mM EDTA or Accutase at room temperature or 37°C for 6-8 minutes.

[0085] Preferred, well-defined vascular cell spheroid induction medium is CDM3-V (RIPA1640 as the basal medium, supplemented with 1 mg / ml BSA, 150...). g / ml LAA). Add 6 g / ml LAA for the first two days of differentiation. M CHIR99021, then change the culture medium and add 100 ng / ml VEGF and 50 ng / ml FGF2 to induce until day 10, changing the medium every two days.

[0086] Preferably, a well-defined induction system is constructed to induce EBs into vascular cell spheres;

[0087] Preferably, the islets can be extracted from mice or other animals such as humans;

[0088] Preferably, this operation is simple and highly repeatable, thus enabling the batch generation of the required number of vascularized pancreatic islet organoids.

[0089] Preferably, vascularized pancreatic islet organoids can be cultured for an extended period of time.

[0090] Example 3

[0091] This invention also provides a method for preparing collagen-coated vascularized islet organoids, wherein the vascular spheres are derived from EB-induced vasculature, the islet spheres are extracted from mouse islets, and the collagen is derived from commercially available rat tail collagen, comprising the following steps:

[0092] (1) The hiPSCs were digested into a single-cell suspension and passaged into an ultra-low adsorption plate at a ratio of 1:3. The cells were cultured in stem cell culture medium for 1 day to induce pluripotent stem cells to form embryoid bodies (EBs).

[0093] (2) One of the EBs was induced to form vascular spheroids for 10 days after 2 days using a well-defined vascular spheroid induction system;

[0094] (3) Mouse islets were extracted from the C57 / BL6 strain and cultured in low-adsorption well plates;

[0095] (4) Mix 5-10 pancreatic islet globules and 1 vascular cell globule in 200 ml of water. Add collagen to one well of a 48-well plate;

[0096] (5) Place in an incubator and wait for the collagen to coagulate;

[0097] (6) Add pancreatic organoid culture medium and supplement with VEGF, FGF2 and 10% FBS;

[0098] (7) Vascularized pancreatic islet organoids (e.g., on day 3 after inoculation) can be obtained. Figure 15 ), and can be continuously cultivated.

[0099] Preferably, hiPSC can be any pluripotent induced stem cell line.

[0100] Preferably, hiPSCs are cultured at a density of 80% before passage using stem cell culture medium.

[0101] Preferably, the stem cell culture medium is Essential 8 or mTeSR.

[0102] Preferably, hiPSCs are digested into a single-cell suspension using 0.5 mM EDTA or Accutase at room temperature or 37°C for 6-8 minutes.

[0103] Preferred, well-defined vascular cell spheroid induction medium is CDM3-V (RIPA1640 as the basal medium, supplemented with 1 mg / ml BSA, 150...). g / ml LAA). Add 6 g / ml LAA for the first two days of differentiation. M CHIR99021, then change the culture medium and add VEGF and 50 ng / ml FGF2 to induce induction until day 10, changing the medium every two days.

[0104] Example 4

[0105] This invention also provides a method for preparing collagen-coated vascularized neural organoids, wherein the vascular spheres are derived from EB-induced neuronal spheres, the neural cell spheres are derived from EB-induced neuronal spheres, and the collagen is derived from commercially available rat tail collagen, comprising the following steps:

[0106] (1) hiPSCs were digested into a single-cell suspension and passaged into ultra-low adsorption plates at a ratio of 1:3. The cells were cultured in stem cell culture medium for 1 day to induce pluripotent stem cells to form embryoid bodies (EBs) (e.g. Figure 2 ), and then the EBs were divided into two parts;

[0107] (2) One of the EBs was induced to form vascular spheroids using a well-defined vascular spheroid induction system for 10 days after 2 days. Fluorescent staining revealed the expression of the vascular-specific marker CD31 (e.g. Figure 3 and 4 ), and has a cavity structure (such as Figure 5 );

[0108] (3) Another batch of EBs began 10-day induction of neural spheres using a well-defined neural sphere induction system 2 days later;

[0109] (4) Identification of differentiated nerve cells showed that the nerve cell clusters were filled with neurons (tuj1 positive), and the cell bodies and distal axons were clearly visible (e.g. Figure 16 (a and b)

[0110] (5) Differentiated nerve cell clusters were embedded in collagen. After 4 days of culture, nerve axons elongated into the matrix, forming a well-defined axonal plexus (e.g., Figure 16 c and d);

[0111] (6) Staining of nerve axons on day 8 revealed axons (tuj1 positive) and glial cells (S100- Positive cells are interconnected, reproducing cell types and distribution patterns in neural networks under physiological conditions. Figure 16 e and f);

[0112] (7) When two clusters of nerve cells are embedded in collagen, it can be observed that the elongated axons in different cell spheres are interconnected, and the nerve synapses interact with each other. Figure 16 g);

[0113] (8) After differentiation, one neuronal cell sphere and one vascular cell sphere were seeded into one well of a 96-well plate with ultra-low adsorption.

[0114] (5) Add culture medium with clearly defined vascularized neural organoids and 20% fetal bovine serum (FBS) to the culture medium for the first 3 days after inoculation to promote cell survival and cell spheroid fusion;

[0115] (6) Starting on day 4, reduce the FBS content in the culture medium for vascularized cardiac organoids to 2.5%;

[0116] (7) Vascularized neural organoids can be obtained on the 4th day after inoculation. These organoids are then embedded in rat tail collagen and cultured for approximately 3 days to observe obvious vascular neural networks (e.g., Figure 17 a).

[0117] (8) Immunofluorescence results showed that in the formed vascular neural network, blood vessels were located at the anterior end of the budding process, and nerves accompanied blood vessel growth (e.g. Figure 17 b)

[0118] Preferably, hiPSC can be any pluripotent induced stem cell line.

[0119] Preferably, hiPSCs are cultured at a density of 80% before passage using stem cell culture medium.

[0120] Preferably, the stem cell culture medium is Essential 8 or mTeSR.

[0121] Preferably, hiPSCs are digested into a single-cell suspension using 0.5 mM EDTA or Accutase at room temperature or 37°C for 6-8 minutes.

[0122] Preferred, well-defined vascular cell spheroid induction medium is CDM3-V (RIPA1640 as the basal medium, supplemented with 1 mg / ml BSA, 150...). g / ml LAA). Add 6 g / ml LAA for the first two days of differentiation. M CHIR99021, then change the culture medium and add 100 ng / ml VEGF and 50 ng / ml FGF2 to induce until day 10, changing the medium every two days.

[0123] The preferred, well-defined neuronal spheroid induction medium is NDM (RPMI 1640 as the basal medium, supplemented with 1×B27, 1×NEAA, and 1×GlutaMAX). 10% of the RPMI 1640 is added throughout the differentiation process. M CHIR99021, 10nM LDN193189, 50 Neuronal induction was performed using MSB431542 (50 ng / ml), BMP4 (50 ng / ml), and Noggin, with the solution changed every two days.

[0124] Preferably, a well-defined induction system is constructed to induce EBs into vascular cell spheres and nerve cell spheres, respectively.

[0125] Preferably, the differentiation of the two types of cell spheres ends on the same day, which facilitates the subsequent fusion of cell spheres to form organoids.

[0126] Preferably, this operation is simple and highly repeatable, thus enabling the batch generation of the required number of vascularized neural organoids.

[0127] The preferred culture medium for vascularized neural organoids is NDM supplemented with 50 ng / ml VEGF and 25 ng / ml FGF2, and 20% FBS is specified to promote cell fusion and cell survival.

[0128] Preferably, the vascularized cardiac organoid culture medium with a well-defined composition has an FBS content of 2.5% to ensure the impact of uncertain components in serum on the research in subsequent applications.

[0129] Preferably, the vascularized neural organoids can generate vascular neural networks in collagen, with blood vessels at the anterior end and nerves growing alongside the blood vessels, fully simulating the structure and development pattern of vascular neural networks in vivo, providing a good in vitro model for subsequent research on vascular neural development and other related fields.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of preparing a vascularized organoid, characterized by, The organoid is a neural organoid, and the steps include: Step 1: Obtaining vascular cell spheres: Inducing pluripotent stem cells to form embryoid bodies, and starting the 10-day induction of vascular cell spheres using a well-defined vascular cell sphere induction system 2 days later; Among them, the well-defined vascular cell spheroid induction system used the well-defined vascular cell spheroid induction medium CDM3-V. 6 μM CHIR99021 was added for the first two days of differentiation, followed by changing the medium and adding 100 ng / ml VEGF and 50 ng / ml FGF2 to induce differentiation until day 10, with the medium changed every two days. The defined vascular cell spheroid induction medium CDM3-V is based on RIPA1640 medium with the addition of 1 mg / ml BSA, 150 g / ml LAA; Step 2, Obtaining Neural Cell Spheres: Pluripotent stem cells are induced to form embryoid bodies. Two days later, a neural cell sphere induction system with clearly defined components is used to begin a 10-day induction process. The differentiated neural cells are then identified. The neural cell clusters are filled with neurons, and the cell bodies and distal axons are clearly visible. wherein the defined neural cell sphere induction system uses a defined neural cell sphere induction medium NDM, and 10 M CHIR99021, 10 nM LDN193189, 50 M SB431542, 50 ng / ml BMP4, 50 ng / ml Noggin for neural induction, medium change every two days; The well-defined neural spheroid induction medium NDM is based on RPMI 1640 with the addition of 1×B27, 1×NEAA, and 1×GlutaMAX. Step 3, Cell Sphere Fusion and Culture: After differentiation, one neural cell sphere and one vascular cell sphere were seeded into one well of a 96-well plate with ultra-low adsorption. For the first 3 days after seeding, NDM medium with well-defined vascularized neural organoids was added, along with 50 ng / ml VEGF, 25 ng / ml FGF2 and 20% fetal bovine serum. Starting from day 4, the content of fetal bovine serum in the medium was reduced to 2.5%. Vascularized neural organoids were obtained on day 4 after seeding.

2. The method for preparing vascularized organoids according to claim 1, characterized in that, In step 1, the presence of the vascular-specific marker CD31 through fluorescent staining, along with a hollow structure, indicates that this step is complete.

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