Tumor organ model and culture method thereof

Abstract

The invention relates to the technical field of biology, in particular to a tumor organoid model and a culture method thereof. Microvascular fragments and two kinds of gel serve as extracellular matrixes of tumor spheres, a dynamic gas-liquid interaction culture mode of a four-layer structure is formed by the extracellular matrixes, the porous structure and the culture medium, the in-vivo angiogenesis biological process is fully simulated, dynamic culture medium perfusion provides shear stress for the interiors of blood vessels derived from the microvascular fragments, and the in-vivo angiogenesis biological process is simulated. Therefore, a perfusion effect is generated. The perfusion of blood vessels provides blood flow stimulation for epithelial tumor spheres in the hydrogel above, provides physical and chemical dual effects for the tumor spheres, and finally effectively promotes the growth and maturation of the tumor spheres.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a tumor organoid model and a culture method thereof. Background Art

[0002] The existence of cell spheres is to simulate the interaction relationship between cells or between cells and tissues, and to make up for the problems that cannot be solved by two-dimensional cultured cells. Normal organoids (refer to 3D multicellular clusters self-assembled in vitro by induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and adult stem cells (ASCs) or isolated organ progenitor cells under the induction of growth factors) or primary tumor tissues are extracted to construct tumor organoids. Compared with the sphere structure formed by single cells, organoids can better simulate the microphysiological or pathological functions of specific tissues from the molecular to the organ level in vitro. Organoids have become an ideal model for simulating the functions of in vivo organs in vitro. However, the central region in the organoids will rapidly necrosis due to the increasing metabolic demand during the long-term development process, and the cultured organoids always have the defects of immature morphological structure and function compared with the target organs in vivo. On the one hand, the growth of normal tissues and organs is inseparable from the mechanical stress generated by blood flow in vivo and the mechanical stimulation of interstitial fluid force in the blood vessels and the interstitial around the organs. On the other hand, the growth and maturation of organs are inseparable from the interaction with vascular endothelial cells. Therefore, when culturing organoids in vitro, the addition of vascular endothelial cells and the mechanical stimulation of fluid can promote the maturation of organoids. In a study published in "Nature Methods", it disclosed a microfluidic culture system for constructing a co-culture model of vascular endothelial cells and kidney organoids, and demonstrated an article that in vitro flow enhances the vascularization and maturation of kidney organoids. These evidences indicate that the existence of fluid force and blood vessels should be considered in the influence of cell spheres or organoids in vitro culture models.

[0003] Microfluidic organ chips that can generate mechanical stimulation have been designed to construct a co-culture model of blood vessels and organoids to explore the influence of fluid on the maturation of blood vessels and organoids, and the important role of mechanical factors in inducing and maintaining the functions of vascularized organoids / cell spheres has been confirmed by using microfluidic organ chips. However, the existing gas-liquid interaction culture plates cannot achieve dynamic perfusion.

[0004] The air-liquid interaction culture mode is one of the epithelial organoid culture methods. Compared with the traditional organoid culture mode, the air-liquid interaction culture mode can more fully simulate the functional state of in-vivo tissues and organs. It refers to using a well plate equipped with a chamber (referred to as the outer chamber) as the culture carrier, placing the hydrogel wrapped with cell spheres / organoids in the chamber, adding a culture medium volume in the well plate that does not submerge the hydrogel. At this time, the upper part of the hydrogel is in contact with the gas, and nutrients provide nutrients for the cells in the hydrogel through the osmosis of the porous membrane at the bottom, forming an air-liquid interaction mode. An article published in "Cell" successfully cultured tumor microenvironment organoids of different disease subtypes in vitro through the organoid construction technology of the air-liquid interaction microenvironment. It was confirmed that in this culture mode, the tumor microenvironment can retain the stromal layer of the tumor tissue, and at the gene level, it was verified that tumor organoids can retain the inherent fibrous matrix and diverse immune components in the tissue. It shows that culturing patient-derived tumor organoids by the air-liquid interaction method reproduces the patient's tumor immune microenvironment.

[0005] However, the existing air-liquid interaction culture uses static culture, lacking dynamic fluid perfusion of organoids / tumor spheres, and unable to provide mechanical stimulation of interstitial fluid force for tumor growth. When co-culturing blood vessels with tumor spheres / organoids by air-liquid interaction, using traditional endothelial cells as angiogenesis materials, the blood vessels generated by endothelial cells in vitro have immature functions and cannot simulate in-vivo blood vessels and other defects. Constructing a dynamic air-liquid interaction culture mode of blood vessels-mammary tumor spheres, fully simulating the morphological structure of blood vessels-epithelial organs in vivo, and reproducing the tumor microenvironment of patients' tumors have important value and significance in many aspects such as basic research, precision medicine, drug development, prognosis evaluation, and immunotherapy, providing great significance for the diagnosis, treatment, and research of tumors for tumor patients. Summary of the Invention

[0006] In view of this, the present invention provides a tumor organoid model and a culture method thereof. This culture method is more in line with the growth environment of breast tumors, reduces necrosis in the center of tumor spheres, promotes the growth of tumor spheres and the improvement of their functions and structures, and is beneficial to studying the growth, invasion, and metastasis mechanisms of tumor cells in vivo, developing and researching new anti-tumor drugs and treatment methods.

[0007] The culture method of tumor organoids includes the following steps:

[0008] Prepare a second gel containing tumor cells and a first gel containing microvascular fragments and thrombin in sequence on the upper and lower layers of the porous structure, add a culture medium to below the upper interface of the second gel, and perform dynamic perfusion culture at a flow rate of 1-10 ml / min;

[0009] The first gel includes a fibrin gel;

[0010] The second gel includes at least one of fibrin gel, polysaccharide-based gel or Matrigel.

[0011] In the present invention, the interface of the culture medium is lower than the lower interface of the second gel, so that the upper interface of the second gel is in contact with air; the porous structure and the first gel are completely immersed in the culture medium.

[0012] The present invention has no special limitation on the mode of dynamic perfusion culture, and perfusion culture with a flow rate of 1 to 10 ml / min can be achieved. In specific embodiments, the present invention performs perfusion of the culture medium through a hose and a peristaltic pump. The peristaltic pump communicates with the lower chamber through the hose, and the fluid culture medium is pumped out by the peristaltic pump and then perfused into the lower chamber through the hose to achieve dynamic gas-liquid interactive co-culture.

[0013] In the present invention, the preparation method of the first gel includes:

[0014] Mix fibrinogen, the first solvent and microvascular fragments, resuspend and mix evenly to obtain a first solution containing microvascular fragments;

[0015] Mix the first solution and thrombin evenly to form a gel, and obtain the first gel.

[0016] Further, the first solvent includes PBS buffer solution, high-glucose DMEM culture medium or a culture medium commonly used in the art to promote angiogenesis.

[0017] In the first solution, the concentration of fibrin is 10 mg / ml, and the concentration of microvascular fragments is 8000 / ml.

[0018] The volume ratio of the first solution to the thrombin is 1:1.

[0019] The concentration of the thrombin is 10 unit / ml.

[0020] In some embodiments, the second gel is a polysaccharide-based gel.

[0021] Further, the preparation method of the second gel includes:

[0022] Mix N-carboxyethyl chitosan and the second solvent, add the centrifuged tumor cells, resuspend and mix evenly, then add oxidized hyaluronic acid, and let it stand to form a gel.

[0023] In some embodiments, the mass ratio of N-carboxyethyl chitosan (CEC) to oxidized hyaluronic acid (OHA) is (1-2):10, preferably 1.5:10. In some specific embodiments, the mass concentration of N-carboxyethyl chitosan is 1%-2%, specifically 1.5%. The mass concentration of the oxidized hyaluronic acid is 10%.

[0024] In the present invention, the second solvent provides a suitable growth environment for the encapsulated tumor cells or spheroids. Among them, the second solvent is PBS buffer or a culture medium suitable for the growth of tumor cells, such as DMEM / F12 medium.

[0025] In the present invention, the porous structure is a culture structure with a porous membrane at the bottom. In the present invention, the porous structure is a culture device with a porous membrane at the bottom and can be embedded inside a culture plate, such as a transwell chamber.

[0026] In some specific embodiments, the above four-layer structure is realized by means of a culture plate containing a transwell (see Figure 7 the cross-sectional view of the shown dynamic gas-liquid interaction culture plate). Specifically, the first gel is made on the outer bottom (i.e., the surface after the upper chamber is flipped) of the upper chamber (i.e., the chamber in Figure 7 ), the second gel is made on the inner bottom (i.e., the bottom of the inner cavity) of the upper chamber, and then the upper chamber carrying the two gels is placed in the lower chamber, and a culture medium is added to the lower chamber to obtain a four-layer structure culture system.

[0027] The present invention uses two hydrogels as the extracellular matrix of tumor spheroids and blood vessels to form a four-layer culture mode. The top layer is a CEC-OHA hydrogel encapsulating tumor spheroids. The upper layer of the hydrogel is in contact with air, the rest of the hydrogel is in contact with the culture medium, and is in contact with blood vessels through a porous membrane to form a gas-liquid interaction mode; the second layer is a porous structure (such as a transwell with a porous membrane at the bottom), serving as a medium for the interaction between factors. The third layer is a fibrin hydrogel encapsulating microvascular fragments, providing nutrients and growth-promoting factors for tumor spheroids. The fourth layer is a flowing culture medium that covers the third layer of fibrin hydrogel, aiming to provide a perfusion function for the blood vessels in the fibrin hydrogel to generate a fluid effect of blood flow and provide richer nutrients for the growth of tumor spheroids.

[0028] In the present invention, before performing dynamic gas-liquid interaction culture, it also includes a step of static culture first to ensure that the first gel adheres sufficiently to the transwell surface. In a specific embodiment of the present invention, the time of the static culture is 1 d.

[0029] In the present invention, there are no special restrictions on the source of microvascular fragments, which can be autologous microvascular fragments or allogeneic microvascular fragments. The characteristics of the microvascular fragments include: expressing the CD31 endothelial marker, the VE-cadherin endothelial tight junction marker, and the α-SMA smooth muscle cell marker, and being microvascular fragments that can sprout blood vessels from the original blood vessel fragments. Research shows that compared with the self-organization of endothelial cells to form a vascular network, the biological process of microvascular fragment-derived capillaries in vitro is more in line with the in vivo angiogenesis process. The specific sources of the microvascular fragments include adipose tissue after liposuction surgery, etc. In a specific embodiment of the present invention, the microvascular fragments are adipose-derived microvascular fragments.

[0030] In the present invention, the microvascular fragments are not directly adhered to the bottom surface of the transwell, but fibrin hydrogel is used to wrap the blood vessel fragments to obtain a fibrin hydrogel-wrapped vascular network, which can not only provide support for the surrounding matrix of the blood vessels, enable blood vessel sprouting to connect with each other to form a vascular network, and enable the blood vessels to form a perfusion function; but also support the interaction between cells, and can more fully simulate the in vivo vascular growth environment, providing richer nutrients and protein factors for the tumor spheroids.

[0031] The present invention uses microvascular fragments combined with fibrin hydrogel, which contains various structures of arterioles, venules and capillaries, and contains characteristic markers of blood vessels. The microvascular fragments derive a capillary network from the original blood vessel fragments, fully simulating the in vivo angiogenesis biological process. The dynamic perfusion of the culture medium provides shear stress inside the microvascular fragment-derived blood vessels, thereby generating a perfusion effect. The perfusion of the blood vessels provides a blood flow stimulus for the epithelial tumor spheroids in the upper hydrogel, providing a dual physical and chemical effect on the tumor spheroids, and ultimately promoting the growth and maturation of the tumor spheroids.

[0032] In the present invention, the tumor cells include at least one of the cells of various tumors such as breast cancer, bladder cancer, lung cancer, colon cancer, etc. In some embodiments, the tumor cells are breast tumor cells; the breast tumor cells include human triple-negative breast cancer cells; the triple-negative breast cancer cells include MDA-MB-231 cells and SUM-159 cells. In some specific embodiments, the tumor cells are SUM-159 cells.

[0033] The present invention uses a dynamic gas-liquid interaction culture method to culture epithelial tumor cells. Two hydrogels are used as the extracellular matrix of tumor spheroids and blood vessels, forming a four-layer culture mode. After culturing, the tumor cells grow into tumor spheroids. The top layer (i.e., the first layer) is the hydrogel that wraps the tumor spheroid. The upper layer of the hydrogel is in contact with air. The second layer is the porous membrane structure of transwell, serving as a medium for the interaction between factors. The third layer is a fibrin hydrogel wrapped with microvascular fragments, providing nutrients and growth-promoting factors for the tumor spheroid. The fourth layer is a flowing culture medium. Among them, the lower layer of the first layer is in contact with the culture medium and the blood vessels of the second layer, forming a gas-liquid interaction mode; the purpose is to provide a perfusion function for the blood vessels in the fibrin hydrogel to generate the fluid effect of blood flow, and to provide richer nutrients for the growth of tumor spheroids.

[0034] The present invention also provides a tumor organoid model obtained by the culture method described in any one of the above.

[0035] The present invention also provides a product for culturing tumor organoids (or preparing a tumor organoid model), which includes reagent A, reagent B, and a culture medium.

[0036] In the present invention, the raw materials of reagent A include: thrombin, microvascular fragments, fibrin, and a first solvent.

[0037] In the present invention, the raw materials of reagent B include: N-carboxyethyl chitosan, oxidized hyaluronic acid, and a second solvent.

[0038] In the present invention, in reagent A, the first solvent includes PBS buffer, high-glucose DMEM medium, or a medium promoting angiogenesis.

[0039] In the present invention, in reagent B, the second solvent provides a suitable growth environment for the encapsulated tumor cells or spheroids. Among them, the second solvent is PBS buffer or a medium suitable for the growth of tumor cells, such as DMEM / F12 medium.

[0040] In the present invention, the culture medium includes a complete medium. In some embodiments, the complete medium includes DMEM / F12 medium.

[0041] The present invention also provides the application of the above-mentioned product in culturing tumor organoids or preparing a tumor organoid model.

[0042] The present invention has the following advantages:

[0043] (1) In the present invention, the fibrin hydrogel encapsulating microvascular fragments and the CEC-OHA hydrogel encapsulating tumor spheroids are indirectly co-cultured through a transwell, enabling the interaction between the secretory factors of blood vessels and tumor spheroids and the interaction between cells, making the tumor spheroids more mimic the in-vivo environment. Compared with the simple endothelial cells adhered to the transwell, there is a richer extracellular environment.

[0044] (2) The present invention uses microvascular fragments as materials for in-vitro angiogenesis. The blood vessel structures generated by the microvascular fragments express mature blood vessel markers such as α-SMA, VE-cadherin, and CD31, gathering the characteristics of in-vivo blood vessels. Compared with endothelial cells, it can rapidly generate a blood vessel network in-vitro, promote the growth and maturation of tumor spheroids, and ultimately promote the invasion of tumor spheroids.

[0045] (3) The culture method of the present invention is dynamic gas-liquid interactive co-culture, providing mechanical stimulation of interstitial fluid force for vascularized breast tumor spheroids, better mimicking the physical microenvironment during tumor growth, and promoting the angiogenesis ability and invasion and metastasis ability of breast tumor spheroids. Description of the Drawings

[0046] Figure 1 Light microscope images of tumor spheroids under six groups of culture modes.

[0047] Figure 2 Comparison results of the diameters of tumor spheroids under six groups of culture modes.

[0048] Figure 3 Expression levels of VEGF-A protein secreted by tumor spheroids under six groups of culture modes.

[0049] Figure 4 Expression levels of MMP-9 protein secreted by tumor spheroids under six groups of culture modes.

[0050] Figure 5 Fluorescence detection images of vimentin secreted by tumor spheroids under four groups of culture modes.

[0051] Figure 6 Analysis results of the fluorescence intensity of vimentin secreted by tumor spheroids under four groups of culture modes.

[0052] Figure 7 Cross-sectional view of the dynamic gas-liquid interactive culture plate.

[0053] Figure 8 Overall structure diagram of the dynamic system.

[0054] Figure 9Detection diagrams of α-SMA and VE-cadherin, which are mature vascular markers for microvascular fragments, and fluorescence co-localization analysis diagrams.

[0055] Figure 10 Characterization diagram of the perfusable function of microvascular fragments. Detailed implementation manners

[0056] The present invention provides a tumor organoid model and a cultivation method thereof. Those skilled in the art can draw on the content herein and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related 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.

[0057] The test materials adopted in the present invention are all ordinary commercially available products and can all be purchased in the market.

[0058] In the present invention, the dynamic culture plate (or static culture plate) includes a transwell and a lower chamber. Among them, the transwell, that is, the upper chamber or the small chamber of the culture plate. The small holes on the culture plate are also called the lower chamber of the culture plate. Among them, the transwell can be embedded in the lower chamber of the culture plate, and the specific structure is as Figure 7 shown.

[0059] The following further elaborates the present invention in conjunction with embodiments:

[0060] Example 1

[0061] 1.1 Preparation of fibrin hydrogel

[0062] Human fibrinogen (F3879-250MG) and human thrombin (100unit T6884-100UN) are both purchased from sigma. 1. Weigh 40 mg of fibrinogen and add it to 4 ml of PBS, dissolve it at 37 °C for 1 hour until there is no turbidity in the liquid to form a fibrin stock solution. The original unit of thrombin is 100 unit, which is pre-divided into 10 unit / ml and placed at -20 °C to avoid repeated freezing and thawing. 3. Add the fibrin stock solution and thrombin to the culture plate in a ratio of 1:1, and place it in the incubator for 10 min to form a fibrin hydrogel.

[0063] 1.2 Extraction of microvascular fragments

[0064] (1) Cut the rat adipose tissue into pieces until the fat becomes milky, measure the volume using a 15 ml centrifuge tube, and transfer it to a beaker containing a magnetic stir bar.

[0065] (2) Add collagenase NB4G with a volume twice that of the sample into a beaker. Place the beaker on a magnetic stirrer, set the temperature to 37°C, the time to 3 minutes, and the rotation speed to 440 rpm / min to initially decompose the fat. Then transfer it to a 2-ml centrifuge tube and centrifuge at 4°C for 3 minutes at a rotation speed of 660 xg / min.

[0066] (3) After centrifugation, collect the supernatant and continue enzymatic digestion at 37°C for 5 minutes at a rotation speed of 440 rpm / min. After centrifugation, collect the precipitate, resuspend it after adding 0.1% BSA-PBS, and filter out the undigested large fibrous tissues using a filter membrane with a pore size of 500 μm.

[0067] (4) After collecting the liquid, centrifuge at 4°C for 3 minutes at a rotation speed of 660 xg / min, collect the precipitate, and observe it under a microscope as microvascular fragments for standby.

[0068] 1.3 Throw the fibrin hydrogel wrapped with MVF onto the bottom surface of the transwell close to the 12-well plate.

[0069] After centrifuging 8000 microvascular fragments, add 75 μl of fibrin stock solution to the precipitate of microvascular fragments, resuspend and mix well. Use a pipette to drop the fibrin stock solution mixed with MVF onto the outer bottom surface of the inverted transwell, then drop an equal volume of thrombin (75 μl), mix well, and let it stand for 10 minutes. Then invert and nest the upper chamber of the transwell into the lower chamber of the culture plate.

[0070] 1.4 Place the polysaccharide-based hydrogel wrapped with tumor cells in the transwell.

[0071] 1.4.1 Resuscitate and culture SUM-159 cells. After the cell number increases by 80%-90%, digest and centrifuge to collect the tumor cells. Add the N-carboxyethyl chitosan stock solution to the centrifuged tumor cells, resuspend and mix well, and then drop it into the transwell (also known as the upper chamber, small chamber) of the dynamic culture well plate (see Figure 7 ). Then add the oxidized hyaluronic acid stock solution and mix well in a 1:1 ratio, and place it in a 37°C incubator and let it stand for 10 minutes to form a gel.

[0072] 1.4.2 After gel formation, take out the dynamic culture plate containing the transwell, add DMEM high-glucose medium and DMEM / F12 medium to the lower chamber in a volume ratio of 1:1. The medium should not exceed the CEC-OHA hydrogel interface to ensure that the hydrogel is in contact with air, and then place it in a 37°C incubator.

[0073] 1.4.3 Incubate statically for one day to ensure that the fibrin hydrogel adheres sufficiently to the transwell surface of the dynamic culture plate. Then insert the hose into the side hole of the lower chamber of the dynamic culture plate and connect it to a peristaltic pump (see Figure 8 ), and perform dynamic gas-liquid interaction co-culture of vascularized tumor spheroids.

[0074] After static incubation for one day, set the flow rates of the peristaltic pump to 1 ml / min and 10 ml / min respectively. Different flow rates generate different shear forces. Compare the growth states of tumor spheroids under gas-liquid interaction culture modes with different shear forces and evaluate the functions of tumor spheroids.

[0075] Example 2

[0076] In this example, the experimental grouping and culture process are as follows:

[0077] Static gas-liquid single culture: Wrap SUM-159 cell spheroids in the hydrogel according to the steps of 1.4.1 and 1.4.2 in Example 1. After placing the hydrogel in an incubator at 37 °C and waiting for gelation, place the CEC-OHA hydrogel into the transwell, place the transwell into a 12-well static culture plate, add DMEM / F12 medium to ensure that it does not cover the CEC-OHA hydrogel, and perform static gas-liquid interaction single culture.

[0078] Dynamic gas-liquid single culture: Wrap SUM-159 cell spheroids in the hydrogel according to the steps of 1.4.1 and 1.4.2 in Example 1. After placing the hydrogel in an incubator at 37 °C and waiting for gelation, place the CEC-OHA hydrogel into the transwell, place the transwell into a 12-well dynamic culture plate, add DMEM / F12 medium to ensure that it does not cover the CEC-OHA hydrogel. Then connect the dynamic culture plate to a peristaltic pump through a hose. After setting the flow rates to 1 ml / min and 10 ml / min respectively, perform dynamic gas-liquid interaction single culture, which are recorded as 1 ml / min single culture and 10 ml / min single culture respectively.

[0079] Static gas-liquid interaction co-culture: Wrap SUM-159 cells in the CEC-OHA hydrogel according to the experimental steps of 1.4.1. After placing the hydrogel in an incubator at 37 °C and waiting for gelation, place the CEC-OHA hydrogel into the transwell, nest the transwell into a static culture plate, wrap the microvascular fragments mentioned in steps 1.3 and 1.4 inside the fibrin hydrogel and adhere them to the bottom of the transwell close to the 12-well plate. Add DMEM high glucose:DMEM / F12 to the medium at a ratio of 1:1, and perform static gas-liquid interaction co-culture.

[0080] Dynamic gas-liquid interactive co-culture: Perform dynamic gas-liquid interactive culture according to the method of Example 1. Set the peristaltic pump flow rates to 1 ml / min and 10 ml / min respectively, and perform circulating perfusion culture, which are respectively recorded as 1 ml / min co-culture and 10 ml / min co-culture.

[0081] Culture according to the above six culture modes for 7 days, and conduct the following experiments.

[0082] 2.1 ELISA analysis of the expression of VEGF-A and MMP-9 secreted by tumor spheroids cultured under different conditions

[0083] (1) Sample addition: Culture according to the above six culture modes respectively. Collect the supernatant cultured for 7 days and different concentrations of standards, 50 μl each, and add them to the pre-set detection wells.

[0084] (2) Antibody reaction: Add 100 μl of detection antibody to each well containing the sample. Place the entire plate in an incubator at 37 °C and incubate in the dark for 60 minutes to allow the antibody to fully bind to the target protein.

[0085] (3) Washing step: After the antibody binding is completed, wash the wells thoroughly to remove unbound antibodies or other proteins.

[0086] (4) Substrate reaction: Add 50 μl of substrates A and B to each well, and place the plate back in the incubator again. Continue to incubate at a constant temperature in the dark for 15 minutes to promote the color reaction.

[0087] (5) Reaction termination and absorbance measurement: Add 50 μl of termination solution to each well to stop the substrate reaction. Subsequently, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of each well, which reflects the concentration of the target protein.

[0088] 2.2 Vimentin expression of tumor spheroids under different culture conditions

[0089] a) Carefully remove the hydrogel from the dynamic culture plate and place it in a confocal dish. Wash the hydrogel 3 times with DPBS.

[0090] b) Add 200 μl of fixative (formaldehyde) and incubate overnight at 4 °C.

[0091] c) Remove the fixative and wash the hydrogel 3 times with DPBS.

[0092] d) Add 200 μl of permeabilization solution (0.3% triton X-100) and incubate at room temperature for 5 min. Prolonged time will cause the hydrogel to soften or dissolve.

[0093] e) Remove the permeate solution, wash the hydrogel 3 times with DPBS, add the blocking solution (0.5% BSA-PBS), and incubate at room temperature for 60 min.

[0094] f) Add the primary antibody (vimentin 1:150) directly to the blocking solution covering the hydrogel, mix gently, and incubate overnight at 4 °C.

[0095] g) Remove the solution containing the primary antibody, wash the hydrogel 3 times with DPBS, add the secondary antibody (cy3), and incubate at 37 °C for 3 hours.

[0096] h) Remove the solution containing the secondary antibody, wash the hydrogel 3 times with DPBS.

[0097] i) Then add DAPI, cover the hydrogel, and wait for 5 minutes.

[0098] j) Obtain 3D images under an Olympus F-1000v laser confocal microscope, and keep all parameters in the laser confocal consistent during shooting.

[0099] k) Perform three-dimensional reconstruction using Imaris software, then perform fluorescence intensity analysis in Image J software, and obtain the fluorescence intensity value.

[0100] 2.3 Experimental procedures and results of tumor spheroid size comparison

[0101] To prove that the dynamic gas-liquid interaction co-culture model of vascularized tumor spheroids can better simulate the tumor growth microenvironment, we set the static gas-liquid interaction monoculture model of tumor spheroids as the control group, and set the dynamic gas-liquid monoculture of tumor spheroids, the static gas-liquid interaction co-culture model of vascularized tumor spheroids, and the dynamic gas-liquid interaction co-culture model of vascularized tumor gas as the experimental groups, and conducted a comparison of tumor sizes under six culture conditions; analyzed the expression of MMP-9 and VEGF-A proteins by ELISA method; analyzed the expression of vimentin epithelial-mesenchymal transition of tumor spheroids by fluorescence intensity.

[0102] 2.3.1 Tumor spheroid size analysis

[0103] Most of the existing tumor sphere culture environments are static culture conditions. The 3D growth of tumor cells aggregates into spheres. The defect of static culture is that the center of the tumor sphere undergoes central necrosis due to the lack of contact with the culture medium, resulting in the inability of the tumor sphere to grow too large, the failure to achieve functional and structural maturity, and the difficulty in accurately simulating in vivo tumors for drug screening. The growth of in vivo tumors is inseparable from the chemical action of vascular growth factors and the physical stimulation of interstitial fluid shear forces. The vascular secretion of growth factors promotes the growth of tumor spheres, and the perfusion of interstitial fluid shear forces dynamically transports the culture medium for tumor spheres and generates fluid shear forces to promote the growth of tumor spheres. Therefore, we adopt a dynamic vascularized gas-liquid interaction co-culture model for tumor spheres. Research has proven that this culture model is more in line with the growth environment of breast tumors, reduces central necrosis of tumor spheres, promotes the growth of tumor spheres, and promotes the improvement of function and structure.

[0104] Tumor spheres were cultured statically in gas-liquid monoculture, and dynamically in gas-liquid monoculture (perfusion at flow rates of 1 ml / min and 10 ml / min respectively). A static vascularized gas-liquid interaction co-culture model for tumor spheres and a dynamic vascularized gas-liquid interaction co-culture model for tumor gas (perfusion at flow rates of 1 ml / min and 10 ml / min respectively) were established. The sizes of the tumor spheres cultured under the six models on the 7th day were compared. The results are shown in Figures 1 - 2 。

[0105] The results showed that the average diameter of the tumor spheres grown under the co-culture model conditions at a flow rate of 10 ml / min was 145.04 μm, and the maximum diameter reached 185.39 μm. While the average diameter of the tumor spheres grown under the co-culture model conditions at a flow rate of 1 ml / min was 67.005 μm, and the maximum diameter was 81.91 μm. Although both were dynamic co-culture conditions, the diameters of the tumor spheres produced differed by more than a factor of two due to different flow rates, indicating that the shear force generated at 10 ml / min in this dynamic culture plate model was more conducive to promoting the growth of tumor spheres.

[0106] The size of the tumor spheres co-cultured with blood vessels at a flow rate of 1 ml / min was not significantly different from that of the tumor spheres in static co-culture. However, the dynamic perfusion at 1 ml / min made the growth size of the tumor spheres and their distribution more uniform.

[0107] 2.3.2 ELISA was used to measure the protein levels secreted by tumor cell spheres under four groups of culture conditions

[0108] MMP-9 is a matrix metalloproteinase that can participate in the degradation and reconstruction of the extracellular matrix, and is involved in the processes of tumor invasion, metastasis, and angiogenesis. The VEGF-A protein promotes tumor angiogenesis. The expression levels of MMP-9 and VEGF-A were measured under six groups of culture conditions. The results are shown in Figures 3 - 4 。

[0109] By comparing six groups of culture conditions, it was found that the level of MMP-9 secreted by tumor spheres under the gas-liquid interaction culture condition of vascularized tumor spheres under dynamic perfusion at 10 ml / min was the highest, proving that the tumor spheres cultured under this model were more invasive.

[0110] By comparing six groups of culture conditions, it was found that the expression of VEGF-A protein was the highest in the gas-liquid interaction culture condition of vascularized tumor spheres under dynamic perfusion at 10 ml / min, proving that culturing tumors under this condition improved the angiogenesis ability of tumor spheres and was in line with the rich blood supply environment of tumors in vivo.

[0111] The above results showed that compared with several other culture modes, the dynamic gas-liquid interaction culture method (co-culture at 1 ml / min and co-culture at 10 ml / min) of the present invention could significantly promote the growth of tumor spheres and the expression of VEGF-A and MMP-9 proteins, among which the effect of co-culture at 10 ml / min was better.

[0112] 2.3.3 Analyze the expression of vimentin by fluorescence intensity method

[0113] Vimentin is vimentin, a marker of epithelial-mesenchymal transition. Epithelial-mesenchymal transition is a key stage of cancer metastasis. Epithelial cells lose their epithelial polarity and cell-cell junctions here and turn into mesenchymal cells related to invasion. In the gas-liquid interaction co-culture model of breast tumor cell spheres and microvascular fragments, by measuring the fluorescence intensity of vimentin, it was proved that the presence of microvascular fragments would promote the expression of vimentin in tumor spheres, and under co-culture conditions, by setting the culture environment as static and dynamic for comparison, the perfusion of the culture medium promoted the expression of vimentin in tumor spheres in the co-culture model, proving that dynamic vascularized tumor spheres were more invasive and represented the maturation of the function of tumor spheres under this culture condition. Measure the expression of Vimentin in four culture modes: static monoculture, monoculture at 10 ml / min, static co-culture, and co-culture at 10 ml / min. The fluorescence detection and analysis results are shown in Figures 5 - 6 。

[0114] The results showed that compared with the static culture group, the monoculture group at 1 ml / min, and the static co-culture group, the perfusion of the culture medium at 10 ml / min (co-culture at 10 ml / min) of the present invention significantly increased the expression of vimentin in tumor spheres, suggesting that the tumor bodies obtained by the culture of the present invention were more invasive and the function was more mature.

[0115] The above results indicate that the dynamic gas-liquid interaction co-culture at 1-10 ml / min of the present invention significantly promotes the growth of tumor spheroids and the expression of VEGF-A and MMP-9 proteins. Among them, the effect of the dynamic gas-liquid interaction co-culture at 10 ml / min is the best.

[0116] 2.3.4 Analysis of the expression of mature vascular markers of microvascular fragments by fluorescence intensity method Experimental procedure: Similar to the experimental steps in Step 2.2, it includes the following steps:

[0117] a) Carefully remove the hydrogel wrapped with microvascular fragments from the dynamic culture plate and place it in a confocal dish. Wash the hydrogel 3 times with DPBS.

[0118] b) Add 200 μl of fixative (formaldehyde) and incubate at room temperature for 30 min or overnight at 4°C.

[0119] c) Remove the fixative and wash the hydrogel 3 times with DPBS.

[0120] d) Add 200 μl of permeabilization solution (0.3% triton X-100) and incubate at room temperature for 5 min. Too long incubation time will cause the hydrogel to soften or dissolve.

[0121] e) Remove the permeabilization solution, wash the hydrogel 3 times with DPBS, add blocking solution (0.5% BSA-PBS), and incubate at room temperature for 60 min.

[0122] f) Add primary antibodies (α-SMA rabbit-derived 1:150, CD31 direct-labeled primary antibody mouse-derived 1:200, and primary antibody (VE-cadherin rabbit-derived 1:100, CD31 direct-labeled primary antibody mouse-derived 1:200)) to two hydrogels covered with blocking solution respectively, gently mix, and incubate overnight at 4°C.

[0123] g) Remove the solution containing the primary antibody, wash the hydrogel 3 times with DPBS, add secondary antibodies (cy3 goat anti-rabbit, 555 goat anti-rabbit) to the 2 confocal dishes respectively, and incubate at 37°C for 3 hours.

[0124] h) Remove the solution containing the secondary antibody, wash the hydrogel 3 times with DPBS.

[0125] i) Then add DAPI to cover the hydrogel and wait for 5 minutes.

[0126] j) Obtain images under an Olympus F-1000v laser confocal microscope

[0127] k) Use imaris software for 3D reconstruction of the images, and then perform fluorescence co-localization analysis in Image J software.

[0128] Results:

[0129] Identification of vascular markers of the extracted microvascular fragments and co-localization analysis of each marker are shown in Figure 9 .

[0130] Among them, as can be seen from Figure A, the extracted microvascular fragments are Y-shaped and express the CD31 endothelial cell marker and the α-SMA smooth muscle cell marker (green: CD31; red: α-SMA; blue: nucleus).

[0131] Figure B is the co-localization signal map of the α-SMA marker and CD31 expressed by the microvascular fragments. It can be seen that the rising and falling trends of the green and red curves are consistent, indicating that the blood vessels express the endothelial cell marker (CD31) and simultaneously express the smooth muscle cell marker (α-SMA) (red curve: α-SMA; green curve: CD31).

[0132] In Figure C, the microvascular fragments are in a straight line and express the VE-Cadherin endothelial cadherin marker (yellow: VE-cadherin; green: CD31; blue: nucleus).

[0133] Figure D is the co-localization analysis result of the VE-cadherin marker and CD31 expressed by the microvascular fragments. Among them, the rising and falling trends of the green and yellow curves are consistent, indicating that the microvascular fragments express the endothelial cell marker (CD31) and simultaneously express the endothelial cadherin (VE-cadherin) (yellow curve: VE-cadherin; green curve: CD31).

[0134] The above results show that the vascular structures generated by the microvascular fragments express mature vascular markers such as α-SMA, VE-cadherin, and CD31, which is beneficial to promoting the growth and maturation of tumor spheroids and ultimately promoting the invasion of tumor spheroids.

[0135] 2.3.5 Fluorescence analysis of the perfusable function of microvascular fragments

[0136] Experimental procedure:

[0137] 1) Dilute 2-μm fluorescent microspheres in high-glucose DMEM medium at a ratio of 1:50;

[0138] 2) When the MVF is cultured to the 7th day, after removing the high-glucose DMEM medium, perform dynamic perfusion of the diluted fluorescent microspheres for 5 hours;

[0139] 3) After the perfusion is completed, wash the excess fluorescent microspheres with PBS until the turbid medium is no longer visible to the naked eye;

[0140] 4) After the cleaning is completed, perform immunofluorescence staining of primary antibody CD31 (1:100 mouse-derived secondary antibody cy3 goat anti-mouse) similar to the process in step (consistent with the immunofluorescence process of the microvascular fragments in step 2.2).

[0141] 5) Image under a confocal microscope, and then use the surface function in Imaris for blood vessel reconstruction, and use the function in Imaris to process fluorescent microspheres to prove that the fluorescent microspheres are present inside the microvascular fragments.

[0142] Stain the extracted microvascular fragments using the above method respectively. Fluorescent microspheres with a fluorescence of 2μm can be directly observed under a confocal microscope, and the position relationship between the fluorescent microspheres and MVF is presented in multiple layers through Imaris software. The results are shown in Figure 10 .

[0143] Figure 10 In [reference], A shows the lumen characterization result of the microvascular fragments through the z-stack axis after three-dimensional reconstruction of the image using Imaris software. The lumen structure can be seen in both the x-z cross-section and the y-z cross-section (the part indicated by the yellow arrow) (red: vimentin, blue: DAPI).

[0144] Figure 10 In [reference], in Figure B, i shows that the fluorescent microspheres are inside the microvascular fragments (green: fluorescent microspheres μ-Beads, red: CD31, blue: DAPI); ii shows the characterization of the relationship between the fluorescent microspheres and the microvascular fragments through Z-stack (the part selected by the yellow box is the fluorescent microspheres inside the MVF, and the part indicated by the yellow arrow is the fluorescent microspheres inside the MVF); iii shows a cross-section after three-dimensional reconstruction, with the fluorescent microspheres inside the microvascular fragments (the yellow arrow indicates the microvascular fragments, red: CD31, green: fluorescent microspheres); iv shows the fluorescent microspheres inside the microvascular fragments (microsphere rendering in Imaris); v shows the rendering characterization of blood vessels using the surface function in Imaris.

[0145] It can be seen from Figure 10 that the fluorescent microspheres are present inside the lumen of the microvascular fragments, indicating that the microvascular fragments have perfusion function.

[0146] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, 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 method for culturing tumor organoids, characterized in that, It includes the following steps: Prepare a second gel containing tumor cells and a first gel containing microvascular fragments and thrombin successively on the upper and lower layers of the porous structure. Add a culture medium below the upper interface of the second gel and perform dynamic perfusion culture at a flow rate of 1-10 ml / min; The first gel includes a fibrin gel; The second gel includes at least one of a fibrin gel, a polysaccharide-based gel, or Matrigel.

2. The cultivation method according to claim 1, characterized in that, The preparation method of the first gel includes: Mix fibrin, a first solvent, and microvascular fragments, resuspend and mix well to obtain a first solution containing microvascular fragments; Mix the first solution and thrombin to form a gel to obtain the first gel; The second gel is a polysaccharide-based gel, and the preparation method of the second gel includes: Mix N-carboxyethyl chitosan and a second solvent, add the centrifuged tumor cells, resuspend and mix well, then add oxidized hyaluronic acid, let it stand to form a gel to obtain the first gel.

3. The culture method according to claim 2, wherein The first solvent includes PBS buffer or high-glucose DMEM medium; In the first solution, the concentration of fibrin is 10 mg / ml, and the concentration of microvascular fragments is 8000 / ml; The volume ratio of the first solution to the thrombin is 1:1; The concentration of the thrombin is 10 unit / ml.

4. The culture method according to claim 2, wherein The mass ratio of N-carboxyethyl chitosan to oxidized hyaluronic acid is (1-2):10; The second solvent includes PBS buffer or DMEM / F12 medium.

5. The culturing method according to any one of claims 1 to 4, characterized in that, The porous structure is a culture structure with a porous membrane at the bottom.

6. The culturing method according to any one of claims 1 to 5, characterized in that, The source of the microvascular fragments includes adipose tissue.

7. The cultivation method according to any one of claims 1 to 6, characterized in that, The tumor includes at least one of breast cancer, lung cancer, colon cancer, and bladder cancer.

8. A tumor organoid model obtained by the culture method according to any one of claims 1-7.

9. A product for tumor spheroid culture, characterized in that, It includes reagent A, reagent B, and a culture medium; The raw materials of reagent A include: thrombin, microvascular fragments, fibrinogen, and a first solvent; The raw materials of reagent B include: N-carboxyethyl chitosan, oxidized hyaluronic acid, and a second solvent.

10. Use of the product according to claim 9 in culturing tumor organoids or preparing a tumor organoid model.

Images