CN117965450A - Method for constructing tumor organoid, vascularized tumor organoid and application thereof - Google Patents
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Abstract
The application discloses a method for constructing a tumor organoid, a vascularized tumor organoid and application thereof, wherein the method for constructing the tumor organoid comprises the following steps: firstly, constructing a tumor organoid, and then co-culturing the tumor organoid with endothelial cells to obtain the vascularized tumor organoid. In the technical scheme of the application, firstly, a tumor organoid is constructed, then the tumor organoid and endothelial cells are co-cultured through a chip to provide micro-flow and fluid shear force for the endothelial cells, thereby forming a vascular network surrounding the tumor organoid, and the vascularized tumor organoid is successfully constructed; the vascularized tumor organoid is similar to the in vivo tumor angiogenesis mode, so that the in vivo state of the tumor organoid is more truly simulated, and a more reliable biological sample is provided for in vitro research of tumors.
Description
Technical Field
The application relates to the field of biology, in particular to a construction method of a tumor organoid, a vascularized tumor organoid and application thereof.
Background
Angiogenesis is one of the hallmark features of malignant tumors in which cancer cells proliferate faster, and nutrients are required to meet cell proliferation, while metabolic waste is also required, so that new blood vessels are required to be formed, ensuring that tumor tissue grows exponentially.
The tumor organoid model has high simulation, has histological characteristics and functions similar to those of human organs, can better retain the advantages of tumor heterogeneity and the like in the tumor model, and has wide application prospect in the fields of accurate medical treatment, drug screening and the like. However, the static in vitro culture lacks vascular structures and cannot simulate complex microenvironment and dynamic characteristics in vivo, and a vascular network is an essential microenvironment factor for malignant tumor research and has close relation with tumor occurrence, growth and metastasis.
During tumor progression, endothelial cells in the pool or vessel proliferate and migrate to form vessels on or within the tumor tissue. Blood vessels are an important part of the tumor microenvironment, and research has been directed to the vascularization of tumor organoids, such as: chinese patent CN 115927164B discloses a method for culturing vascularized tumor organoids and application thereof, in particular discloses a method for preparing biopsy tissue into mixed cell clusters, then culturing tumor cells and endothelial cells by using the mixed cell clusters, finally placing the tumor cells and endothelial cells in a tumor organoid culture medium containing matrigel for 3D co-culture (culturing in a 48-well plate), culturing vascularized tumor organoids, which have the following defects, forming a vascular network in tumor tissue through angiogenesis after the tumor tissue grows to a certain size in vivo, but the above patent co-cultures the endothelial cells and the tumor cells, which are static culture, lack dynamics characteristics, and cannot accurately represent vascularization of tumor organoids in vivo. Chinese patent CN 111218401A discloses a new blood vessel formation and drug evaluation method based on tumor chip, in particular discloses a channel for respectively inoculating tumor cells and endothelial cells on two sides, wherein matrigel is infused in a middle channel to culture a 3D co-culture system for forming sprouting blood vessels of tumor 3D cells and endothelial cells, and the method can only observe the migration phenomenon of endothelial cells and tumor cells to matrigel and cannot accurately represent vascularization of tumor tissues in vivo. Nashimoto, and the like, by using a 3-channel microfluidic chip, a tumor sphere which is cultured in advance is placed in a middle channel, and endothelial cells are inoculated in other two channels for culturing (Nashimoto,Y.,Okada,R.,Hanada,S.,Arima,Y.,Nishiyama,K.,Miura,T.,&Yokokawa,R.(2020).Vascularized cancer on a chip:The effect of perfusion on growth and drug delivery of tumor spheroid.Biomaterials,229,119547.),, although the method can provide dynamic characteristics, in the culturing process, the microvessels only exist on two side surfaces of the tumor sphere, and microvessels which surround the tumor sphere are not really formed, and the vascularization of tumor tissues cannot be accurately represented.
Therefore, how to provide a tumor organoid solution that more closely mimics the in vivo microenvironment and dynamics is a technical problem to be solved in the art.
Disclosure of Invention
In the existing method for constructing the tumor organoid, the endothelial cells and the tumor cells are co-cultured to realize vascularization of the tumor organoid, which is a conventional thinking and forms a technical prejudice in the industry. The tumor organoids obtained in the traditional way can not truly simulate the microenvironment of tumor tissues in vivo due to lack of dynamic characteristics or the formation of microvessels only on the surface of tumor spheres, and are greatly limited and restricted in application level (such as drug screening, scientific research and the like) of the tumor organoids.
In view of the above, the application provides a method for constructing a tumor organoid, a vascularized tumor organoid and application thereof.
According to one aspect of the present application, there is provided a method of constructing a tumor organoid, the method comprising: firstly, constructing a tumor organoid, and then co-culturing the tumor organoid with endothelial cells to obtain the vascularized tumor organoid.
Specifically, the construction method comprises the following steps: obtaining primary tumor cells; placing the primary tumor cells in a tumor organoid culture medium containing matrigel for culture to obtain a tumor organoid; the tumor organoid is mixed with endothelial cells for culture to obtain vascularized tumor organoid.
In particular, the diameter of the tumour organoids to be cultivated in admixture with said endothelial cells is less than 150 μm, preferably 80 μm-100 μm.
Specifically, the endothelial cells are at least one of HUVEC endothelial cells, endothelial cells obtained by inducing differentiation of pluripotent stem cells and endothelial cells obtained by separating tumor tissues.
In particular, the vascularized tumor organoid comprises at least one of immune cells and cancer-associated fibroblasts.
Specifically, the mixed culture of the tumor organoid and the endothelial cells is to prepare a mixed solution of the tumor organoid, the endothelial cells, thrombin and the protein hydrogel solution.
Specifically, the mixed solution is injected into a tissue culture area, and after the protein hydrogel solution is solidified, the mixed solution is added into a vascularized tumor organoid culture medium for culture, so as to provide micro-flow and fluid shear force for endothelial cells.
Specifically, the protein hydrogel in the mixed solution is fibrinogen hydrogel.
Specifically, the final concentration of thrombin in the mixed solution is 3U, the concentration of fibrinogen is 2.5mg/mL-5.0mg/mL, and the density of endothelial cells is 3X 10 6/mL-5X 10 6/mL.
In particular, the vascularized tumor organoid medium includes tumor organoid medium and endothelial cell medium.
Specifically, the adding ratio of the tumor organoid culture medium to the endothelial cell culture medium is 1:1-1:2.
According to a second aspect of the present application, a vascularized tumor organoid is presented, which is prepared by the above-described method of constructing a tumor organoid.
In particular, the tumor organoids include vascularized solid tumor organoids.
Specifically, the vascularized solid tumor organoid is a vascularized malignant solid tumor organoid. Or the vascularized malignant solid tumor organoid comprises vascularized lymphoma organoid, vascularized lung cancer organoid, vascularized pancreatic cancer organoid, vascularized breast cancer organoid, vascularized ovarian cancer organoid, vascularized cervical cancer organoid, vascularized endometrial cancer organoid, vascularized prostate cancer organoid, vascularized stomach cancer organoid, vascularized kidney cancer organoid, vascularized colon cancer organoid, vascularized rectal cancer organoid, vascularized liver cancer organoid, vascularized bladder cancer organoid, vascularized esophagus cancer organoid, vascularized bile duct cancer organoid, vascularized nasopharyngeal cancer organoid, vascularized glioma organoid, vascularized melanoma organoid, vascularized skin cancer organoid, vascularized head and neck cancer organoid, vascularized thyroid cancer organoid, vascularized osteosarcoma organoid or vascularized cartilage sarcoma organoid.
According to a third aspect of the present application, there is provided the use of vascularised tumour organoids, including in pharmaceutical screening, preparation of in vitro diagnostic reagents and scientific research for in vitro non-therapeutic purposes.
In the application, firstly, a tumor organoid is constructed, then the tumor organoid and endothelial cells are co-cultured through a chip to provide micro-flow and fluid shear force for the endothelial cells, thereby wrapping the vascular network of the tumor organoid and successfully constructing the vascularized tumor organoid; the vascularized tumor organoid is similar to the in vivo tumor angiogenesis mode, so that the in vivo state of the tumor organoid is more truly simulated, and a more reliable biological sample is provided for in vitro research of tumors.
Additional features and advantages of the application will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a simplified flow chart of a method of constructing a tumor organoid of the present application.
FIG. 2 is a flow chart of a method of constructing a tumor organoid of the present application.
FIG. 3 is a flow chart of a method of constructing a tumor organoid according to an embodiment of the application.
Fig. 4 is a schematic diagram of a chip according to an embodiment of the application.
Fig. 5 is a morphology diagram of a vascularized ovarian cancer organoid according to example 1 of the present application in the open field.
FIG. 6 is a morphology of vascularized ovarian cancer organoid immunofluorescence according to example 1 of the present application.
FIG. 7 is a graph of immunofluorescence identification of blood vessels and ovarian cancer organoids in vascularized ovarian cancer organoids in accordance with example 1 of the present application.
FIG. 8 is a diagram of immunofluorescence identification of vascularized ovarian cancer organoids in accordance with example 1 of the present application.
FIG. 9 is an immunofluorescence identification chart of vascularized breast, bladder and liver organoids constructed in comparative example 1.
FIG. 10 is a diagram showing immunofluorescence identification of vascularized breast cancer organoids constructed in comparative example 2.
Detailed Description
The technical scheme of the present application will be described in detail below with reference to the accompanying drawings in combination with embodiments.
The reagents used in the present application are commercially available unless otherwise specified. The experimental procedures for specific implementation conditions are not specified in the examples and are generally carried out according to conventional conditions or conditions recommended by the manufacturer or with reference to the specific procedures listed in the "molecular cloning Experimental guidelines (fourth edition) J.
The inside or the periphery of the tumor in the body is rich in blood vessels, the blood vessels can provide oxygen and nutrient substances for the inside of tumor tissues, and vascular endothelial cells can secrete various cytokines to influence the growth of the tumor. The vascularized tumor organoid in the present application refers to a tumor organoid which is co-cultured with endothelial cells to form a capillary network containing tumor organoid.
According to one aspect of the present application, there is provided a method of constructing a tumor organoid, the method comprising: firstly, constructing a tumor organoid, and then co-culturing the tumor organoid with endothelial cells to obtain the vascularized tumor organoid.
In an in vivo environment, a solid tumor of smaller volume can obtain oxygen and nutrients by diffusion, but when the tumor volume is greater than 2mm 3, new blood vessels need to be formed inside the tumor to satisfy nutrition and metabolism inside tumor tissues. Therefore, on the basis of the tumor organoid formed in vitro, the application vascularizes the tumor organoid, provides a dynamic environment similar to that in vivo, better simulates the angiogenesis of solid tumors in vivo, reduces the microenvironment and growth condition of tumor tissues in vivo, and provides a reliable biological sample for in vitro research of tumors.
According to a preferred embodiment of the present application, the construction method may include: obtaining primary tumor cells; the primary tumor cells can be placed in a tumor organoid medium containing matrigel for culture to obtain a tumor organoid; the tumor organoid is mixed with endothelial cells for culture to obtain vascularized tumor organoid.
In order to simulate the condition of a tumor tissue in vivo, the normal growth and function of the tumor organoid is ensured, in particular, the diameter of the tumor organoid to be cultured in admixture with said endothelial cells is less than 150 μm, preferably 80 μm-100 μm. In mammals, the diffusion depth of oxygen, nutrients and metabolic waste is 100 μm-200 μm, and tumor tissues which cannot obtain oxygen and nutrients can be necrotized, so as to ensure the activity of tumor cells in the tumor organoids, and vascularized tumor organoids are constructed on the tumor organoids with diameters smaller than 100 μm.
In order to form vascular structures in vivo, the endothelial cells can be at least one of HUVEC endothelial cells, endothelial cells obtained by inducing the differentiation of pluripotent stem cells and endothelial cells obtained by separating tumor tissues, preferably HUVEC endothelial cells, and the HUVEC endothelial cells are human umbilical vein endothelial cells, are convenient to obtain materials, have fewer mixed cells, have the potential of stem cells and can be stably passaged.
In order to more realistically mimic the tumor microenvironment, in addition to the blood vessels that may be contained in the tumor tissue, at least one of immune cells and cancer-associated fibroblasts may be included in the vascularized tumor organoid. The immune cells may include at least one cell of B cells, T cells, tumor-associated macrophages, dendritic cells, tumor-associated neutrophils, myeloid-derived suppressor cells, monocytes, mast cells, eosinophils, and NK cells. Immune cells in tumor tissues participate in immunosuppression of tumor microenvironment, help tumor cells realize immune escape, and promote tumor development. The cytokines secreted by the cancer-related fibroblasts can realize the regulation and control of tumor cells and immune cells, and the tumor cells, the immune cells and the cancer-related fibroblasts form a complex information network to participate in the tumor progression process.
According to one embodiment of the present application, the mixed culture of the tumor organoid and the endothelial cells can be specifically prepared as a mixed solution of the tumor organoid and the endothelial cells, thrombin and protein hydrogel solution. Thrombin in the mixed solution can coagulate the protein hydrogel solution to form a hydrogel, thereby coating both the tumor organoid and the endothelial cells in the hydrogel.
More specifically, according to one exemplary embodiment, the mixed solution is injected into a tissue culture zone, and after the protein hydrogel solution solidifies, vascularized tumor organoid medium may be added for culture, providing micro-flow and fluid shear forces to endothelial cells.
As shown in FIG. 4, the chip comprises a tissue culture area 3 (a substance exchange section) and culture medium channels 6 (comprising a culture medium inlet 1 and a culture medium outlet 4) positioned at the left side and the right side of the tissue culture area, when the chip is used, the mixed solution is added into a hydrogel inlet 2, excessive mixed solution can flow out through a hydrogel outlet 5, and after the hydrogel is fixed, vascularized tumor organoid culture medium is added into the culture medium inlet 1. By using the chip, micro-flow and fluid shearing force are provided for endothelial cells through the pressure difference between the culture medium inlet 1 and the culture medium outlet 4, so that the endothelial cells spontaneously or self-assemble to form blood vessels through the fluid shearing force, and the formation of a blood vessel network is promoted.
The chip in the present application may be an existing chip device for 3D culture of cells and capable of providing fluid shear force, such as various culture devices capable of shaking the chip, or various suitable microfluidic chips, such as the chip devices employed in the prior art mentioned in the background.
In order to simulate the extracellular matrix microenvironment of tumor tissue in vivo, in particular, the protein hydrogel in the mixed solution may be a fibrinogen hydrogel. The fibrinogen may be rabbit fibrinogen, preferably bovine fibrinogen. The bovine fibrinogen has higher biocompatibility and can better simulate the in-vivo extracellular matrix microenvironment.
In order to allow vascularization of the tumor organoids, in particular, the final concentration of thrombin in the mixed solution may be 3U, the concentration of fibrinogen may be 2.5mg/mL-5.0mg/mL, and the density of endothelial cells may be 3X 10 6/mL-5X 10 6/mL.
For administration of endothelial cells and tumor organoids, in particular, the vascularized tumor organoid medium may comprise a tumor organoid medium and an endothelial cell medium. The adding ratio of the tumor organoid culture medium to the endothelial cell culture medium is 1:1-1:2.
The tumor organoid culture medium is a basic culture medium which is added according to different choices of the cultured tumor organoids and contains different cytokines, wherein the cytokines can be one or a combination of any several kinds of FGF 2、FGF 7、FGF8、EGF、BMP-4、HGF、Wnt 5A、FGF 10、Y27632、SB202190、A83-01、N-Acetylcysteine、B27、Nicotinamide、GlutaMax、Hepes、Primocin、Noggin、R-Spoondin 1、SB431542, and the cytokines are not limited to the cytokines listed above and can also comprise cytokines which can be beneficial to culturing different tumor organoids; the basal medium may be at least one of DMEM/F12 medium, DMEM medium, RPMI-1640 medium, MEM medium, BME medium, IMDM medium, ham's F-10 medium, 199 medium, mcCoy5A medium and L15 medium, preferably DMEM/F12 medium.
According to a second aspect of the present application, a vascularized tumor organoid is presented, which is prepared according to the above-described method of constructing a tumor organoid.
In particular, the tumor organoid may comprise a vascularized solid tumor organoid.
In particular, the vascularized solid tumor organoid may be a vascularized malignant solid tumor organoid; or the vascularized malignant solid tumor organ may comprise a vascularized lymphoma tumor organ, a vascularized lung cancer organ, a vascularized pancreatic cancer organ, a vascularized breast cancer organ, a vascularized ovarian cancer organ, a vascularized cervical cancer organ, a vascularized endometrial cancer organ, a vascularized prostate cancer organ, a vascularized gastric cancer organ, a vascularized renal cancer organ, a vascularized colon cancer organ, a vascularized rectal cancer organ, a vascularized liver cancer organ, a vascularized bladder cancer organ, a vascularized esophageal cancer organ, a vascularized bile duct cancer organ, a vascularized nasopharyngeal cancer organ, a vascularized glioma organ, a vascularized melanoma organ, a vascularized skin cancer organ, a vascularized head and neck cancer organ, a vascularized thyroid cancer organ, a vascularized osteosarcoma organ, or a vascularized cartilage sarcoma organ.
1. Examples
EXAMPLE 1 construction of vascularized ovarian cancer organoids
The embodiment provides a construction method for constructing vascularized ovarian cancer organoids, which comprises the following steps:
a1: placing ovarian cancer tissues in a cell culture dish, adding a proper amount of PBS solution containing 1% of double antibodies, cleaning for 3-5 times, and removing redundant blood; removing non-tumor tissues (connective tissues and blood vessels) by using an ophthalmic scissors and an ophthalmic forceps, and cutting the tissues into blocks with the size of 1 mm-3 mm;
A2. Placing the tissues into a 50mL centrifuge tube, adding a proper amount of tissue digestion solution (containing 1mg/mL collagenase I solution, 1% double antibody, 10 mu M Rock inhibitor and DMEM F12 basal medium, wherein the Rock inhibitor can be Y27632, preferably Y33075 dihydrochloride) to permeate the tissues, and shearing the tissues into minced meat by using an ophthalmic scissors; adding 10mL of digestive juice, and digesting for 45min at 37 ℃;
A3. After digestion is completed, the digested cell suspension is filtered by a cell sieve with a pore diameter of 100 mu m, the filtrate is transferred to a new sterile 50mL centrifuge tube, and a large amount of PBS solution is added to terminate digestion;
A4. Centrifuging the solution at 4deg.C for 2000RPM for 10min, discarding supernatant (if red blood cells are found after discarding supernatant, adding 1mL of red blood cell lysate, mixing, transferring to 15mL centrifuge tube, standing for 3min after transferring, adding a large amount of PBS solution, stopping lysis, centrifuging the solution at 200g,5min, 4deg.C, discarding supernatant, and retaining precipitate);
A5. adding appropriate amount of ovarian cancer organoid culture medium (Boframe biotechnology Co., ltd., K2168-OC) into the precipitate, and filtering with 200 mesh sterile filter screen; adding a proper volume of matrigel (final matrigel concentration is 70%) into the filtered solution, and blowing on ice for uniform mixing to obtain a mixed solution;
A6. loading the mixed solution into a low-adsorption 96-well cell culture plate, wherein each well is about 20 mu L, and placing the cell culture plate in an incubator for 60min after loading;
A7. adding 100 mu L of ovarian cancer organoid culture medium into each hole, and placing into a 37 ℃ and 5% CO 2 cell incubator for culture;
A8. ovarian cancer organoids were observed every 48 hours for growth, for example: status and size of ovarian cancer organoids; the next test was performed according to the growth conditions.
A9. When the size of the ovarian cancer organoids reaches the requirement, discarding the ovarian cancer organoids culture medium in the hole of the cell culture plate, and adding 1mg/mL of disperse enzyme solution into the hole, wherein 100 mu L of disperse enzyme solution is added into each hole;
A10. placing the cell culture plate in a cell culture box for 40min to dissociate matrigel;
A11. after the matrigel is dissociated, collecting ovarian cancer organoids in the holes in a centrifuge tube, centrifuging for 5min at 200g, and removing the supernatant; adding an ovarian cancer organoid culture medium to resuspend the ovarian cancer organoid, filtering by using a cell sieve, and collecting the ovarian cancer organoid with the diameter of 80-100 mu m;
A12. Uniformly mixing the collected ovarian cancer organoid with endothelial cell suspension and bovine fibrinogen solution to obtain a mixed solution, adding a proper amount of thrombin solution, wherein the final concentration of bovine fibrinogen in the mixed solution is 2.5mg/mL, the final concentration of thrombin solution is 3U, and the density of endothelial cells is 5 multiplied by 10 6/mL;
A13. The mixed solution is quickly injected into a tissue culture area of a chip through a tissue culture area loading port on the chip; placing into a cell incubator for incubation for 15-20 min to polymerize bovine fibrinogen into hydrogel;
A14. After the hydrogel is formed, adding fresh vascularized ovarian cancer organoid culture medium (vascularized ovarian cancer organoid culture medium is ovarian cancer organoid culture medium (primary frame biotechnology Co., ltd., K2168-OC) and endothelial cell culture medium (scientific research laboratory, 1001) 1:1 (v/v)) into a culture medium channel of the chip, and placing the chip into a cell culture box for culture;
A15. fresh vascularized ovarian cancer organoids medium was added daily to the chip and the vascularized state of the ovarian cancer organoids was observed under a microscope.
EXAMPLE 2 construction of vascularized breast cancer organoids
The embodiment provides a construction method for constructing vascularized breast cancer organoids, which comprises the following steps:
a1: placing breast cancer tissues in a cell culture dish, adding a proper amount of PBS solution containing 1% of double antibodies, cleaning for 3-5 times, and removing redundant blood; removing non-tumor tissues (connective tissues and blood vessels) by using an ophthalmic scissors and an ophthalmic forceps, and cutting the tissues into blocks with the size of 1 mm-3 mm;
A2. Placing the tissues into a 50mL centrifuge tube, adding a proper amount of tissue digestion solution (containing 1mg/mL collagenase I solution, 1% diabody and DMEM F12 basic culture medium) to submerge the tissues, and shearing the tissues into minced meat by using an ophthalmic scissors; adding 10mL of digestive juice, and digesting for 45min at 37 ℃;
A3. After digestion is completed, the digested cell suspension is filtered by a cell sieve with a pore diameter of 100 mu m, the filtrate is transferred to a new sterile 50mL centrifuge tube, and a large amount of PBS solution is added to terminate digestion;
A4. Centrifuging the solution at 4deg.C for 2000RPM for 10min, discarding supernatant (if red blood cells are found after discarding supernatant, adding 1mL of red blood cell lysate, mixing, transferring to 15mL centrifuge tube, standing for 3min after transferring, adding a large amount of PBS solution, stopping lysis, centrifuging the solution at 200g,5min, 4deg.C, discarding supernatant, and retaining precipitate);
A5. Adding appropriate amount of breast cancer organoid culture medium (Boframe biotechnology Co., ltd., K2147-BC) into the precipitate, resuspending, and filtering with 200 mesh sterile filter screen; adding a proper volume of matrigel (final matrigel concentration is 70%) into the filtered solution, and blowing on ice for uniform mixing to obtain a mixed solution;
A6. loading the mixed solution into a low-adsorption 96-well cell culture plate, wherein each well is about 20 mu L, and placing the cell culture plate in an incubator for 60min after loading;
A7. 100 mu L of breast cancer organoid culture medium is added into each hole, and the mixture is placed into a cell incubator with 5% CO 2 for culture at 37 ℃;
A8. The growth of breast cancer organoids was observed every 48 hours, for example: the state and size of breast cancer organoids; the next test was performed according to the growth conditions.
A9. When the size of the breast cancer organoid reaches the requirement, discarding the breast cancer culture medium in the hole of the cell culture plate, and adding 1mg/mL of disperse enzyme solution into the hole, wherein 100 mu L of disperse enzyme solution is added into each hole;
A10. placing the cell culture plate in a cell culture box for 40min to dissociate matrigel;
A11. After the matrigel is dissociated, collecting breast cancer organoids in the holes in a centrifuge tube, centrifuging for 5min at 200g, and removing the supernatant; adding a breast cancer organoid culture medium to resuspend the breast cancer organoid, filtering by using a cell sieve, and collecting the breast cancer organoid with the diameter of 80-100 mu m;
A12. Uniformly mixing the collected breast cancer organoid with endothelial cell suspension and bovine fibrinogen solution to obtain a mixed solution, adding a proper amount of thrombin solution, wherein the final concentration of bovine fibrinogen in the mixed solution is 3.5mg/mL, the final concentration of thrombin solution is 3U, and the density of endothelial cells is 3X 10 6/mL;
A13. The mixed solution is quickly injected into a tissue culture area of a chip through a tissue culture area loading port on the chip; placing into a cell incubator for incubation for 15-20 min to polymerize bovine fibrinogen into hydrogel;
A14. After the hydrogel is formed, adding fresh vascularized breast cancer organoid culture medium (vascularized breast cancer organoid culture medium is breast cancer organoid culture medium (primary frame biotechnology Co., ltd., K2147-BC) and endothelial cell culture medium (scientific research laboratory, 1001) 1:1.5 (v/v) into a culture medium channel of the chip, and then placing the chip into a cell culture box for culture;
A15. Fresh vascularized breast cancer organoid medium was added to the chip daily and the vascularized state of the breast cancer organoid was observed under a microscope.
EXAMPLE 3 construction of vascularized renal carcinoma organoids
The embodiment provides a construction method for constructing vascularized kidney cancer organoids, comprising the following steps:
a1: placing kidney cancer tissue in a cell culture dish, adding a proper amount of PBS solution containing 1% of double antibodies, cleaning for 3-5 times, and removing redundant blood; removing non-tumor tissues (connective tissues and blood vessels) by using an ophthalmic scissors and an ophthalmic forceps, and cutting the tissues into blocks with the size of 1 mm-3 mm;
A2. Placing the tissues into a 50mL centrifuge tube, adding a proper amount of tissue digestion solution (0.25% EDTA-pancreatin digestion solution) to submerge the tissues, and shearing the tissues into minced meat by using an ophthalmic scissors; adding 10mL of digestive juice, and digesting for 45min at 37 ℃;
A3. after digestion is completed, the digested cell suspension is filtered by a cell sieve with a pore diameter of 100 mu m, the filtrate is transferred to a new sterile 50mL centrifuge tube, and a large amount of PBS solution is added to stop digestion;
A4. Centrifuging the solution at 4deg.C for 2000RPM for 10min, discarding supernatant (if red blood cells are found after discarding supernatant, adding 1mL of red blood cell lysate, mixing, transferring to 15mL centrifuge tube, standing for 3min after transferring, adding a large amount of PBS solution, stopping lysis, centrifuging the solution at 200g,5min, 4deg.C, discarding supernatant, and retaining precipitate);
A5. Adding appropriate amount of kidney cancer organoid culture medium (Boframe biotechnology Co., ltd., K2171-KC) into the precipitate, resuspending, and filtering with 200 mesh sterile filter screen; adding a proper volume of matrigel (final matrigel concentration is 70%) into the filtered solution, and blowing on ice for uniform mixing to obtain a mixed solution;
A6. loading the mixed solution into a low-adsorption 96-well cell culture plate, wherein each well is about 20 mu L, and placing the cell culture plate in an incubator for 60min after loading;
A7. Adding 100 μl of kidney cancer organoid culture medium into each well, and culturing in a 5% CO 2 cell incubator at 37deg.C;
A8. the growth of the kidney cancer organoids was observed every 48 hours, for example: status and size of kidney cancer organoids; the next test was performed according to the growth conditions.
A9. The medium in the wells of the cell culture plate was discarded, and a dispersion enzyme solution (1 mg/mL) was added to the wells, 100. Mu.L per well;
A10. Placing the cell culture plate into a cell culture box for about 40min to dissociate the matrigel;
A11. After the matrigel is dissociated, collecting kidney cancer organoids in the holes in a centrifuge tube, centrifuging for 5min at 200g, and removing the supernatant; adding kidney cancer organoid culture medium to resuspend organoid, filtering with cell sieve, collecting kidney cancer organoid with diameter of 80-100 μm;
A12. Uniformly mixing the collected organoids with endothelial cell suspension and bovine fibrinogen solution to obtain a mixed solution, adding a proper amount of thrombin solution, wherein the final concentration of bovine fibrinogen in the mixed solution is 4.5mg/mL, the final concentration of thrombin solution is 3U, and the density of endothelial cells is 4 multiplied by 10 6/mL;
A13. The mixed solution is quickly injected into a tissue culture area of a chip through a tissue culture area loading port on the chip; placing into a cell incubator for incubation for 15-20 min to polymerize bovine fibrinogen into hydrogel;
A14. After the hydrogel is formed, adding fresh vascularized kidney cancer organoid medium (vascularized kidney cancer organoid medium is kidney cancer organoid medium (primary frame biotechnology Co., ltd., K2171-KC) and endothelial cell medium (scientific research laboratory, 1001) 1:2 (v/v) into the culture medium channel of the chip, and culturing the chip in a cell culture box;
A15. Fresh vascularized kidney cancer organoid medium was added daily to the chip and the vascularized state of the kidney cancer organoid was observed under a microscope.
Comparative example 1
This comparative example is a construction of vascularized tumor organoids by mixed culture of tumor cells and endothelial cells, and is described in detail in chinese patent CN 115927164B.
Comparative example 2
The contrast example is to construct vascularized tumor balls by utilizing a microfluidic chip, specifically, pre-culturing tumor cells and fibroblasts into tumor balls, inoculating the tumor balls into a middle channel of the microfluidic chip, and planting endothelial cells into channels on two sides, thereby constructing the vascularized tumor balls. Articles with detailed reference Nashimoto et al (2020) Vascularized cancer on a chip:The effect of perfusion on growth and drug delivery of tumor spheroid.
2. Test method
2.1 Morphological observations of vascularized tumor organoids
The vascularization of ovarian cancer organoids in the chip at the bright field was observed daily using an inverted microscope (Mingmei phototechnology Co., guangzhou, inc.), and after vascularization had formed, a photograph was taken.
2.2 Immunological identification of vascularized tumor organoids
After vascularized ovarian cancer organoids in the chip are formed, the culture medium is removed, and the treatment is carried out according to the conventional cell immunofluorescence staining steps, which are simply described as follows, the vascularized ovarian cancer organoids are fixed, the vascular ovarian cancer organoids are transparent, antigen is repaired, primary antibody is incubated, secondary antibody is incubated and sealed, after the sealing, the vascular ovarian cancer organoids are observed and photographed by using an inverted fluorescence confocal microscope (FV 1000, olin Bass Co., ltd.), F-actin is filiform actin, and is commonly used for searching for the distribution and local directional change of filiform actin of cytoskeleton; EPCAM is commonly used to label ovarian cancer cells as one of the biomarkers for early detection and prognosis of ovarian cancer; CD31 is a platelet-endothelial cell adhesion molecule that is widely present in blood cells as well as vascular endothelial cells, and is commonly used to demonstrate the presence of junctions between endothelial cells and to assess angiogenesis; DAPI, 4', 6-diamidino-2-phenylindole, is a fluorescent dye capable of binding strongly to DNA, and is commonly used for locating nuclei.
3. Test results
3.1 Morphological identification of vascularized tumor organoids
The morphology of the vascularized ovarian cancer organoids in example 1 is shown in fig. 5 and 6, wherein fig. 5 and 6 are morphology diagrams of the same vascularized organoids under bright field and immunofluorescence, respectively, the yellow dotted line in fig. 5 is labeled as ovarian cancer organoids, and the green fluorescence signal in fig. 6 represents the labeled protein F-actin of cytoskeleton; blue fluorescent signal indicates DAPI-labeled nuclei. From fig. 5 and 6, it can be seen that endothelial cells are reticulately distributed around the ovarian cancer organoids and have complex interconnecting and bifurcation structures, exhibiting similar characteristics to blood vessels in vivo, indicating that endothelial cells form a vascular network around the ovarian cancer organoids.
3.2 Immunological identification of vascularized tumor organoids
FIGS. 7 and 8 show the distribution of ovarian cancer cells and endothelial cells in a vascularized ovarian cancer organoid according to example 1 of the present application. The green fluorescent signal in fig. 7 represents the marker protein EPCAM of ovarian cancer cells; red fluorescent signal indicates the marker protein CD31 of vascular endothelial cells; blue fluorescent signal indicates DAPI-labeled nuclei; the green fluorescent signal in FIG. 8 represents the cytoskeletal marker protein F-actin; red fluorescent signal indicates the marker protein CD31 of vascular endothelial cells; blue fluorescent signal indicates DAPI-labeled nuclei.
FIG. 9 is a graph showing the vascularized breast, bladder and liver organoids of comparative example 1 (green fluorescent signal for endothelial cells, red fluorescent signal for tumor cells, blue fluorescent signal for nuclei).
FIG. 10 is a vascularized tumor pellet of comparative example 2 (red fluorescent signal for endothelial cells, yellow fluorescent signal for MCF-7 breast cancer cells, blue fluorescent signal for fibroblasts).
As can be seen from fig. 7, in the vascularized ovarian cancer organoids of example 1, the expression of the marker protein CD31 of vascular endothelial cells was observed, more at the ovarian cancer organoids; from fig. 8, it can be seen that endothelial cells elongate in shape and interconnect, migrate, and accumulate more in ovarian carcinoma organoids. The ovarian cancer organoid of the application is wrapped by a microvascular network, so that the vascularized ovarian cancer organoid is successfully constructed; in fig. 9, it can be seen that the tumor cells and endothelial cells in comparative example 1 are symbiotic and cross each other to form a structure similar to blood vessels on the surface and inside of organoids, but the dynamic process of stimulating endothelial cell migration in vivo due to fluid shear force and micro-flow cannot be represented by static co-culture of tumor cells and endothelial cells, and the formation difference of vascular network in vivo tumor tissue is large, and the formation of vascular network on in vivo tumor tissue cannot be represented; as can be seen from fig. 10, the formation of microvessels is seen on both surfaces of the tumor sphere, and it is difficult to form a vascular network surrounding the tumor sphere, and furthermore, the tumor sphere constructed from a tumor cell line and fibroblasts, which lacks tumor heterogeneity, is unable to simulate the in vivo tumor stimulated angiogenesis. Therefore, the vascularized tumor organoid constructed by the application is similar to angiogenesis of in-vivo tumor tissues, a vascular network surrounding the tumor organoid can be formed, the tumor heterogeneity is well reserved by the tumor organoid surrounded by the microvessels, and the construction method of the vascularized tumor organoid is further proved to be feasible.
In the application, firstly, a tumor organoid is constructed, then the tumor organoid and endothelial cells are co-cultured through a chip to provide micro-flow and fluid shear force for the endothelial cells, thereby forming a vascular network surrounding the tumor organoid, and the vascularized tumor organoid is successfully constructed; the vascularized tumor organoid is similar to the in vivo tumor angiogenesis mode, so that the in vivo state of the tumor organoid is more truly simulated, and a more reliable biological sample is provided for in vitro research of tumors.
According to a third aspect of the present application, there is provided the use of vascularised tumour organoids, including in pharmaceutical screening, preparation of in vitro diagnostic reagents and scientific research for in vitro non-therapeutic purposes.
The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the scope of the technical concept of the present application, and all the simple modifications belong to the protection scope of the present application.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the application can be made without departing from the spirit of the application, which should also be considered as disclosed herein.
Claims (15)
1. The method for constructing the tumor organoid is characterized by comprising the following steps: firstly, constructing a tumor organoid, and then co-culturing the tumor organoid with endothelial cells to obtain the vascularized tumor organoid.
2. The method of constructing a tumor organoid according to claim 1, comprising: obtaining primary tumor cells; placing the primary tumor cells in a tumor organoid culture medium containing matrigel for culture to obtain a tumor organoid; the tumor organoid is mixed with endothelial cells for culture to obtain vascularized tumor organoid.
3. The method of claim 1, wherein the diameter of the tumor organoid to be mixed with the endothelial cells is less than 150 μm, preferably 80 μm-100 μm.
4. The method of claim 1, wherein the endothelial cells are at least one of HUVEC endothelial cells, endothelial cells obtained by inducing differentiation of pluripotent stem cells, and endothelial cells obtained by isolating tumor tissue.
5. The method of claim 1, wherein the vascularized tumor organoid comprises at least one of immune cells, cancer-associated fibroblasts, and adipocytes.
6. The method of claim 1, wherein the step of culturing the tumor organoid in a mixed solution with endothelial cells comprises mixing the tumor organoid with a hydrogel solution of endothelial cells, thrombin and protein.
7. The method of claim 5, wherein the mixed solution is injected into a tissue culture zone, and the protein hydrogel solution is added to the vascularized tumor organoid medium after solidification to provide micro-flow and fluid shear force to endothelial cells.
8. The method of claim 5, wherein the protein hydrogel in the mixed solution is fibrinogen hydrogel.
9. The method according to claim 5, wherein the final concentration of thrombin in the mixed solution is 3U, the concentration of fibrinogen is 2.5mg/mL-5.0mg/mL, and the density of endothelial cells is 3X 10 6/mL-5X 10 6/mL.
10. The method of claim 5, wherein the vascularized tumor organoid medium comprises a tumor organoid medium and an endothelial cell medium.
11. The method of claim 9, wherein the tumor organoid medium and endothelial cell medium are added in a ratio of 1:1-1:2.
12. A vascularized tumor organoid prepared according to the method of constructing a tumor organoid of any of claims 1-11.
13. The vascularized tumor organoid of claim 12, comprising a vascularized solid tumor organoid.
14. The vascularized tumor organoid of claim 13,
The vascularized solid tumor organoid is a vascularized malignant solid tumor organoid; or alternatively
The vascularized malignant solid tumor organ comprises vascularized lymphoma organ, vascularized lung cancer organ, vascularized pancreas cancer organ, vascularized breast cancer organ, vascularized ovary cancer organ, vascularized cervical cancer organ, vascularized endometrium cancer organ, vascularized prostate cancer organ, vascularized stomach cancer organ, vascularized kidney cancer organ, vascularized colon cancer organ, vascularized rectum cancer organ, vascularized liver cancer organ, vascularized bladder cancer organ, vascularized esophagus cancer organ, vascularized bile duct cancer organ, vascularized nasopharyngeal cancer organ, vascularized glioma organ, vascularized melanoma organ, vascularized skin cancer organ, vascularized head and neck cancer organ, vascularized thyroid cancer organ, vascularized osteosarcoma organ or vascularized cartilage sarcoma organ.
15. Use of a vascularized tumor organoid according to claim 12 or 13 or 14 in drug screening, preparation of in vitro diagnostic reagents and scientific research for in vitro non-therapeutic purposes.
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