Brain glioma organ chip with adjustable tumor microenvironment gradient and method for mediating formation of brain glioma organoid by brain glioma organ chip

Abstract

The invention discloses a brain glioma organ chip with adjustable tumor microenvironment gradient and a method for mediating formation of brain glioma organoid by the brain glioma organ chip, and belongs to the technical field of organ chips. The preparation method comprises the following steps: firstly, preparing a glioma organ chip with adjustable tumor microenvironment gradient by utilizing a prepolymer solution containing glioma cells and vascular endothelial cells and a prepolymer solution containing biochemical molecules; then, the obtained brain glioma organ chip with the adjustable tumor microenvironment gradient is subjected to three-dimensional dynamic co-culture under the perfusion condition, three-dimensional culture and co-culture of brain glioma cells and vascular endothelial cells are achieved at the same time, endothelial-glioma multicellular tumor spheres are formed, and construction of brain glioma organs is achieved. The brain glioma organoid established by the invention successfully summarizes key characteristics of a tumor microenvironment, including biophysical factors simulated by physiological fluid and biochemical factors of three-dimensional culture microgel with adjustable concentration gradient of biochemical molecules.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ chips, and in particular relates to a glioma organ chip with adjustable tumor microenvironment gradient and a method for mediating glioma organoid formation. Background Art

[0002] Tumor cells exist in a tumor microenvironment (TME) with different gradients, which involves complex biochemical and biophysical factors, as well as specific physical and chemical properties (oxygen content, temperature, pH and osmotic pressure), soluble factors, cell-to-cell contacts and extracellular matrix (ECM) interactions. The tumor microenvironment plays a vital role in tumor development, cancer metastasis and the formation of drug resistance. Among them, the main component of the tumor microenvironment is the ECM, which not only provides structural support, but also regulates a series of pathological activities such as phenotypic changes of tumor cells (such as differentiation, transformation, etc.). During tumor spread and metastasis, the ECM acts as a medium for transmitting external forces and even cell signals from environmental cues, thereby affecting cell migration and anchoring behavior. The ECM is composed of many different biochemical components, including proteins, glycoproteins, proteoglycans and polysaccharides. When these components are arranged in a specific way, cells are endowed with various biochemical, biomechanical and physical properties through adhesion points.

[0003] Traditional tumor microenvironment construction reveals essential information about cellular behavior and function in vitro. However, because existing methods cannot accurately simulate the in vitro TME and the constructed models cannot accurately represent the cells in vitro, their application in pathological research, drug evaluation, and the mechanisms of tumor development and progression is limited. Furthermore, while animal models have been used to recapitulate the tumor microenvironment, they are time-consuming and expensive, and species-specific differences inevitably exist between animals and humans. Animal models cannot completely replace humans; for example, mice can tolerate higher concentrations of drugs than human patients. Furthermore, ethical concerns regarding animal models have significantly limited their use in preclinical drug analysis in recent years. Organ-on-a-chips are an emerging scientific technology that integrates biological, chemical, and engineering laboratories to simulate and construct tissue and organ microenvironments in vitro, with widespread applications in disease modeling and new drug discovery. Combining organ-on-a-chips with constructed tumor microenvironments to develop tumor-on-a-chips offers new strategies for studying tumor development, progression, and drug resistance, as well as for developing novel oncology drugs.

[0004] Constructing a TME in a tumor chip requires consideration of both biochemical and biophysical factors. Among these biochemical factors, the ECM is the most important, and can include natural or synthetic ECMs such as basement membranes, collagen, and hydrogels. Hydrogels are widely used in three-dimensional cell culture because they share some of the characteristics of natural ECMs used for 3D in vitro culture, such as ease of formation into various shapes and the presence of biologically relevant chemical and physical signals and biochemical properties. Based on their biocompatibility, biodegradability, bioactive functional groups, and compatibility with fabrication strategies, various natural polymers and synthetic hydrogels have been used to mimic the ECM. These hydrogels possess mechanical strength and can be used for cell immobilization, attachment, tissue formation, and highly permeable porous structures for nutrient and oxygen delivery. In addition to the ECM, concentration gradients of other biochemical molecules can also influence tumor cell growth and migration. By reproducing in vivo concentration gradients, cell migration, differentiation, and other responses can be simulated in vitro, potentially facilitating drug screening and therapeutic optimization. The biophysical factors in the TME are primarily fluid shear forces caused by blood flow or interstitial fluid flow. However, there are no reports in the prior art on how to use organ chips to mediate the formation of brain glioma organoids. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a glioma organ chip with adjustable tumor microenvironment gradient and a method for mediating glioma organoid formation.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a method for forming non-therapeutic glioma organoids mediated by a glioma organ chip with adjustable tumor microenvironment gradient, comprising the following steps:

[0008] (1) A glioma organ-on-a-chip with adjustable tumor microenvironment gradient was prepared using a prepolymer solution containing glioma cells and vascular endothelial cells and a prepolymer solution containing biochemical molecules;

[0009] (2) The glioma organ chip with adjustable tumor microenvironment gradient obtained in step (1) is subjected to three-dimensional dynamic co-culture under perfusion conditions to form endothelial-glioma multicellular tumor spheroids, thereby realizing the construction of glioma organoids.

[0010] Furthermore, in step (1), the total density of the glioma cells and vascular endothelial cells in the prepolymer solution containing glioma cells and vascular endothelial cells is 1×10 5 ~1×10 7 cells / mL; the density ratio of the brain glioma cells to the vascular endothelial cells is 1:2.

[0011] Furthermore, in step (2), the perfusion flow rate is 0.01-10 μL / min.

[0012] Furthermore, in step (2), the temperature of the three-dimensional dynamic co-culture is 37° C., and the time is more than 5 days.

[0013] The present invention also provides a method for preparing a glioma organ chip with adjustable tumor microenvironment gradient, comprising the following steps:

[0014] A. injecting a prepolymer solution containing glioma cells and vascular endothelial cells into a microfluidic chip until the microchannels and micropores of the microfluidic chip are completely filled; then, introducing air to discharge the solution in the microchannels;

[0015] B. The prepolymer solution containing biochemical molecules is injected into the microfluidic chip again, and the injection is stopped after the prepolymer solution containing biochemical molecules flows through the last micropore. After standing, air is introduced again to expel the residual solution in the microchannel. After solidification, the brain glioma organ chip with adjustable tumor microenvironment gradient is obtained in each micropore.

[0016] Furthermore, in step A, the prepolymer solution containing glioma cells and vascular endothelial cells includes glioma cells, vascular endothelial cells, a polymer and a photoinitiator; and the solvent of the prepolymer solution is PBS buffer.

[0017] Furthermore, the polymer is selected from one or more of gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), and N-isopropylacrylamide (NIPAM), with gelatin methacrylate (GelMA) being more preferred. The polymer of the present invention can form a microgel by photocuring under the action of a photoinitiator, which can be used to simulate the ECM.

[0018] Furthermore, the photoinitiator is selected from photoinitiator I2959.

[0019] Furthermore, the mass concentration of the polymer in the prepolymer solution containing brain glioma cells and vascular endothelial cells is 1-20% (ie, the mass of the polymer added to each 1 mL of PBS buffer is 0.01-2 g), and the mass concentration of the photoinitiator is 0.1-2%.

[0020] Furthermore, in step A, the injection speed of the prepolymer solution is 8 μL / min.

[0021] Furthermore, in step A, the speed of introducing air is 30 μL / min.

[0022] Furthermore, in step B, the biochemical molecules in the prepolymer solution containing biochemical molecules are selected from dyes, collagen or proteins.

[0023] Furthermore, the biochemical molecules in the prepolymer solution containing biochemical molecules are selected from FITC-dextran or fibronectin.

[0024] Furthermore, the concentration of the FITC-dextran in the prepolymer solution containing the biochemical molecule is 0.1-10 mg / mL, more preferably 2 mg / mL.

[0025] Furthermore, the concentration of the fibronectin in the prepolymer solution containing biochemical molecules is 10 ng / mL-10 μg / mL, and more preferably 200 ng / mL.

[0026] Furthermore, in step B, the injection speed of the prepolymer solution containing biochemical molecules is 2 μL / min.

[0027] Furthermore, in step B, the speed of introducing air is 30 μL / min.

[0028] Furthermore, in step B, the standing time is 5-120 min, more preferably 30-60 min.

[0029] Furthermore, in step B, the curing is: irradiating under 365nm ultraviolet light for 1-120s, more preferably irradiating under 365nm ultraviolet light for 45-90s.

[0030] Furthermore, the method for preparing the brain glioma organ chip with adjustable tumor microenvironment gradient comprises the following steps:

[0031] (1) A prepolymer solution containing glioma cells and vascular endothelial cells is injected into the microchannel from the inlet of the microfluidic chip to fill the entire microfluidic chip.

[0032] (2) Air is introduced in the reverse direction from the outlet of the microfluidic chip to discharge the solution in the microchannel in the reverse direction. Due to the structure of the microchannel, the solution in the micropores will be restricted and will not be squeezed out.

[0033] (3) The prepolymer solution containing biochemical molecules is injected into the microchannel from the inlet of the microfluidic chip again, and the injection is stopped immediately after the solution flows through the last micropore (#48). The newly injected prepolymer solution containing biochemical molecules will exchange substances with the prepolymer solution containing glioma cells and vascular endothelial cells stored in the micropores as it flows through each micropore, and will be diluted due to the continuous loss of biochemical molecules. The prepolymer solution containing glioma cells and vascular endothelial cells in the original micropores will be mixed with different amounts of biochemical molecules, thereby forming a concentration gradient.

[0034] (4) Let it stand for 30 minutes. This allows the material exchange in the micropores to stabilize.

[0035] (5) Air is introduced again from the outlet of the microfluidic chip in the reverse direction to discharge the residual solution in the microchannel. After photocuring, a brain glioma organ chip with adjustable tumor microenvironment gradient is obtained in each microwell.

[0036] The present invention also provides a brain glioma organ chip with adjustable tumor microenvironment gradient, which is prepared according to the preparation method described in the above technical solution.

[0037] Compared with the prior art, the present invention has the following advantages and technical effects:

[0038] The present invention first uses a prepolymer solution containing glioma cells and vascular endothelial cells and a prepolymer solution containing biochemical molecules to prepare a glioma organ chip with an adjustable tumor microenvironment gradient. The obtained glioma organ chip with an adjustable tumor microenvironment gradient is then subjected to three-dimensional dynamic co-culture under perfusion conditions, simultaneously achieving three-dimensional culture and co-culture of glioma cells and vascular endothelial cells, forming endothelial-glioma multicellular tumor spheroids, and realizing the construction of glioma organoids. The glioma organoids established by the present invention successfully summarize the key features of the tumor microenvironment, including the biophysical factors of physiological fluid simulation and the biochemical factors of three-dimensional culture microgels with adjustable concentration gradients of biochemical molecules.

[0039] The experimental results show that under physiological fluid perfusion culture, glioblastoma and vascular endothelial cells maintained a high survival rate and showed good proliferation. Under the co-culture of glioblastoma and vascular endothelial cells, the study found that the concentration of biochemical molecules can affect the formation of self-organized endothelial cell-glioma multicellular spheroids (brain glioma organoids). By quantitatively analyzing the probability and number of occurrence of multicellular spheroids under different conditions and the volume distribution of spheroids, and characterizing the relevant tumor markers vimentin, a-smooth muscle actin, and CD133 by immunofluorescence staining and fluorescent quantitative PCR, the presence of tumor stem cells was demonstrated. In addition, compared with the MTT assay of traditional 96-well plates, the multicellular spheroids (brain glioma organoids) in the present invention have enhanced resistance to the chemotherapy drug temozolomide, highlighting the potential of the brain glioma organoids to study drug responses in a more physiologically relevant model. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0041] Figure 1 Schematic diagram of the method for forming brain glioma organoids for non-therapeutic purposes mediated by a brain glioma organ chip with adjustable tumor microenvironment gradient provided by the present invention;

[0042] Figure 2 This is a physical picture of the microfluidic chip in the present invention;

[0043] Figure 3 This is a fluid dynamics simulation diagram;

[0044] Figure 4 This is a PI staining image of one of the microwells of the uniform, additive-free GelMA microgel cell culture chip prepared in Example 1 after culturing glioma cells for 3 days;

[0045] Figure 5 This is a panoramic fluorescence image of glioma cells cultured on the uniform, additive-free GelMA microgel cell culture chip prepared in Example 1 on day 0;

[0046] Figure 6 The MTT test results of glioma cells cultured on the uniform GelMA microgel cell culture chip prepared in Example 1 after 0 days, 3 days, and 5 days;

[0047] Figure 7 The immunofluorescence staining results of brain glioma cells cultured on the uniform GelMA microgel cell culture chip prepared in Example 1 after 7 days of culture;

[0048] Figure 8This is a panoramic fluorescence image of vascular endothelial cells cultured on the uniform, additive-free GelMA microgel cell culture chip prepared in Example 1 at day 0;

[0049] Figure 9 The results of live and dead cell staining after culturing vascular endothelial cells for 3 days on the uniform GelMA microgel cell culture chip prepared in Example 1;

[0050] Figure 10 The MTT test results of vascular endothelial cells cultured on the uniform GelMA microgel cell culture chip prepared in Example 1 after 0 days, 3 days, and 5 days;

[0051] Figure 11 The expression of HUVECs cytoskeleton proteins F-actin and CD31 in well 1 and well 28 after culturing vascular endothelial cells on the GelMA microgel cell culture chip with adjustable FN concentration gradient prepared in Example 2 for 18 hours and 3 days;

[0052] Figure 12 The expression of HUVECs cytoskeleton protein F-actin in wells 8#, 18#, 36#, and 46# after culturing vascular endothelial cells on the GelMA microgel cell culture chip with adjustable FN concentration gradient prepared in Example 2 for 3 days;

[0053] Figure 13 This is a panoramic fluorescence image of the brain glioma organ chip with adjustable tumor microenvironment gradient constructed in Example 3;

[0054] Figure 14 This is a fluorescence image of vascular endothelial cells and brain glioma cells in wells 7#, 10#, 18#, and 28# after 5 days of three-dimensional dynamic co-culture in Example 4;

[0055] Figure 15 The probability of appearance (A), number (B), size (C), and volume distribution (D) of endothelial-glioma multicellular tumor spheroids after 5 days of three-dimensional dynamic co-culture in Example 4;

[0056] Figure 16 The expression results of a-SMA in the endothelioma-glioma multicellular tumor spheroids formed in Example 4;

[0057] Figure 17 The CD133 staining results of the endothelial-glioma multicellular tumor spheroids formed in Example 4;

[0058] Figure 18 The expression of VIM, a-SMA, and CD133 in the total mRNA extracted from the co-cultured cells on day 0 (control group) and day 5 (chip group) in Example 5;

[0059] Figure 19 IC of U87-eGFP cells to temozolomide in 96-well plate assay 50 ;

[0060] Figure 20 These are the drug resistance results of U87-eGFP-no drug group, U87-eGFP-TMZ group, U87-eGFP+HUVECs-no drug group and U87-eGFP+HUVECs-TMZ group. DETAILED DESCRIPTION

[0061] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] Three-dimensional dynamic co-culture: By simulating the in vivo microenvironment in a three-dimensional environment, cells can grow and interact in an environment closer to physiological conditions. Using gel materials to provide a three-dimensional growth space for cells, microfluidic chips simulate the shear forces of fluids in the body and the dynamic supply of nutrients, allowing different cell types to be co-cultured.

[0064] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0065] In the following examples, the microfluidic chip used consists of an inlet, an outlet, 48 micropores with the same structure, and microchannels for connecting the micropores; wherein the microchannel has a height of 225 μm and a width of 200 μm; the diameter of the micropore is 900 μm; the front gap width between the micropore and the microchannel is 200 μm, the narrow gap width is 40 μm, and the narrow gap length is 140 μm. See the actual picture of the microfluidic chip for details. Figure 2 The 48 micropores with the same structure are numbered 1#, 2#, 3#...46#, 47#, 48#.

[0066] In order to match the physiological model and achieve three-dimensional dynamic cell culture, perfusion is used to provide continuous fluid stimulation to the cells, simulate the biophysical factors of the cells, facilitate material exchange, maintain the normal physiological function of the cells, and achieve the goal of multi-day culture on the cell chip. The present invention uses COMSOL software to perform fluid dynamics simulation to obtain the fluid flow rate distribution within the chip. When the perfusion flow rate is 0.1 μL / min, the fluid flow rate in the microchannel can reach 0.7×10 -3 m / s, which matches the flow rate of physiological fluids in capillaries, while the flow rate in micropores is extremely low and can be used to simulate interstitial fluid flow. Figure 3 .

[0067] Example 1

[0068] A method for preparing a uniform, additive-free GelMA microgel cell culture chip, comprising the following steps:

[0069] PBS buffer containing 5% (w / v) GelMA and 0.5% (w / v) photoinitiator I2959 (i.e., 0.05 g GelMA and 0.005 g photoinitiator I2959 were added to each 1 mL PBS buffer) was used as the prepolymer solution. Cells were cultured at a rate of 2×10 6 Cells were added to the prepolymer solution at a density of cells / mL to obtain a cell-containing prepolymer solution. The cell-containing prepolymer solution was injected from the inlet of the microfluidic chip at a flow rate of 8 μL / min through Tygon tubing connected to a syringe pump until the microchannels and micropores of the microfluidic chip were completely filled. Subsequently, air was introduced from the outlet of the microfluidic chip at a flow rate of 30 μL / min to drain the solution in the microchannels in the reverse direction. The solution was then irradiated with 365 nm UV light for 45 seconds and solidified to form a uniform, additive-free GelMA microgel cell culture chip.

[0070] The cells in Example 1 are brain glioma cells U87-eGFP. The uniform GelMA microgel cell culture chip obtained in Example 1 was dynamically cultured at 37°C. On the third day of culture, the cells were stained for viability using PI staining. The results are shown in FIG. Figure 4 On the 0th day of culture, panoramic fluorescence imaging was performed, and the results are shown in Figure 5 MTT assay was performed on the 0th, 3rd and 5th day of culture, and the results are shown in Figure 6 On the 7th day of culture, immunofluorescence staining of U87-eGFP vimentin (VIM) and a-SMA was performed. Figure 7 .

[0071] Figure 4This is a PI staining image of one of the microwells of the uniform GelMA microgel cell culture chip prepared in Example 1 after culturing glioma cells for 3 days. Figure 4 It can be seen that in the PI staining results on the third day, dead cells are red and living cells are green, and the survival rate after 3 days of dynamic culture is above 95%.

[0072] Figure 5 This is a panoramic fluorescence image of the uniform GelMA microgel cell culture chip prepared in Example 1 after culturing glioma cells on day 0. Figure 5 It can be seen that since U87-eGFP cells themselves express green fluorescent protein, the cells in the panoramic photo show green fluorescence.

[0073] Figure 6 The MTT test results of the uniform GelMA microgel cell culture chip prepared in Example 1 after culturing glioma cells for 0 days, 3 days and 5 days. Figure 6 It can be seen that the absorbance A490 on the 0th, 3rd and 5th day of culture increased by 1.68 times and 2.44 times respectively, which shows that the cells are in a good three-dimensional dynamic culture state in the uniform and additive-free GelMA microgel cell culture chip.

[0074] Figure 7 The immunofluorescence staining results of the uniform GelMA microgel cell culture chip prepared in Example 1 after culturing glioma cells for 7 days. Figure 7 It can be seen that after 7 days of three-dimensional dynamic culture of brain glioma cells U87-eGFP, the cells showed obvious expression of vimentin (red and superimposed yellow) at the connection close to the fluid perfusion channel, and there were also sporadic cells expressing obvious a-SMA (red and superimposed yellow).

[0075] The cells in Example 1 are vascular endothelial cells (HUVECs). The uniform GelMA microgel cell culture chip obtained in Example 1 was dynamically cultured at 37°C. On the 0th day of culture, the vascular endothelial cells (HUVECs) were labeled with a live cell tracking dye and the chip was subjected to panoramic fluorescence imaging. The results are shown in FIG. Figure 8 On the third day of culture, the viability of endothelial cells was detected by using dead cell staining (calcein AM / PI staining). Figure 9 MTT assay was performed on the 0th, 3rd and 5th day of culture, and the results are shown in Figure 10 .

[0076] Figure 8 This is a panoramic fluorescence image of vascular endothelial cells cultured on the uniform GelMA microgel cell culture chip prepared in Example 1 at day 0. Figure 8It can be seen that the vascular endothelial cells are well loaded in the microgel, there is no obvious cell residue in the microchannel, and the difference in cell density between microwells is small.

[0077] Figure 9 The results of live and dead cell staining after culturing vascular endothelial cells for 3 days on the uniform GelMA microgel cell culture chip prepared in Example 1. Figure 9 It can be seen that the cell survival rate was greater than 95% after 3 days of culture.

[0078] Figure 10 The MTT test results of the homogeneous GelMA microgel cell culture chip prepared in Example 1 after culturing vascular endothelial cells for 0 days, 3 days and 5 days. Figure 10 It can be seen that by comparing the absorbance A490 on day 0, day 3, and day 5, the A490 of cells increased by 1.57 times and 2.36 times, respectively, as the number of culture days increased, indicating good proliferation activity.

[0079] Example 2

[0080] A method for preparing a GelMA microgel cell culture chip with adjustable FN concentration gradient, comprising the following steps:

[0081] (1) PBS buffer containing 5% (w / v) GelMA and 0.5% (w / v) photoinitiator I2959 was used as the prepolymer solution. Cells were cultured at a rate of 2×10 6 Cells were added to the prepolymer solution at a density of cells / mL to obtain a prepolymer solution containing cells. The cell-containing prepolymer solution was injected into the inlet of the microfluidic chip at a flow rate of 8 μL / min through Tygon tubing connected to a syringe pump until the microchannels and micropores of the microfluidic chip were completely filled. Subsequently, air was introduced into the outlet of the microfluidic chip at a flow rate of 30 μL / min to drain the solution in the microchannels in the reverse direction.

[0082] (2) A PBS buffer solution containing 5% (w / v) GelMA, 0.5% (w / v) photoinitiator I2959, and 200 ng / mL FN (i.e., 0.05 g GelMA, 0.005 g photoinitiator I2959, and 200 ng FN were added to each 1 mL of PBS buffer) as a prepolymer solution containing FN. The prepolymer solution containing FN was injected from the inlet of the microfluidic chip at a flow rate of 2 μL / min through a Tygon tube connected to a syringe pump. The injection was stopped after the solution flowed through the last micropore (#48) and the solution was allowed to stand for 30 min. Subsequently, air was introduced from the outlet of the microfluidic chip again at a flow rate of 30 μL / min to discharge the residual solution in the microchannel. The chip was then solidified after irradiation with 365 nm ultraviolet light for 45 s to obtain a GelMA microgel cell culture chip with adjustable FN concentration gradient.

[0083] The cells in Example 2 are human vascular endothelial cells (HUVECs). The GelMA microgel cell culture chip with adjustable FN concentration gradient obtained in Example 2 was dynamically cultured at 37°C. On the third day of culture, HUVECs were imaged by staining for cytoskeletal protein F-actin and immunofluorescence staining for CD31. The 48 wells in the organ chip were divided into C max(1#-13#) Group C sub-max(14#-27#) Group C mid(28#-36#) Group and C min(37#-48#) Group, see the results Figure 11 and Figure 12 .

[0084] Figure 11 The expression of HUVECs cytoskeleton proteins F-actin and CD31 in well 1 and well 28 after culturing vascular endothelial cells for 18 hours and 3 days on the GelMA microgel cell culture chip with adjustable FN concentration gradient prepared in Example 2. max(1#-13#) Well 1 in group (calculated FN concentration is about 61.2 ng / mL) and C mid(28#-36#) The morphology and protein expression of HUVECs in well 28 of the group (calculated FN concentration is approximately 35.2 ng / mL) after 18 hours and 3 days of culture clearly show that after 18 hours of culture, the HUVECs in well 1 took on a polygonal shape with extended pseudopodia and strong CD31 expression. After 3 days of culture, the HUVECs completely spread out like cobblestones, with CD31 expressed at tight junctions between cells, forming a grid-like pattern. In contrast, the overall morphology of HUVECs in well 28 after 18 hours and 3 days of culture was not much different.

[0085] Figure 12The expression of HUVECs cytoskeleton protein F-actin in wells 8#, 18#, 36# and 46# after culturing vascular endothelial cells on the GelMA microgel cell culture chip with adjustable FN concentration gradient prepared in Example 2 for 3 days. max(1#-13#) The performance of the 8# hole in the group is similar to that of the 1# hole, while the C sub-max(14#-27#) Well 18 in the group showed some transitional states, with a small number of cells showing polygonal shapes. mid(28#-36#) Group 36# hole and C min(37#-48#) The 46# hole of the group is the same as the C mid(28#-36#) This suggests that the addition of FN helps promote the extension of endothelial cells, which may help increase direct contact between cells and improve the ability of cell interaction.

[0086] Example 3

[0087] A method for preparing a glioma organ-on-a-chip with adjustable tumor microenvironment gradients, comprising the following steps:

[0088] (1) PBS buffer containing 5% (w / v) GelMA and 0.5% (w / v) photoinitiator I2959 (i.e., 0.05 g GelMA and 0.005 g photoinitiator I2959 were added to each 1 mL PBS buffer) as the prepolymer solution. Brain glioma cells (U87-eGFP) and vascular endothelial cells (HUVECs) were cultured at a rate of 2×10 6 A total density of 100 cells / mL was added to the prepolymer solution (the density ratio of glioma cells to vascular endothelial cells was 1:2) to obtain a prepolymer solution containing glioma cells and vascular endothelial cells. The prepolymer solution containing glioma cells and vascular endothelial cells was injected from the inlet of the microfluidic chip at a flow rate of 8 μL / min through Tygon tubing connected to a syringe pump until the microchannels and micropores of the microfluidic chip were completely filled. Subsequently, air was introduced from the outlet of the microfluidic chip at a flow rate of 30 μL / min to drain the solution in the microchannels in the reverse direction.

[0089] (2) A PBS buffer solution containing 5% (w / v) GelMA, 0.5% (w / v) photoinitiator I2959, and 200 ng / mL FN (i.e., 0.05 g GelMA, 0.005 g photoinitiator I2959, and 200 ng FN were added to each 1 mL PBS buffer) as a prepolymer solution containing FN. The prepolymer solution containing FN was injected from the inlet of the microfluidic chip at a flow rate of 2 μL / min through a Tygon tube connected to a syringe pump. The injection was stopped after the solution flowed through the last micropore (#48) and the solution was allowed to stand for 30 min. Subsequently, air was introduced from the outlet of the microfluidic chip again at a flow rate of 30 μL / min to discharge the residual solution in the microchannel. The chip was then cured after being irradiated with 365 nm ultraviolet light for 45 s to obtain a brain glioma organ chip with adjustable tumor microenvironment gradient.

[0090] Panoramic fluorescence imaging was performed on the brain glioma organ chip with adjustable tumor microenvironment gradient constructed in Example 3. The results are shown in Figure 13 .from Figure 13 It can be seen that vascular endothelial cells are labeled with red live cell tracking dye, and U87-eGFP itself expresses green fluorescent protein.

[0091] Example 4

[0092] A method for forming non-therapeutic glioma organoids mediated by a glioma organ-on-a-chip with adjustable tumor microenvironment gradients, comprising the following steps:

[0093] The brain glioma organ chip with adjustable tumor microenvironment gradient constructed in Example 3 was subjected to three-dimensional dynamic co-culture under perfusion conditions with a flow rate of 0.1 μL / min. The temperature of the three-dimensional dynamic co-culture was 37°C. After 5 days of culture, endothelial-glioma multicellular tumor spheres were formed in the group with a higher FN concentration, realizing the construction of brain glioma organoids.

[0094] Figure 14 The fluorescence images of vascular endothelial cells and brain glioma cells in wells 7#, 10#, 18# and 28# after 5 days of three-dimensional dynamic co-culture in Example 4 are shown. Figure 14 It can be seen that after 5 days of three-dimensional dynamic co-culture, max(1#-13#) Group and C sub-max(14#-27#) An obvious cell self-assembly phenomenon was found in the group. Endothelial cells could not spread spontaneously as they did when cultured alone, but instead formed obvious endothelial-glioma multicellular tumor spheres with U87-eGFP cells.

[0095] Figure 15 The probability (A), number (B), size (C) and volume distribution (D) of endothelial-glioma multicellular tumor spheroids after 5 days of three-dimensional dynamic co-culture in Example 4 are shown. Figure 15It can be seen that the C max(1#-13#) The probability, number and volume of multicellular tumor spheroids in group C were higher than those in other groups, and as the FN concentration decreased, the subsequent group C sub-max(14#-27#) and C mid(28#-36#) are gradually decreasing, C min(37#-48#) No multicellular tumor spheroids appeared in the group. The volume estimation size distribution of all spheroids found in the experiment was statistically analyzed, and the estimated size distribution was 1.4×10 6 μm 3 The probability of occurrence before is relatively smooth, and the average volume estimate is 0.769×10 6 μm 3 . The above results show that the appearance of endothelial-glioma multicellular tumor spheroids is closely related to the FN concentration in the ECM. When the FN concentration reaches a certain amount, vascular endothelial cells are more likely to stretch and the interaction between cells is enhanced, which may be conducive to the self-assembly of multicellular tumor spheroids. This phenomenon may be due to FN regulating cytoskeleton reorganization and collective migration behavior through integrin-mediated cell adhesion; at the same time, FN may also synergistically promote the co-aggregation of tumor-endothelial cells and the formation of vasculogenic mimicry by changing the mechanical properties of ECM (such as increased stiffness). These findings are consistent with previous studies reporting the role of FN in tumor cell aggregation, angiogenesis and microenvironment remodeling, indicating that FN concentration is a key factor in regulating the self-organization of glioma multicellular three-dimensional structures.

[0096] In order to verify that the endothelial-glioma multicellular tumor spheroids formed in Example 4 have tumor stem cell characteristics, the related markers a-SMA and CD133 were detected by immunofluorescence staining. The results are shown in Figure 4. Figure 16 and 17 .

[0097] Figure 16 The expression results of a-SMA in the endothelial-glioma multicellular tumor spheroids formed in Example 4 are compared with the previous results ( Figure 7 ) As can be seen, after 7 days of 3D dynamic cell culture of U87-eGFP cells alone, a small amount of a-SMA expression was observed. However, in co-cultures of U87-eGFP and endothelial cells, significant a-SMA expression was observed after 5 days of co-culture, and a-SMA expression became even more pronounced after the generation of multicellular endothelial-glioma tumor spheroids. In the fluorescence images, U87-eGFP cells appear green due to their natural expression of green fluorescent protein, while a-SMA staining is red. Endothelial cells were not stained and are only shown in the magnified brightfield inset.

[0098] In C max(1#-13#) Group (8# hole) and C sub-max(14#-27#)In the group (well 15), endothelial-glioma multicellular tumor spheroids with different relative sizes were selected for CD133 staining. The results are shown in Figure 17 CD133 is a cell surface glycoprotein that is generally considered a characteristic marker of cancer stem cells. In scientific research, CD133 expression levels are associated with tumor biological behaviors such as proliferation, metastasis, and drug resistance. Figure 17 The CD133 staining results of the endothelial-glioma multicellular tumor spheroids formed in Example 4. Figure 17 It can be seen that both well 8# and well 15# clearly express CD133, indicating that the self-organized endothelioma-glioma multicellular tumor spheroids of the present invention contain tumor stem cells and can be regarded as tumor organoids.

[0099] Example 5

[0100] A method for forming non-therapeutic glioma organoids using a glioma organ-on-chip with a homogenous tumor microenvironment. The optimized concentration of FN for glioma organoid formation can be obtained according to Example 4. Under these conditions, a glioma organ-on-chip with a homogenous tumor microenvironment is prepared and mediated to form non-therapeutic glioma organoids. The specific steps are as follows:

[0101] (1) PBS buffer containing 5% (w / v) GelMA, 0.5% (w / v) photoinitiator I2959, and 50 ng / mL FN was used as the prepolymer solution (i.e., 0.05 g GelMA, 0.005 g photoinitiator I2959, and 50 ng FN were added to each 1 mL PBS buffer). Brain glioma cells (U87-eGFP) and vascular endothelial cells (HUVECs) were cultured at a rate of 2×10 6 The prepolymer solution was added to the prepolymer solution (with a density ratio of 1:2) at a total density of glioma cells / mL to vascular endothelial cells, resulting in a prepolymer solution containing glioma cells and vascular endothelial cells. The prepolymer solution was injected from the inlet of the microfluidic chip at a flow rate of 8 μL / min through Tygon tubing connected to a syringe pump until the microchannels and micropores of the microfluidic chip were completely filled. Subsequently, air was introduced from the outlet of the microfluidic chip at a flow rate of 30 μL / min, and the solution in the microchannels was discharged in the reverse direction. The solution was then cured by irradiation with 365 nm ultraviolet light for 45 seconds to form a glioma organ-on-a-chip with a uniform tumor microenvironment and a final FN concentration of 50 ng / mL.

[0102] (2) The glioma organ chip with a uniform tumor microenvironment obtained in step (1) was subjected to three-dimensional dynamic co-culture under perfusion conditions at a flow rate of 0.1 μL / min. The temperature of the three-dimensional dynamic co-culture was 37°C. After 5 days of culture, endothelial-glioma multicellular tumor spheres were formed, thereby realizing the construction of glioma organoids.

[0103] The expression of VIM, a-SMA and CD133 in the total mRNA extracted from the co-cultured cells on day 0 (control group) and day 5 (chip group) in Example 5 was compared. Figure 18 .

[0104] Figure 18 The expression of VIM, a-SMA and CD133 in the total mRNA extracted from the co-cultured cells on day 0 (control group) and day 5 (chip group) in Example 5 was analyzed. Figure 18 It can be seen that the expression of VIM, a-SMA, and CD133 in the chip group were significantly increased, by 2.0 times, 45.8 times, and 559.0 times, respectively, further demonstrating the presence of tumor stem cells in the self-organized endothelial-glioma multicellular tumor spheroids of the present invention, which can be regarded as tumor organoids.

[0105] Since the appearance of multicellular tumor spheroids of endothelial-glioma and the expression of indicators such as CD133 are related to tumor organoids and tumor stem cells, which are closely related to tumor drug resistance, the present invention further carried out drug evaluation of the anti-tumor drug temozolomide. Temozolomide is an oral chemotherapy drug used in the treatment of brain gliomas. It can spontaneously degrade to produce active metabolites MTIC. Existing literature reports that temozolomide has an effect on the cell IC of U87-eGFP. 50 The values ​​were mostly between 30-300 μM. The IC values ​​of U87-eGFP cells to temozolomide were detected by the traditional 96-well plate-based MTT assay. 50 (Drug action time 2 days) is about 154μM, see Figure 19 .

[0106] Drug evaluation:

[0107] Grouping: U87-eGFP in the uniform GelMA microgel cell culture chip without additives prepared in Example 1 without drug group (U87-eGFP-no drug group); U87-eGFP in the uniform GelMA microgel cell culture chip without additives prepared in Example 1 treated with 150 μM temozolomide for two days (U87-eGFP-TMZ group); U87-eGFP co-cultured with HUVECs in the glioma organ chip with a uniform tumor microenvironment of 50 ng / mL in Example 5 without drug group (U87-eGFP+HUVECs-no drug group); U87-eGFP co-cultured with HUVECs in the glioma organ chip with a uniform tumor microenvironment of 50 ng / mL in Example 5 with 150 μM temozolomide for two days (U87-eGFP+HUVECs-TMZ group). Results are shown in Figure 20 .

[0108] Figure 20 The drug resistance results of U87-eGFP-no drug group, U87-eGFP-TMZ group, U87-eGFP+HUVECs-no drug group and U87-eGFP+HUVECs-TMZ group are shown. Figure 20 As can be seen, the survival rate (CV) of U87-eGFP cells in the U87-eGFP-TMZ group was approximately 59% compared to the U87-eGFP-no drug group, a result higher than the 50% observed in 96-well plate culture conditions, indicating that the three-dimensional dynamic environment can induce drug resistance in U87-eGFP cells. The overall cell survival rate in the U87-eGFP+HUVECs-TMZ group was approximately 72% of that in the U87-eGFP+HUVECs-no drug group, indicating that the formation of tumor organoids further enhances tumor drug resistance.

[0109] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for forming non-therapeutic glioma organoids using a glioma organ-on-a-chip with adjustable tumor microenvironment gradients, characterized in that: The following steps are involved: (1) A glioma organ-on-a-chip with adjustable tumor microenvironment gradient was prepared using a prepolymer solution containing glioma cells and vascular endothelial cells and a prepolymer solution containing biochemical molecules; (2) The glioma organ chip with adjustable tumor microenvironment gradient obtained in step (1) is subjected to three-dimensional dynamic co-culture under perfusion conditions to form endothelial-glioma multicellular tumor spheroids, thereby realizing the construction of glioma organoids.

2. The method for forming non-therapeutic glioma organoids mediated by a glioma organ chip with adjustable tumor microenvironment gradient according to claim 1, characterized in that: In step (1), the total density of the glioma cells and vascular endothelial cells in the prepolymer solution containing glioma cells and vascular endothelial cells is 1×10 5 ~1×10 7 cells / mL; the density ratio of the brain glioma cells to the vascular endothelial cells is 1:

2.

3. The method for forming non-therapeutic glioma organoids mediated by a glioma organ chip with adjustable tumor microenvironment gradient according to claim 1, characterized in that: In step (2), the perfusion flow rate is 0.01-10 μL / min.

4. The method for forming non-therapeutic glioma organoids mediated by a glioma organ chip with adjustable tumor microenvironment gradient according to claim 1, characterized in that: In step (2), the temperature of the three-dimensional dynamic co-culture is 37° C. and the time is more than 5 days.

5. A method for preparing a glioma organ chip with adjustable tumor microenvironment gradient, characterized in that: The following steps are involved: A. Inject the prepolymer solution containing glioma cells and vascular endothelial cells into the microfluidic chip until the microchannels and micropores of the microfluidic chip are completely filled; Then, air is introduced to discharge the solution in the microchannel; B. The prepolymer solution containing biochemical molecules is injected into the microfluidic chip again, and the injection is stopped after the prepolymer solution containing biochemical molecules flows through the last micropore. After standing, air is introduced again to expel the residual solution in the microchannel. After solidification, the brain glioma organ chip with adjustable tumor microenvironment gradient is obtained in each micropore.

6. The method for preparing a glioma organ chip with adjustable tumor microenvironment gradient according to claim 5, characterized in that: In step A, the prepolymer solution containing glioma cells and vascular endothelial cells comprises glioma cells, vascular endothelial cells, polymer and photoinitiator; and / or, The polymer is selected from one or more of methacrylated gelatin, polyethylene glycol diacrylate and N-isopropylacrylamide; and / or, The mass concentration of the polymer in the prepolymer solution containing brain glioma cells and vascular endothelial cells is 1-20%, and the mass concentration of the photoinitiator is 0.1-2%.

7. The method for preparing a glioma organ chip with adjustable tumor microenvironment gradient according to claim 5, characterized in that: In step B, the biochemical molecules in the prepolymer solution containing biochemical molecules are selected from dyes, collagen or proteins.

8. The method for preparing a glioma organ chip with adjustable tumor microenvironment gradient according to claim 7, characterized in that: The biochemical molecules in the prepolymer solution containing biochemical molecules are selected from FITC-dextran or fibronectin; and / or, The concentration of the FITC-dextran in the prepolymer solution containing the biochemical molecule is 0.1-10 mg / mL; and / or, The concentration of the fibronectin in the prepolymer solution containing the biochemical molecules is 10 ng / mL-10 μg / mL.

9. The method for preparing a glioma organ chip with adjustable tumor microenvironment gradient according to claim 5, characterized in that: In step B, the standing time is 5-120 min; and / or, The curing step is: irradiating with 365 nm ultraviolet light for 1-120 seconds.

10. A glioma organ chip with adjustable tumor microenvironment gradient prepared according to the preparation method according to any one of claims 5-9.

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