An osteosarcoma in vitro culture chip that mimics bone microenvironment and its construction method and application

Abstract

The present invention discloses an osteosarcoma in vitro culture chip that mimics a bone microenvironment, and a construction method and application thereof, which belong to the field of medical technology. The osteosarcoma in vitro culture chip comprises a carrier chip and a cover plate; the carrier chip comprises two inlets, a concentration mixing channel area, a concentration gradient forming area, a tissue culture area, an outlet, and a liquid storage area; the concentration mixing channel area is provided with a concentration mixing channel; the concentration mixing channel area, the concentration gradient forming area, the tissue culture area, and the liquid storage area are arranged in steps from high to low; the cover plate comprises two baffles, corresponding to the tissue culture area and the liquid storage area of ​​the carrier chip; the present invention also constructs a bone-skeletal muscle tissue interface including bone tissue and vascularized muscle tissue, improves the simulation, and enables tumor cells to grow in a real environment that interacts with bone tissue, thereby providing an effective platform for the later study of the metastasis mechanism of osteosarcoma.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular to an osteosarcoma in vitro culture chip that mimics a bone microenvironment, and a construction method and application thereof. Background Art

[0002] In recent years, in tissue engineering and cancer biology research, organ chip technology, as an emerging in vitro model, has gradually become an important tool for studying tumor microenvironment, drug screening and biological response mechanisms. Osteosarcoma is one of the common malignant tumors in adolescents. The study of its metastasis mechanism and treatment effect faces the challenge that traditional models cannot provide an accurate physiological microenvironment. Therefore, the development of an organ chip that can more realistically simulate the bone tissue microenvironment is crucial for studying the biological characteristics and drug response of osteosarcoma.

[0003] Existing osteosarcoma models mainly rely on two-dimensional cell culture or small animal models, which have obvious limitations in reproducing the complex interaction between tumors and the microenvironment. Two-dimensional cell culture cannot reflect the spatial relationship between cells and their impact on tumor growth, while small animal models are often limited by ethical, time and economic costs. In order to solve these problems, some research teams at home and abroad have gradually adopted three-dimensional bioprinting technology to construct more complex bone tissue models. For example, recent studies have successfully constructed biomimetic bone tissue models using composite materials of polymers and cells. These models can effectively mimic the mechanical properties and biological activity of bone tissue.

[0004] The Chinese invention patent application with application number 202210839348.4 discloses an osteosarcoma organoid model, which includes at least a biological scaffold and osteosarcoma cells and exosomes derived from bone marrow mesenchymal stem cells loaded on the biological scaffold; the biological scaffold is a criss-cross three-dimensional structure constructed by a hydrogel preparation, and the hydrogel preparation includes a decellularized matrix and fibrin. The invention uses the dOsEM-Fibrin hydrogel loading system combined with BMSC-EV to integrate the "component-structure" multi-level biomimetic optimization of osteosarcoma organoid chips on a microfluidic chip by using 3D bioprinting technology.

[0005] However, these models often lack material transport and mixing efficiency and cannot achieve effective concentration gradient control, which limits their application in drug screening. In addition, existing models fail to simulate the bone-meat tissue interface and are difficult to truly simulate the growth microenvironment of osteosarcoma, which affects the application and development of existing in vitro models. Summary of the invention

[0006] In order to solve the above problems, the osteosarcoma organ chip provided by the present invention adopts an integrated multilayer structure, which can effectively form a stable material concentration gradient; in order to improve the material mixing efficiency, the present invention designs an S-bend mixing channel and sets a reverse herringbone bone structure therein. This design not only increases the turbulence of the fluid, but also effectively promotes the uniform mixing of different substances; at the same time, in order to prevent the backflow of the outlet liquid, the present invention designs an anti-backflow structure to ensure that the material concentration in the culture area remains stable. This innovative design makes the chip provided by the present invention significantly superior to the existing bone tissue model in terms of material transfer efficiency. In addition, the chip provided by the present invention designs multiple stepped spaces in the culture area, which further enhances the stability of the material concentration gradient. This stepped design helps to simulate the natural microenvironment of bone tissue, allowing cells to grow and interact under conditions closer to physiology. Through 3D bioprinting technology, the present invention constructs a bone-skeletal muscle tissue interface and improves the simulation of the model. This design enables tumor cells to grow in a real environment that interacts with bone tissue, thereby providing a platform for determining drug safety concentration research.

[0007] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0008] An osteosarcoma in vitro culture chip that mimics a bone microenvironment, comprising a carrier chip 2 and a cover plate 1; the carrier chip 2 comprises two inlets 21, a concentration mixing channel area 22, a concentration gradient forming area 23, a tissue culture area 24, an outlet 26, and a liquid storage area 25 designed to prevent liquid backflow and destroy the concentration gradient; the concentration mixing channel area 22 is provided with a concentration mixing channel 221; the concentration mixing channel area 22, the concentration gradient forming area 23, the tissue culture area 24 and the liquid storage area 25 are arranged in a stepped manner from high to low; the cover plate 1 comprises two baffles 11, which correspond to the tissue culture area 24 and the liquid storage area 25 of the carrier chip respectively.

[0009] Two liquids of different concentrations enter the concentration mixing channel area 22 from two inlets 21. The mixed liquids enter the concentration gradient forming area 23. After the concentration gradient is formed and stabilized, they flow into the tissue culture area 24. The tumor spheres are placed in the tissue culture area 24. Multiple tumor spheres can be evenly dispersed in the tissue culture area, and different drug concentrations can be cultured for the same batch of tumor spheres at the same time. Finally, the liquid enters the liquid storage area 25 and flows out from the outlet 26.

[0010] Preferably, the concentration mixing channel 221 is provided with at least three sections, and each channel in each section is S-shaped; the first section is provided with two S-shaped channels, one end of the two S-shaped channels of the first section is respectively connected to the two inlets 21, and the other end is connected to the S-shaped channel of the next section through a transversely arranged channel, and the number of channels increases by one each time entering the next section. This design allows the liquid to be fully mixed and uniform in a limited area, and the number and number of layers of the S-shaped channels of the concentration mixing channel area 22 can be designed and adjusted according to experimental requirements to obtain drug concentrations of different gradients.

[0011] Preferably, the concentration mixing channel 221 is provided with a reverse herringbone bone 2211. The addition of the reverse herringbone bone can further ensure the uniformity of liquid mixing.

[0012] Preferably, the concentration gradient forming area 23 is additionally provided with a baffle 231, and the baffle 231 is arranged on the concentration gradient forming area 23 corresponding to the middle point of the outlets of two adjacent S-shaped channels.

[0013] Preferably, the osteosarcoma in vitro culture chip is provided with an overflow port 27 , and the overflow port 27 is arranged above the outlet 26 , and the height of the overflow port 27 is lower than the horizontal height of the tissue culture area 24 .

[0014] Preferably, the raw materials of the carrier chip 2 and the cover plate 1 include polylactic acid, photosensitive resin or polydimethylsiloxane (PDMS), and are made by 3D printing technology.

[0015] In chip preparation, the present invention uses 3D printing technology, which has significant advantages. Traditional photolithography or high-precision digital machine tool processing is limited by the process and it is difficult to construct a multi-dimensional structure. 3D printing can break through this bottleneck. The materials can be PLA, photosensitive resin or PDMS, and the printing methods include extrusion printing and DLP printing. It can not only adapt to the needs of different scenarios, but also rely on their respective advantages to accurately create complex and multi-dimensional chip structures and improve chip performance. Instant adjustment and reprinting according to actual needs, fast speed and high efficiency; the polylactic acid or photosensitive resin used for printing is low-cost and non-toxic.

[0016] Preferably, the cover plate 1 covers the carrier chip 2 and is then fixed with biological glue.

[0017] An in vitro osteosarcoma organ culture system comprises a medicine bottle I and a medicine bottle II, two peristaltic pumps III and IV respectively connected to the two medicine bottles, and the osteosarcoma in vitro culture chip V and the waste liquid bottle connection VII which are sequentially connected through a rubber tube VI.

[0018] A method for constructing an in vitro osteosarcoma culture chip using the osteosarcoma in vitro culture chip comprises the following steps:

[0019] S1, dissolving the raw material of bioprinting ink, methacryloyl gelatin (GelMa), in a cell culture medium to prepare a methacryloyl gelatin solution;

[0020] S2, dissolving fibrinogen in physiological saline to prepare a fibrinogen solution;

[0021] S3, mixing equal volumes of the methacrylylated gelatin solution in step S1 and the fibrinogen solution in step S2, adding phenyl-2,4,6-trimethylbenzoyl lithium phosphate (LAP) and osteosarcoma cells; after mixing evenly, removing bubbles, loading into the needle tube of a bioprinter for printing, and obtaining osteosarcoma tumor spheres of uniform size and shape;

[0022] S4, irradiating the osteosarcoma tumor spheres obtained in step S3 with ultraviolet light (ultraviolet light wavelength is 405 nm) to solidify the methacrylylated gelatin, then soaking them with thrombin to solidify the fibrinogen, and washing them with phosphate buffered saline (PBS) after soaking;

[0023] S5. Place the osteosarcoma tumor spheres processed in step S4 between the bone tissue and the vascularized skeletal muscle tissue in the bone-skeletal muscle tissue and then place them in the tissue culture area of ​​the osteosarcoma in vitro culture chip, cover them with a cover plate, and culture them using the osteosarcoma in vitro organ culture system.

[0024] The present invention adopts a double-linking method to improve the stability of tumor spheres. If the tumor spheres are only linked by methacrylamide photo-linking, they are easy to lyse after 3 days of culture. In addition to methacrylamide photo-linking, the present invention adds secondary linking of fibrinogen and thrombin, which effectively avoids the lysis of tumor spheres. In addition, the present invention also provides a complete osteosarcoma microenvironment, improves the simulation, and enables tumor cells to grow in a real environment that interacts with bone tissue.

[0025] Preferably, the mass volume concentration of the methacrylated gelatin solution in step S1 is 10% to 20% (m / v).

[0026] Preferably, the mass volume concentration of the fibrinogen solution in step S2 is 1% to 10% (m / v).

[0027] Preferably, the mass volume concentration of phenyl-2,4,6-trimethylbenzoyl lithium phosphate in step S3 is 0.1% to 1% (m / v).

[0028] Preferably, the density of osteosarcoma cells in step S3 is 1×10 6 ~10×10 6 cell / mL.

[0029] Preferably, the particle size of the osteosarcoma tumor spheres described in step S3 is 20-1000 μm.

[0030] Preferably, the ultraviolet irradiation duration in step S4 is 20 to 100 seconds.

[0031] Preferably, the thrombin soaking time in step S4 is 30 to 300 seconds.

[0032] Preferably, the bone-skeletal muscle tissue in vitro culture construction method described in step S5 comprises the following steps:

[0033] P1. Mix gelatin, sodium alginate and hydroxyapatite to prepare an aqueous solution, completely melt and mix, and then transfer to a printing needle tube for multi-layer mesh structure extrusion printing to prepare a bone tissue body;

[0034] P2, soaking and hardening the product extruded and printed in step P1 in a calcium chloride solution to obtain a bone tissue body with a high hardness and a multi-layered network structure, and forming bone tissue after culturing;

[0035] P3, mixing methacrylated gelatin, type I rat tail collagen, skeletal muscle cells and human umbilical vein endothelial cells to prepare a suspension, performing extrusion printing on the suspension and then curing it under ultraviolet light to obtain a muscle tissue body, wherein the structure of the muscle tissue body forms a nested structure with the structure of the bone tissue body, and culturing the muscle tissue body for a period of time to form a vascularized skeletal muscle tissue;

[0036] P4. Embedding the bone tissue obtained in step P2 in the vascularized skeletal muscle tissue obtained in step P3, wherein the vascularized skeletal muscle tissue covers the bone tissue, thereby obtaining the bone-skeletal muscle tissue.

[0037] Further preferably, in the aqueous solution described in step P1, the mass volume concentration of sodium alginate is 2% to 8% (w / v), the mass volume concentration of gelatin is 5% to 15% (w / v), and the mass volume concentration of hydroxyapatite is 5% to 20% (w / v). The bone tissue prepared in this way is relatively hard and has very good biocompatibility. After culturing osteoblasts in it for a period of time, they will mineralize by themselves, thereby enhancing the hardness of the bone.

[0038] More preferably, osteoblasts or bone marrow stem cells are added to the aqueous solution in step P1. Hydroxyapatite is one of the main components of bones, and the additional addition of osteoblasts or bone marrow stem cells can increase the density of bone tissue.

[0039] More preferably, the amount of osteoblasts or bone marrow stem cells added is 1×10 6 ~10×10 6 cell / mL.

[0040] More preferably, the mass volume concentration of the calcium chloride solution described in step P2 is 2% to 7% (m / v).

[0041] More preferably, the soaking time in step P2 is 20 to 80 minutes.

[0042] More preferably, the mass volume concentration of the methacryloyl gelatin in step P3 is 5% to 8% (w / v), the mass volume concentration of type I rat tail collagen is 1% to 5% (w / v), and the density of skeletal muscle cells is 1×10 6 ~10×10 6 cell / mL, the density of human umbilical vein endothelial cells is 1×10 6 ~10×10 6 / mL. After being cultured for a period of time, the human umbilical vein endothelial cells spontaneously formed a microvascular network.

[0043] The application of the osteosarcoma in vitro culture chip in the study of the metastasis mechanism of osteosarcoma.

[0044] The osteosarcoma in vitro culture chip is used in studying the safe concentration of drugs or studying the effect of drug concentration on tumors.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] 1. The osteosarcoma in vitro culture chip provided by the present invention adopts an integrated multi-layer structure that can effectively form a stable material concentration gradient, providing cells with a culture environment that is closer to physiological conditions; and the integrated design simplifies experimental operations, reduces external interference factors, and improves the accuracy and repeatability of the experiment.

[0047] 2. The osteosarcoma in vitro culture chip provided by the present invention adopts an S-shaped concentration mixing channel and a reverse herringbone structure, which increases the turbulence of the fluid, promotes the uniform mixing of different substances, and improves the efficiency of material transfer; it utilizes the principles of fluid mechanics to optimize the mixing effect by changing the channel shape and adding specific structures.

[0048] 3. The osteosarcoma in vitro culture chip provided by the present invention is provided with an anti-backflow structure to prevent the backflow of the outlet liquid and ensure that the concentration of the substance in the culture area remains stable; it is crucial to maintain the stability of the experimental conditions and avoid concentration fluctuations and experimental errors caused by backflow.

[0049] 4. The osteosarcoma in vitro culture chip provided by the present invention adopts multiple stepped spaces, which further enhances the stability of the substance concentration gradient and helps to simulate the natural microenvironment of bone tissue; it not only increases the structural complexity of the chip, but also improves its simulation, allowing cells to grow and interact under physiological conditions that are closer to reality.

[0050] 5. The present invention uses 3D bioprinting technology to construct the bone-skeletal muscle tissue interface and improve the simulation of the model. This design enables tumor cells to grow in a real environment that interacts with bone tissue, thus providing an effective platform for later research on the metastasis mechanism of osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 A 3D model diagram of the osteosarcoma in vitro culture chip provided in Example 1;

[0052] Figure 2 A schematic diagram of the structure of the carrier chip of the osteosarcoma in vitro culture chip provided in Example 1;

[0053] Figure 3 A schematic structural diagram of the cover plate of the osteosarcoma in vitro culture chip provided in Example 1;

[0054] Figure 4 for Figure 2 A magnified view of the S-shaped channel in;

[0055] Figure 5 This is a 3D model diagram of the osteosarcoma in vitro culture chip after closure provided in Example 1;

[0056] Figure 6 A 3D model diagram of the osteosarcoma in vitro culture chip provided in Example 2;

[0057] Figure 7 A schematic diagram of the structure of the carrier chip of the osteosarcoma in vitro culture chip provided in Example 2;

[0058] Figure 8 A schematic diagram of an in vitro osteosarcoma organ culture system provided by the present invention;

[0059] Fig. 9 It is the concentration gradient simulation diagram;

[0060] Fig.10 This is a physical picture of osteosarcoma tumor spheres;

[0061] Fig.11 This is a micrograph of vascularized skeletal muscle tissue;

[0062] Fig.12 Extrusion printing model for bone-skeletal muscle tissue;

[0063] In the figure: 1, cover plate; 11, baffle plate; 2, chip; 21, inlet; 22, concentration mixing channel area; 221, concentration mixing channel; 23, concentration gradient forming area; 231, baffle bar; 24, tissue culture area; 25, liquid storage area; 26, outlet; 27, overflow port. DETAILED DESCRIPTION

[0064] The technical scheme of the present invention is further described below by way of examples. The raw materials used in the examples can be purchased from the market or prepared by conventional methods.

[0065] Example 1

[0066] A 3D printed osteosarcoma in vitro culture chip IV, e.g. Figure 1 to Figure 5 As shown, a 3D printed osteosarcoma in vitro culture chip IV, as Figure 6 to Figure 8 As shown, the osteosarcoma in vitro culture chip IV includes a carrier chip 2 and a cover plate 1; the carrier chip includes two inlets 21, a concentration mixing channel area 22, a concentration gradient forming area 23, a tissue culture area 24, an outlet 26 and a liquid storage area 25 designed to prevent liquid backflow and destroy the concentration gradient; the concentration mixing channel area 22 is provided with an S-shaped concentration mixing channel 221; the concentration mixing channel 221 is a three-section S-shaped channel, the first section is two S-shaped channels, one end of the two S-shaped channels of the first section is connected to the two inlets 21 respectively, and the other end is connected to the three S-shaped channels of the second section through a transversely arranged channel, and the three S-shaped channels of the second section are connected to the four S-shaped channels of the third section through a transversely arranged channel; a plurality of reverse herringbone bones 2211 are evenly distributed in each S-shaped channel; the tumor sphere is placed in the tissue culture area 26; the cover plate includes two baffles 11, corresponding to the tissue culture area 24 and the liquid storage area 25 of the carrier chip 2.

[0067] Two liquids of different concentrations enter the concentration mixing channel 221 from two inlets. The concentration mixing channel 221 is a three-section S-shaped channel. The first section is two S-shaped channels. One end of the two S-shaped channels in the first section is connected to the two inlets 21 respectively, and the other end is connected to the three S-shaped channels in the second section through a transverse channel. The three S-shaped channels in the second section are connected to the four S-shaped channels in the third section through a transverse channel. Multiple reverse herringbone bones 2211 are evenly distributed in each S-shaped channel. This design allows the liquid to be fully mixed in a limited area. The liquid mixed in the three-section S channel enters the concentration gradient forming area 23, and flows into the tissue culture area 24 after the concentration gradient is formed and stabilized. Multiple osteosarcoma tumor spheres are evenly and dispersedly placed in the tissue culture area 24, and the same batch of tumor spheres are cultured at different drug concentrations at the same time. Finally, the liquid enters the liquid storage area 25 and flows out from the outlet 26.

[0068] The carrier chip 2 and the cover plate 1 are both made of polylactic acid. The carrier chip and the cover plate of this embodiment can be realized by 3D printing technology.

[0069] This embodiment uses 3D printing technology to print the carrier chip 2 and the cover plate 1, so that the in vitro model is easier to meet actual needs. The number and number of layers of the S-shaped channels 4 in the concentration mixing channel area can be designed and adjusted according to experimental needs to obtain drug concentrations of different gradients. Instant adjustment and printing according to the actual map are fast and efficient. The polylactic acid material used for printing is low-cost and non-toxic.

[0070] Example 2

[0071] A 3D printed osteosarcoma in vitro culture chip IV, e.g. Figure 6-7 As shown, the osteosarcoma in vitro culture chip IV comprises a carrier chip 2 and a cover plate 1; the carrier chip comprises two inlets 21, a concentration mixing channel area 22, a concentration gradient forming area 23, a tissue culture area 24, an outlet 26, and a liquid storage area 25 designed to prevent liquid backflow and destroy the concentration gradient; the concentration mixing channel area 22 is provided with an S-shaped concentration mixing channel 221; the concentration mixing channel 221 is a three-section S-shaped channel, the first section is two S-shaped channels, one end of the two S-shaped channels of the first section is connected to the two inlets 21 respectively, and the other end is connected to the two inlets 21 respectively. The transversely arranged channel is connected with the three S-shaped channels arranged in the second section, and the three S-shaped channels arranged in the second section are connected with the four S-shaped channels arranged in the third section through the transversely arranged channel; a plurality of reverse herringbone bones 2211 are evenly distributed in each S-shaped channel; the concentration gradient forming area 23 is additionally provided with a baffle 231, and the baffle 231 is arranged on the concentration gradient forming area 23 corresponding to the midpoint of the outlets of two adjacent S-shaped channels; the tumor sphere is placed in the tissue culture area 26; the cover plate includes two baffles 11, corresponding to the tissue culture area 24 and the liquid storage area 25 of the carrier chip 2.

[0072] Two liquids of different concentrations enter the concentration mixing channel 221 from two inlets. The concentration mixing channel 221 is a three-section S-shaped channel. The first section is two S-shaped channels. One end of the two S-shaped channels of the first section is connected to the two inlets 21 respectively, and the other end is connected to the three S-shaped channels of the second section through a transverse channel. The three S-shaped channels of the second section are connected to the four S-shaped channels of the third section through a transverse channel. A plurality of reverse herringbone bones 2211 are evenly distributed in each S-shaped channel. This design allows the liquid to be fully mixed in a limited area. The liquid mixed through the three-section S channel enters the concentration gradient forming area 23. The baffle 231 can further ensure the temperature of the concentration gradient. After the concentration gradient is formed and stabilized, it flows into the tissue culture area 24. A plurality of osteosarcoma tumor spheres are evenly and dispersedly placed in the tissue culture area 24, and the same batch of tumor spheres are cultured with different drug concentrations at the same time. Finally, the liquid enters the liquid storage area 25 and flows out from the outlet 26. There is an overflow port 27 to prevent the liquid from flowing back.

[0073] The carrier chip 2 and the cover plate 1 are both made of polylactic acid. The carrier chip and the cover plate of this embodiment can be realized by 3D printing technology.

[0074] This embodiment uses 3D printing technology to print the carrier chip 2 and the cover plate 1, so that the in vitro model is easier to meet actual needs. The number and number of layers of the S-shaped channels 4 in the concentration mixing channel area can be designed and adjusted according to experimental needs to obtain drug concentrations of different gradients. Instant adjustment and printing according to the actual map are fast and efficient. The polylactic acid material used for printing is low-cost and non-toxic.

[0075] Example 3

[0076] An in vitro organ culture system for osteosarcoma, such as Figure 8 As shown, it includes medicine bottle I and medicine bottle II, two peristaltic pumps III and IV respectively connected to the two medicine bottles, and an osteosarcoma in vitro culture chip V prepared in Example 1 and connected to a waste liquid bottle VII in sequence through a rubber tube VI.

[0077] In this embodiment, drugs of two concentrations, 1 μg / mL and 10 μg / mL, are placed in medicine bottles I and II, respectively, and are pumped into carrier chip IV through peristaltic pumps III and IV, respectively, and enter concentration mixing channel 221 from two inlets 21 of osteosarcoma in vitro culture chip IV, and after being fully mixed through three sections of S-shaped channels, flow into concentration gradient forming area 22, where a stable concentration gradient is formed, and then flow into tissue culture area 24. At this time, the drug concentration gradient in the tissue culture area is clear and uniform, as shown in the concentration gradient simulation diagram. Figure 7 As shown, it provides great convenience for testing drug concentration in tumor spheres and the experimental results are stable. Finally, the liquid flows into the liquid storage area 25 and flows out from the outlet 26 of the osteosarcoma in vitro culture chip IV.

[0078] like Fig. 9 The concentration gradient simulation diagram of the osteosarcoma in vitro culture chip V prepared in Example 1 shows that different gradients of drug concentrations are obtained.

[0079] In addition, the number and segments of the S-shaped channels can be adjusted at any time according to the experimental requirements to form the drug concentration and concentration quantity that meet the requirements. 3D printing makes chip adjustment simple and convenient, and the polylactic acid material is non-toxic to cells and does not affect the properties of the drug.

[0080] Example 4

[0081] A method for constructing an in vitro osteosarcoma culture chip using the osteosarcoma in vitro culture chip comprises the following steps:

[0082] S1, dissolving the raw material of bioprinting ink GelMa in the cell culture medium to prepare a GelMa solution with a mass volume concentration of 14% (m / v);

[0083] S2, dissolving fibrinogen in physiological saline to prepare a fibrinogen solution with a mass volume concentration of 10% (m / v);

[0084] S3, the GelMa solution in step S1 and the fibrinogen solution in step S2 were mixed in equal volumes, and LAP and osteosarcoma cells were added; the mass volume concentration of LAP was 1% (m / v), and the density of osteosarcoma cells was 10×10 6 cell / mL, mix well, remove bubbles, and load into the needle of the bioprinter;

[0085] S4. The required tumor sphere particle size is 300 μm and the number is 10. Set the parameters of the bioprinter inkjet printing, install the needle in step S3 on the printer for printing, and obtain osteosarcoma tumor spheres of uniform size and shape.

[0086] S5, the osteosarcoma tumor spheres obtained in step S4 were irradiated with ultraviolet light of wavelength 405 nm for 55 seconds, and then soaked in thrombin for 120 seconds. After soaking, the osteosarcoma tumor spheres were washed with PBS. Fig.10 As shown;

[0087] S6. Place the osteosarcoma tumor spheres processed in step S5 between the bone tissue and the vascularized skeletal muscle tissue in the bone-skeletal muscle tissue and then place them in the tissue culture area of ​​the osteosarcoma in vitro culture chip, cover them with a cover plate, and culture them using the osteosarcoma in vitro organ culture system.

[0088] The bone-skeletal muscle tissue in vitro culture construction method described in step S6 comprises the following steps:

[0089] P1. Mix gelatin, sodium alginate and hydroxyapatite to prepare an aqueous solution, wherein the mass volume concentration of gelatin is 5%, the mass volume concentration of sodium alginate is 3%, and the mass volume concentration of hydroxyapatite is 15%. After completely melting and mixing, transfer the mixture to a printing needle tube for multi-layer mesh structure extrusion printing to prepare a bone tissue body;

[0090] P2, soaking the product extruded and printed in step P1 in a calcium chloride solution with a volume concentration of 3% (m / v) for 20 minutes for hardening treatment to obtain a bone tissue body with a high hardness and a multi-layered reticular structure, and obtaining bone tissue after culturing;

[0091] P3, GelMa, rat tail collagen type I, skeletal muscle cells and human umbilical vein endothelial cells were mixed to prepare a suspension, wherein the mass volume concentration of GelMa in the suspension was 5% (w / v), the mass volume concentration of rat tail collagen type I was 2% (w / v), and the density of skeletal muscle cells was 2×10 6cell / mL, the density of human umbilical vein endothelial cells was 1×10 6 / mL is extrusion-printed and then cured by ultraviolet light with a wavelength of 405nm to obtain a muscle tissue body, wherein the structure of the muscle tissue body forms a nested structure with the structure of the bone tissue body, and the muscle tissue body is cultured for a period of time to form vascularized skeletal muscle tissue, wherein the vascularized skeletal muscle tissue is as follows Fig.11 As shown;

[0092] P4, embedding the bone tissue obtained in step P2 in the vascularized skeletal muscle tissue obtained in step P3, wherein the vascularized skeletal muscle tissue covers the bone tissue to obtain the bone-skeletal muscle tissue, wherein the bone-skeletal muscle tissue is as follows: Fig.12 shown.

[0093] It should be understood that the above embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. An osteosarcoma in vitro culture chip that mimics bone microenvironment, characterized in that: The invention comprises a carrier chip (2) and a cover plate (1); the carrier chip (2) comprises two inlets (21), a concentration mixing channel area (22), a concentration gradient forming area (23), a tissue culture area (24), an outlet (26), and a liquid storage area (25) designed to prevent liquid from flowing back and destroying the concentration gradient; the concentration mixing channel area (22) is provided with a concentration mixing channel (221); the concentration mixing channel area (22), the concentration gradient forming area (23), the tissue culture area (24) and the liquid storage area (25) are arranged in a step-by-step manner from high to low; the cover plate (1) comprises two baffles (11), and the baffles (11) respectively correspond to the tissue culture area (24) and the liquid storage area (25) of the carrier chip.

2. The osteosarcoma in vitro culture chip mimicking bone microenvironment according to claim 1, characterized in that: The concentration mixing channel (221) is provided with at least three sections, and each channel in each section is S-shaped; the first section is provided with two S-shaped channels, one end of the two S-shaped channels of the first section is connected to the two inlets (21) respectively, and the other end is connected to the S-shaped channel of the next section through a transversely arranged channel, and the number of channels increases by one each time entering the next section.

3. The osteosarcoma in vitro culture chip simulating bone microenvironment according to claim 1, characterized in that: The concentration mixing channel (221) is provided with a reverse herringbone (2211).

4. The osteosarcoma in vitro culture chip mimicking bone microenvironment according to claim 1, characterized in that: The raw materials of the carrier chip (2) and the cover plate (1) include polylactic acid, photosensitive resin or polydimethylsiloxane, and are manufactured by 3D printing technology.

5. An in vitro organ culture system for osteosarcoma, characterized in that: The invention comprises a medicine bottle (I) and a medicine bottle (II), two peristaltic pumps (III) and a peristaltic pump (IV) respectively connected to the two medicine bottles, and an osteosarcoma in vitro culture chip (V) as described in any one of claims 1 to 4 and connected to a waste liquid bottle (VII) in sequence through a rubber tube (VI).

6. A method for constructing an in vitro osteosarcoma culture chip using any one of claims 1 to 4, characterized in that: The following steps are involved: S1, dissolving methacryloyl gelatin, a raw material of biological printing ink, in a cell culture medium to prepare a methacryloyl gelatin solution; S2, dissolving fibrinogen in physiological saline to prepare a fibrinogen solution; S3, mixing equal volumes of the methacrylylated gelatin solution in step S1 and the fibrinogen solution in step S2, adding phenyl-2,4,6-trimethylbenzoyl lithium phosphate and osteosarcoma cells; after mixing evenly, removing bubbles, loading into the needle tube of a bioprinter for printing, and obtaining osteosarcoma tumor spheres of uniform size and shape; S4, irradiating the osteosarcoma tumor spheres obtained in step S3 with ultraviolet light to solidify the methacrylylated gelatin, then soaking them with thrombin to solidify the fibrinogen, and washing them with phosphate buffered saline after soaking; S5. Place the osteosarcoma tumor spheres processed in step S4 between the bone tissue and the vascularized skeletal muscle tissue in the bone-skeletal muscle tissue and then place them in the tissue culture area of ​​the osteosarcoma in vitro culture chip, cover them with a cover plate, and culture them using the osteosarcoma in vitro organ culture system.

7. The method for constructing an osteosarcoma in vitro culture chip for culturing osteosarcoma in vitro according to claim 6, characterized in that: The mass volume concentration of the methacrylated gelatin solution in step S1 is 10% to 20%; the mass volume concentration of the fibrinogen solution in step S2 is 1% to 10%.

8. The method for constructing an osteosarcoma in vitro culture chip for culturing osteosarcoma in vitro according to claim 6, characterized in that: The mass volume concentration of phenyl-2,4,6-trimethylbenzoyl lithium phosphate in step S3 is 0.1% to 1%; the density of osteosarcoma cells in step S3 is 1×10 6 ~10×10 6 cell / mL; the particle size of the osteosarcoma tumor spheres described in step S3 is 20 to 1000 μm; the ultraviolet irradiation time described in step S4 is 20 to 100 s; the thrombin immersion time described in step S4 is 30 to 300 s.

9. The method for constructing an osteosarcoma in vitro culture chip for culturing osteosarcoma in vitro according to claim 6, characterized in that: The bone-skeletal muscle tissue in vitro culture construction method described in step S5 comprises the following steps: P1. Mix gelatin, sodium alginate and hydroxyapatite to prepare an aqueous solution, completely melt and mix, and then transfer to a printing needle tube for multi-layer mesh structure extrusion printing to prepare a bone tissue body; P2, soaking and hardening the product extruded and printed in step P1 in a calcium chloride solution to obtain a bone tissue body with a high hardness and a multi-layered network structure, and forming bone tissue after culturing; P3, mixing methacrylylated gelatin, type I rat tail collagen, skeletal muscle cells and human umbilical vein endothelial cells to prepare a suspension, performing extrusion printing on the suspension and then curing it under ultraviolet light to obtain a muscle tissue body, wherein the structure of the muscle tissue body forms a nested structure with the structure of the bone tissue body, and culturing the muscle tissue body for a period of time to form a vascularized skeletal muscle tissue; P4. Embedding the bone tissue obtained in step P2 in the vascularized skeletal muscle tissue obtained in step P3, wherein the vascularized skeletal muscle tissue covers the bone tissue, thereby obtaining the bone-skeletal muscle tissue.

10. The method for constructing an osteosarcoma in vitro culture chip for culturing osteosarcoma in vitro according to claim 9, characterized in that: In the aqueous solution described in step P1, the mass volume concentration of sodium alginate is 2% to 8%, the mass volume concentration of gelatin is 5% to 15%, and the mass volume concentration of hydroxyapatite is 5% to 20%; the mass volume concentration of the calcium chloride solution described in step P2 is 2% to 7%; the soaking time described in step P2 is 20 to 80 minutes; the mass volume concentration of the methacryloyl gelatin described in step P3 is 5% to 8%, the mass volume concentration of type I rat tail collagen is 1% to 5%, and the density of skeletal muscle cells is 1×10 6 ~10×10 6 cell / mL, the density of human umbilical vein endothelial cells is 1×10 6 ~10×10 6 / mL.

11. Use of the osteosarcoma in vitro culture chip according to any one of claims 1 to 4 in the study of the metastasis mechanism of osteosarcoma.

12. Use of the osteosarcoma in vitro culture chip according to any one of claims 1 to 4 in studying the safe concentration of drugs or studying the effect of drug concentration on tumors.

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