Method for culturing vascularized tumor organoids

Abstract

The invention relates to the technical field of biological tissue culture, in particular to a vascularized tumor organoid culture method. Through the 3D printing technology, the pourable vascularized tumor organoid model with the tumor distribution rule is obtained, the problem of nutrient competition in traditional culture is solved, and the pourable vascularized tumor organoid model obtained through the method can be used for exploring the physiological interaction between microvessels and solid tumor tissues, and has the advantages of being high in practicability and the like. The invention has important application value in the fields of oncology and cancer therapeutics.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue culture, and particularly to a method for culturing vascularized tumor organoids. Background Art

[0002] Most tumors with a thickness exceeding 200 μm will generate blood vessels to ensure sufficient nutrient and oxygen supply. And the generation of tumor angiogenesis is a challenge in the current field of tissue engineering. In traditional 2D cell models, cells grow in a single layer, lacking the interaction between cell-cell and cell-extracellular matrix, and unable to simulate the three-dimensional structure of the in vivo tumor tissue microenvironment. Due to species differences and the lack of immune response in some experimental animals, the results of animal experiments cannot accurately predict the therapeutic effects in humans, which are not conducive to the study of the tumor angiogenesis mechanism.

[0003] In view of the limitations of the above 2D culture and animal models, more and more scholars have begun to use 3D models to study the occurrence and development mechanism of tumor blood vessels. The most common model starts with co-culturing endothelial cells and tumor cells to produce heterotypic tumor spheroids, using the growth factors secreted by tumor cells themselves or by adding exogenous vascular growth factors to attract endothelial cells, self-assemble, and invade the tumor spheroids.

[0004] However, these models still have some defects. Direct mixing and culturing in a three-dimensional environment is likely to cause nutrient competition between the two types of cells during growth, and does not conform to the distribution law of in vivo tumors. How to explore the physiological interaction between microvessels and solid tumor tissues and construct a vascularized tumor organoid model is of great significance for understanding oncology and cancer therapeutics. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for culturing vascularized tumor organoids.

[0006] The present invention provides a perfusable vascularized tumor organoid model, which includes:

[0007] A hollow cylindrical hydrogel conduit;

[0008] An endothelial cell layer 1 covering the inner side of the hollow cylindrical hydrogel conduit;

[0009] A laminin layer covering the outer side of the hollow cylindrical hydrogel conduit;

[0010] An endothelial cell layer 2 covering the laminin layer.

[0011] The raw materials of the hollow cylindrical hydrogel conduit include: protein hydrogel, sodium alginate, and tumor organoids.

[0012] Further, in the raw materials of the hollow cylindrical hydrogel conduit,

[0013] the protein hydrogel includes at least one or a combination of two or more of collagen, matrix gel, and fibrin; specifically, it is type I rat tail collagen;

[0014] the high molecular compound includes one or a combination of two or more of sodium alginate, gelatin, chitosan, collagen, and hyaluronic acid, specifically, it is sodium alginate;

[0015] the tumor organoids include Matrigel microspheres containing primary cancer cells; the Matrigel microspheres containing primary cancer cells are obtained by culturing a mixture of Matrigel and primary tumor cells in a tumor organoid complete medium in an ultra-low attachment 96-well plate; the diameter of the tumor organoids obtained after culturing is preferably 60 μm to 200 μm; in the mixture, the addition ratio of Matrigel to primary tumor cells is 1×10 4 ~2×10 5 primary tumor cells per 1 mL of Matrigel.

[0016] Furthermore, in the raw materials of the hollow cylindrical hydrogel conduit,

[0017] the concentration of the protein hydrogel is 3 mg / mL to 15 mg / mL. Specifically, the protein hydrogel is collagen, and the collagen is type I rat tail collagen with a concentration of 3 mg / mL;

[0018] the concentration of the high molecular compound is 1 to 10 mg / mL. Specifically, the high molecular compound is sodium alginate with a concentration of 2 mg / mL sodium alginate;

[0019] the concentration of the Matrigel microspheres containing primary cancer cells is 1×10 3 ~5×10 3 per mL;

[0020] In the perfusable vascularized tumor organoid model of the present invention,

[0021] in the endothelial cell layer 1, the concentration of endothelial cells is 1×10 6 to 6×10 6 per mL; specifically, it is 2×10 6 per mL; the endothelial cells include at least one of HUVEC endothelial cells, endothelial cells isolated from tumor tissues, and / or primary HUVEC endothelial cells; in a specific embodiment of the present invention, they are HUVEC endothelial cells.

[0022] In the endothelial cell layer 2, the concentration of endothelial cells is 1×10 6 cells / mL to 6×10 6 cells / mL; specifically, it is 2×10 6 cells / mL; the endothelial cells include at least one of HUVEC endothelial cells, endothelial cells obtained by separating tumor tissues, and / or primary HUVEC endothelial cells; in a specific embodiment of the present invention, they are HUVEC endothelial cells.

[0023] In the laminin layer, the concentration of laminin is 0.1 mg / mL to 1 mg / mL, specifically 0.5 mg / mL.

[0024] In the perfusable vascularized tumor organoid model of the present invention,

[0025] the inner diameter of the hollow cylindrical hydrogel conduit is 150 μm to 250 μm;

[0026] the wall thickness of the hollow cylindrical hydrogel conduit is 100 μm to 300 μm.

[0027] The present invention provides a method for preparing the perfusable vascularized tumor organoid model, and the preparation method includes the following steps:

[0028] Step 1, prepare a hollow cylindrical hydrogel conduit;

[0029] Step 2, crosslink laminin on the outer side of the hollow cylindrical hydrogel conduit to form a laminin layer;

[0030] Step 3, bring the inner side of the hollow cylindrical hydrogel conduit and the laminin layer into contact with endothelial cells to obtain an endothelial cell layer 1 on the inner side of the hollow cylindrical hydrogel conduit and an endothelial cell layer 2 on the surface of the laminin layer.

[0031] In the present invention, the hollow cylindrical hydrogel conduit is obtained by using a bioprinting technique, specifically a 3D coaxial coextrusion technique; in a specific embodiment of the present invention, the preparation method of the hollow cylindrical hydrogel conduit includes: connecting a mixture containing type I rat tail collagen, sodium alginate, and tumor organoids to the outer channel of a coaxial pillow, connecting a CaCl2 solution 1 to the inner channel of the coaxial pillow, controlling the co-discharge of the mixture and the CaCl2 solution 1, and the liquid passes through the coaxial needle to form a hollow tube structure and enters a CaCl2 solution 2 for shaping to obtain the hollow cylindrical hydrogel conduit. After obtaining the hollow cylindrical hydrogel conduit, it also includes a cleaning step, and the cleaning solution is a PBS buffer solution.

[0032] The concentration of CaCl2 in the CaCl2 solution 1 is 2 mg / mL to 3 mg / mL, specifically 2 mg / mL;

[0033] The concentration of CaCl₂ in the CaCl₂ solution 2 is 1 to 3 mg / mL;

[0034] In the present invention, the diameter a of the inner cavity of the coaxial needle is 150 to 250 μm, and the diameter b of the outer cavity is 500 to 860 μm; the wall thickness of the hollow hydrogel tube is 100 to 300 μm.

[0035] In the preparation method of the present invention, the cross-linking time is 15 min.

[0036] The culture conditions are 37 °C, with flipping once every 24 h.

[0037] With the aid of 3D printing technology, and through the screening of raw materials, the optimization of raw material ratios, and the optimization of step parameters involved in the preparation, etc., the present invention obtains a perfusable vascularized tumor organoid model with a tumor distribution pattern. The perfusable vascularized tumor organoid model solves the problem of nutritional competition in traditional culture, realizes continuous perfusion, simulates the in-vivo nutrient / oxygen gradient distribution, eliminates the central necrosis phenomenon caused by diffusion limitation in traditional static culture, and precisely reproduces the penetration kinetics of drugs in tumor tissues.

[0038] The present invention provides the application of the described perfusable vascularized tumor organoid model and / or the perfusable vascularized tumor organoid model prepared by the described preparation method in the study of in-vivo and / or in-vitro tumor angiogenesis and / or development mechanisms.

[0039] The present invention provides a method for studying in-vivo and / or in-vitro tumor angiogenesis and / or development mechanisms, which includes using the perfusable vascularized tumor organoid model of the present invention and / or the perfusable vascularized tumor organoid model prepared by the preparation method of the present invention.

[0040] Through 3D printing technology, the present invention obtains a perfusable vascularized tumor organoid model with a tumor distribution pattern, solves the problem of nutritional competition in traditional culture, and the perfusable vascularized tumor organoid model obtained by the present invention can be used to explore the physiological interaction between microvessels and solid tumor tissues, and has important application value in the fields of oncology and cancer therapeutics. Description of the Drawings

[0041] Figure 1 Schematic diagram showing the construction of a vascularized tumor organoid, where a is the diameter of the inner cavity and b is the diameter of the outer cavity. Detailed Embodiments

[0042] The present invention provides a method for culturing vascularized tumor organoids. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0043] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The following further elaborates the present invention in conjunction with embodiments:

[0044] Example 1 Printing Hollow Hydrogel Tubes Loaded with Tumor Organoids

[0045] S1. Prepare materials

[0046] The commercially available type I rat tail collagen solution is diluted in 10×PBS containing phenol red to prepare a stock solution with a concentration of 5 mg / ml for standby; sodium alginate powder is dissolved in PBS to prepare a stock solution with a concentration of 3 mg / mL for standby; CaCl2 powder is dissolved in PBS to prepare a stock solution with a concentration of 3 mg / mL for standby;

[0047] Obtaining tumor organoids: Prepare a mixed solution containing Matrigel and primary tumor cells; the mixed solution is dropped into an ultra-low attachment 96-well plate and cultured with complete tumor organoid medium to obtain the required tumor organoids; the diameter of the tumor organoids is preferably 60 μm to 200 μm;

[0048] S2. Printing

[0049] (1) Prepare Solution 1 and Solution 2 respectively according to the following formula:

[0050] Solution 1: Contains type I rat tail collagen with a final concentration of 3 mg / mL, a 2 mg / mL sodium alginate solution, and a density of 1×10 3 ~5×10 3 tumor organoids / mL;

[0051] Solution 2: Contains a 2 mg / mL CaCl2 solution;

[0052] (2) Vertically arrange the coaxial needle above a 1 - 3 mg / mL CaCl2 solution. Solution 1 is connected to the outer cavity of the coaxial needle, and Solution 2 is connected to the inner cavity of the coaxial needle;

[0053] (3) Use a micro-injection pump to control the simultaneous outflow of Solution 1 and Solution 2. The liquid forms a hollow tube structure through the coaxial needle and enters the CaCl2 solution for shaping;

[0054] (4)Collect the hollow hydrogel conduit and wash the hollow hydrogel conduit with PBS.

[0055] S3. Culture

[0056] Add the purchased complete culture medium for tumor organoids and culture in an incubator.

[0057] Example 2 Construction of vascularized tumor organoids

[0058] S1. Refer to Example 1 to prepare a hollow hydrogel conduit loaded with tumor organoids;

[0059] S2. Dissolve laminin powder in PBS to prepare a solution with a concentration of 0.5 mg / mL for standby; spin-coat laminin onto the outer wall of the collagen hollow hydrogel conduit, place it in an incubator at 37 °C and incubate for crosslinking for 15 min; remove the excess laminin solution.

[0060] S3. Inoculate HUVEC endothelial cells at a density of 2×10 6 cells / mL outside the hollow hydrogel conduit, and perfuse HUVEC endothelial cells at a density of 2×10 6 cells / mL into the hollow hydrogel conduit, place it in an incubator for continuous culture, and turn it over every 24 h to facilitate the uniform distribution of endothelial cells in the lumen.

[0061] S4. After the endothelial cells are evenly attached, complete the culture to obtain the required tumor organoids containing a dense endothelial layer.

[0062] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A perfusable vascularized tumor organoid model, characterized in that, Comprising: A hollow cylindrical hydrogel conduit; An endothelial cell layer 1 covering the inner side of the hollow cylindrical hydrogel conduit; A laminin layer covering the outer side of the hollow cylindrical hydrogel conduit; An endothelial cell layer 2 covering the laminin layer; The raw materials of the hollow cylindrical hydrogel conduit include: protein hydrogel, polymer compound and tumor organoids.

2. The perfusable vascularized tumor organoid model according to claim 1, wherein The protein hydrogel includes at least one or a combination of two or more of collagen, basement membrane matrix and fibrin; The polymer compound includes one or a combination of two or more of sodium alginate, gelatin, chitosan, collagen and hyaluronic acid; The tumor organoids include Matrigel microspheres containing primary cancer cells.

3. The perfusable vascularized tumor organoid model according to claim 1, characterized in that, Among the raw materials of the hollow cylindrical hydrogel conduit, The concentration of the protein hydrogel is 3mg / mL - 15mg / mL; The concentration of the polymer compound is 1mg / mL - 10mg / mL; The concentration of the Matrigel microspheres containing primary cancer cells is 1×10 3 cells / mL to 5×10 3 cells / mL.

4. The perfusable vascularized tumor organoid model according to claim 3, wherein In the endothelial cell layer 1, the concentration of endothelial cells is 1×10 6 cells / mL to 6×10 6 cells / mL; In the endothelial cell layer 2, the concentration of endothelial cells is 1×10 6 cells / mL to 6×10 6 cells / mL.

5. The perfusable vascularized tumor organoid model according to claim 4, characterized in that, In the laminin layer, the concentration of laminin is 0.1mg / mL - 1mg / mL.

6. The perfusable vascularized tumor organoid model according to claim 5, wherein The inner diameter of the hollow cylindrical hydrogel conduit is 150μm - 250μm; The wall thickness of the hollow cylindrical hydrogel conduit is 100μm - 300μm.

7. The method for preparing the perfusable vascularized tumor organoid model according to any one of claims 1 to 6, characterized in that, Including the following steps: Step 1, prepare and obtain a hollow cylindrical hydrogel conduit; Step 2, crosslink laminin on the outer side of the hollow cylindrical hydrogel conduit to form a laminin layer; Step 3, culture a layer of endothelial cells on the inner side of the hollow cylindrical hydrogel conduit and the surface of the laminin layer respectively, form an endothelial cell layer 1 on the inner side of the hollow cylindrical hydrogel conduit, and obtain an endothelial cell layer 2 on the surface of the laminin layer.

8. The preparation method according to claim 7, wherein The crosslinking time is 15 min; The culture conditions are 37°C, and it is flipped once every 24 h.

9. The application of the perfusable vascularized tumor organoid model according to claims 1 - 6 and / or the perfusable vascularized tumor organoid model prepared by the preparation method according to claim 7 or 8 in the study of the mechanism of tumor angiogenesis and / or development in vivo and / or in vitro.

10. A research method for the mechanism of in vivo and / or in vitro tumor angiogenesis and / or development, characterized in that, Including using the perfusable vascularized tumor organoid model according to claims 1 - 6 and / or the perfusable vascularized tumor organoid model prepared by the preparation method according to claim 7 or 8.

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