WO2024012073A1 - Osteosarcoma organoid model, construction method, and use - Google Patents

Abstract

Provided are an osteosarcoma organoid model and a construction method therefor. The osteosarcoma organoid model at least comprises a biological scaffold, osteosarcoma cells, and exosomes derived from mesenchymal stem cells, wherein the osteosarcoma cells and the exosomes are loaded on the biological scaffold, the biological scaffold is of a crisscrossed three-dimensional structure constructed by a hydrogel preparation, and the hydrogel preparation comprises a decellularized extracellular matrix and fibrin.

Description

An osteosarcoma organoid model, construction method and application Technical field

The invention relates to the field of medicine, and in particular to an osteosarcoma organoid model, construction method and application.

Background technique

Currently, tumor-related biomedical research mainly relies on in vitro cultured human cells or rodent models. Although research using cells cultured in vitro can directly reflect the effects of drugs, the response in physiological functions is still significantly different from the real physiological functions of the human body. In contrast, animal models can simulate the physiological functions of a certain organ or multiple organs, but their limitation is that the inherent differences in physiological functions between animals and humans will put some drug candidates that are effective against tumors at risk of failure in human trials. . Biomimetic systems, especially organ-on-chips, are important models that have emerged in recent years to study tissue physiology and pathological functions in vitro. It refers to a micro-engineered biomimetic body tissue system built on a microfluidic chip. It can combine the advantages of human cell in vitro simulation models and rodent models to cultivate human tissues by simulating the effects of the human blood circulation system and construct structures similar to human tissues. organizational structure. By developing and utilizing these microcirculatory systems, in vitro tissue physiology and pathological disease models can be established to understand biological mechanisms and screen potential drugs. The above studies demonstrate the great potential of these microfluidic systems in biomedical, pharmaceutical and toxicological applications in the medical field.

Although organ-on-a-chip has huge application prospects in clinical applications. Due to the influence of processing technology, the existing 3D osteosarcoma organ chips are still unable to microscopically control the spatial structure and cell distribution of the osteosarcoma matrix, nor can they accurately control the exchange of oxygen and nutrients in the matrix, so they cannot be used at the microscopic level. Accurately simulating the characteristics of high proliferation activity, high cell density and strong matrix-cell interaction of osteosarcoma tissue, there is still a certain gap between it and active osteosarcoma tissue in vivo. Moreover, single organ chips currently only study the impact of mechanical stimulation of the circulatory system on physiological and pathological behaviors, which greatly ignores the relationship between the biological functions and metabolic behaviors of other solid organs and tumor development and treatment, because it cannot simulate the in vivo well. biological processes.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an osteosarcoma organoid model, construction method and application.

Through the combined application of "3D bioprinting technology" and "organoid chip model", the present invention (1) 3D prints dOsEM-Fibrin hydrogel scaffold on the microfluidic chip to load patient osteosarcoma cells and BMSC-EV to construct A multi-level biomimetic organoid dynamic culture model that can simulate the composition-structure of the osteosarcoma stromal microenvironment in vivo. And screen the therapeutic potential of drug combination regimens for osteosarcoma with high proliferative activity through 3D multi-level bionic organoid chips.

The present invention builds a multi-level scaffold that can simulate the high proliferation and metastasis activity of osteosarcoma in vivo by 3D printing a hydrogel scaffold of bone tissue decellularized matrix (dOsEM-Fibrin) on a microfluidic chip and combining it with osteosarcoma cells and BMSC-EV. Biomimetic osteosarcoma organoid dynamic culture model. We also explore the therapeutic effect of multiple inhibitors combined with first-line chemotherapy drugs for osteosarcoma on highly proliferative osteosarcoma, and clarify the application value of this model in personalized diagnosis and treatment of osteosarcoma.

This invention will help to deeply understand the specific effects and mechanisms of physicochemical components and physical structures in the physiological and pathological microenvironment on osteosarcoma cell proliferation and adhesion behavior, and clarify the multi-level bionic tumor organ chip detection based on 3D bioprinting. The platform is of great significance for in vitro drug screening of osteosarcoma, and provides a preclinical drug evaluation platform closer to the human body for current new biotherapy, immunotherapy and targeted therapy.

A first aspect of the present invention provides an osteosarcoma organoid model, which osteosarcoma model organoid model at least includes a biological scaffold and exosomes derived from osteosarcoma cells and bone marrow mesenchymal stem cells loaded on the biological scaffold; The bioscaffold is a crisscross three-dimensional structure constructed from a hydrogel preparation that includes an acellular matrix and fibrin.

A second aspect of the present invention provides a method for constructing an osteosarcoma organoid model, which at least includes the following steps:

Exosomes derived from bone marrow mesenchymal stem cells and osteosarcoma cells are loaded on the biological scaffold to obtain an osteosarcoma organoid model; the biological scaffold is a crisscross three-dimensional structure constructed with a hydrogel preparation, and the water Gel formulations include acellular matrix and fibrin.

A third aspect of the present invention provides a microfluidic chip, which contains:

An organoid culture area, the organoid culture area contains the aforementioned osteosarcoma organoid model;

a liquid inlet, connected to the organoid culture area, for pumping in fluid;

A liquid outlet is connected to the organoid culture area and is used to discharge fluid;

Microfluidic channel is used to connect the organoid culture area, liquid inlet and liquid outlet.

A fourth aspect of the present invention provides a method for preparing a microfluidic chip, which at least includes the following steps: placing the aforementioned osteosarcoma organoids in an organoid culture area; injecting fluid through a liquid inlet into microfluidic channels and organoid culture area to obtain the microfluidic chip.

The fifth aspect of the present invention provides a dynamic culture model of osteosarcoma model organoids. The dynamic culture model includes the aforementioned microfluidic chip and a microfluidic pump. The microfluidic pump is connected to the liquid inlet in the microfluidic chip. The port is connected to the liquid outlet and is used to provide power for fluid flow in the microfluidic chip.

A sixth aspect of the present invention provides the application of the aforementioned osteosarcoma organoid model or the aforementioned microfluidic chip or the aforementioned osteosarcoma model organoid dynamic culture model in anti-osteosarcoma drug screening.

As mentioned above, the osteosarcoma organoid model, construction method and application of the present invention have the following beneficial effects:

(1) This invention combines the advantages of the most cutting-edge 3D bioprinting technology in the world and the organoid chip screening platform. point, and further deepened and innovated it. For the first time, the dOsEM-Fibrin hydrogel loading system was combined with BMSC-EV to integrate the "component-structure" multi-level bionic optimization on the microfluidic chip using 3D bioprinting technology. The osteosarcoma organoid chip constructs a dynamic organoid culture model that can simulate the osteosarcoma stromal microenvironment in vivo, and also lays an ideal model foundation for the subsequent in vitro 3D culture of physiological and pathological bone tissue.

(2) The present invention uses bone matrix mesenchymal stem cell exosomes (BMSC-EV) as the research object to innovatively explore how BMSC-EV activates the CXCL12/CXCR4 signaling axis to regulate osteosarcoma cell proliferation and invasion in the osteosarcoma stromal microenvironment. specific mechanism of action. Systematically clarified that the 3D osteosarcoma organoid chip better simulates the regulatory role of CXCL12/CXCR4 under the in vivo osteosarcoma matrix-cell interaction than the 2D culture model, revealing the potential application value of this model. In addition, this project innovatively revealed that BMSCs jointly affect the osteosarcoma stromal microenvironment through secretion of exosomes and matrix components, and regulate the proliferation and metastasis behavior of osteosarcoma cells.

(3) The present invention innovatively studies the therapeutic effect of CXCR4-specific inhibitor Plerixafor combined with first-line osteosarcoma chemotherapy drugs on highly proliferative osteosarcoma through multi-level bionic organoid chips, and further verifies the 3D printing of multi-level bionic osteosarcoma organoid chips. The advantage of simulating drug effects in vivo has established an effective in vitro evaluation platform for the screening and optimization of personalized multi-drug treatment plans for osteosarcoma.

Organ-on-a-chip has strong clinical application value for personalized drug evaluation. It can accurately and quickly observe the toxicity of drugs to the patient's target organs and other multiple organs in real time. The present invention uses brand-new technical means to provide a brand-new diagnosis and treatment strategy and treatment evaluation means for the diagnosis and treatment of osteosarcoma. The invention provides a powerful tool for clinical research and treatment of bone tumors, thereby filling the gap in in vitro models of osteosarcoma, and has good economic benefits. On the other hand, it can provide new research ideas for finding relevant therapeutic targets for osteosarcoma, which is of great value.

Description of drawings

Figure 1: Schematic flow diagram of the present invention.

Figure 1-1: Exploded view of a microfluidic chip according to an embodiment of the present invention.

Figure 1-2: Structural diagram of a microfluidic chip according to an embodiment of the present invention.

Figure 1-3: Preparation process of a microfluidic chip according to an embodiment of the present invention.

Figure 2: Characterization of BMSC-EVs. (A) Cell morphology diagram of BMSCs. (B) Flow cytometric analysis of surface markers of BMSCs. (C) TEM micromorphology image of BMSC-EV. (D) Particle size distribution diagram of BMSC-EV. (E) Cellular uptake diagram of BMSC-EV. (F) West Blot analysis results of specific markers of BMSC-EV.

Figure 3: BMSC-EV promotes the proliferation and migration of MG63. (A) MTT method detects the proliferation of BMSC-EV at different concentrations. (B,C) Scratch test detects the migration of BMSC-EV at different concentrations. (D) BMSC-EV enhances MG63 expression of bone-related genes.

Figure 3-1: Analysis of the active ingredients of dOsEM using protein biomass spectrometry.

Figure 4: Preparation of osteosarcoma acellular matrix dOsEM and dOsEM-Fibrin hydrogel.

Figure 5: Characterization of osteosarcoma acellular matrix dOsEM and dOsEM-Fibrin hydrogel. (A) Protein spectrum identifies active ingredients in dOsEM. (B) TEM identifies the micromorphology within dOsEM. (C) Quantitative analysis of DNA, Collagen and GAG components in dOsEM, showing the effects of osteosarcoma acellular matrix treatment on their respective components.

Figure 6: Rheological properties of dOsEM-Fibrin hydrogel. (A) Dependence of storage modulus (G′) and loss modulus (G″) on shear strain of dOsEM-Fibrin hydrogel materials with different concentrations. (B) dOsEM-Fibrin hydrogels with different concentrations Dependence of material viscosity on shear strain.

Figure 7: (A) Schematic diagram of the manufacturing process of 3D printed on-chip osteosarcoma model. (B) Optical micrograph and macro view of the 3D printed osteosarcoma-on-chip model. (C) 3D printed osteosarcoma chip model for drug screening applications.

Figure 8: (A) Compared with Alginate hydrogel, the organoid chip constructed from 3D bioprinted dOsEM-Fibrin hydrogel can significantly improve the in vitro cell adhesion activity of osteosarcoma cells (top A: phalloidin immunofluorescence staining) and proliferation activity (bottom A: immunofluorescence staining of live and dead cells). (B) Cck8 detection results of bone tissue cell proliferation. dOsEM-Fibrin hydrogel can significantly promote the proliferation of physiological bone tissue-related cells. (C) Cck8 detection results of osteosarcoma cell proliferation. dOsEM-Fibrin hydrogel can significantly promote the proliferation of pathological osteosarcoma cells.

Figure 9: (A) Use 3D printed osteosarcoma organoid chips to construct a drug screening platform; B is the effect of different drug treatments on the activity of cells inside osteosarcoma tissue (B is the result of live-death fluorescence staining); C is the effect on cells in B Quantitative statistics of activity. (B, C) It is clear that the combination of Plerixafor and Dox can significantly increase the killing effect of Dox on osteosarcoma cells with high proliferative activity.

Figure 10: Metabolic function of 3D bioprinted dOsEM-Fibrin hydrogel scaffold. To compare the 3D hydrogel chip (3D chip) group with the conventional 2D single cell culture (2D Mono) group, the cell viability was measured using MTT to obtain the 50% lethal dose (IC50) results of the cells for different drugs.

Figure 11: Technical roadmap of the present invention.

Figure 12: Flow chart of the application method of the present invention.

1-Organoid culture area

2-Liquid inlet

3-Liquid outlet

4-Microfluidic channel

5-Cover

6-Bottom

7-Adhesive film

Detailed ways

3D bioprinting technology to construct micro-tumor tissue

First of all, 3D printing technology is an effective way to construct tissue biological scaffolds. Compared with other 3D culture models, it can accurately arrange cells and extracellular matrix, control the local tissue microenvironment, and then construct a similar morphology and mechanical environment of in vivo tissues, simulating the interaction between cells, cells and extracellular matrix in the body. interaction. In addition, it can accurately control the porosity inside the scaffold by blending the materials and cells in advance and arranging them regularly after extrusion, which facilitates the transmission of oxygen and nutrients and the exchange of metabolic waste, thereby maintaining the survival of cells inside the scaffold. In recent years, 3D bioprinting active scaffolds constructed with biohydrogel-loaded stem cells as printing ink have shown high potential in tissue regeneration and repair. It mainly includes three organic components: functional cells, biological scaffold materials, and loaded bioactive components. The selection of bioscaffold materials and loaded bioactive components is the core and difficulty of this technology. Loaded bioactive ingredients. Moreover, the unique high-precision processing technology of 3D bioprinting technology can better realize the construction of micro-tissues and better simulate the microscopic composition and structure of tissues to alleviate the shortcomings of traditional tissue chips in this field.

In addition, 3D bioprinting technology has also demonstrated its unique advantages in constructing solid tumor models. The traditional 2D method to construct tumor tissue is extremely limited. The reason is that the two-dimensional monolayer cell model lacks the natural microenvironment characteristics of tumor tissue in vivo. On the other hand, rodent tumor-bearing models established in immunocompromised mice cannot well simulate the development and progression of tumors in humans. In order to overcome the above obstacles, more and more studies are beginning to use in vitro 3D tumor models based on human cancer cells to accurately simulate the characteristics of human cancer tissue, and then study their effects on tumor cell morphology, proliferation, drug metabolism, gene expression, and protein synthesis. . Related technologies such as multi-cell spheroid technology, 3D scaffold seeding technology, hydrogel embedding technology, microfluidic chips and cell patterning have also been developed for the construction of 3D in vitro tumor models. Although these studies have revealed the great application potential of in vitro 3D tumor models, it is still difficult to simulate the complex 3D tumor microenvironment in most of the above models due to limitations in manufacturing technology. 3D biotechnology provides more possibilities for studying the pathogenesis of diseases and the discovery of new drugs by constructing tumor cells and the interaction of extracellular matrix (ECM) materials. Compared with traditional 2D flat culture models, 3D printed scaffolds have higher tumor proliferation activity and colony formation ability. 3D bioprinting technology can better simulate the interaction between tumor cells and extracellular matrix (ECM), accurately control the transport of nutrients inside the scaffold, and more accurately simulate the pathological microenvironment of tumor tissues with high proliferative activity in the body.

3D bioprinting technology creates multi-level bionic organoids with composition-structure that can simulate the osteosarcoma matrix microenvironment in vivo ecological culture model

However, some chemical treatments involved in preparing dOsEM acellular matrix may destroy bioactive proteins in the matrix microenvironment, especially exosomes (BMSC-EV) represented by bone marrow mesenchymal stem cells (BMSC) and signaling molecules. etc., therefore, the organoid chip constructed based on dOsEM-Fibrin hydrogel cannot well simulate the interaction between BMSCs and osteosarcoma cells in the matrix. BMSC, as an important component of the bone microenvironment, are cells in the bone marrow with multi-directional differentiation potential and immune regulatory functions. Studies have shown that they are involved in the proliferation and migration of tumor tissues, the formation of the tumor microenvironment, and their interaction with tumor cells. interactions, etc., and BMSCs have also been shown to promote tumor cell growth, metastasis and immune evasion by secreting a series of cytokines such as IL-6, VEGF, TNF-a, etc. Among them, exosomes (BMSC-EV) secreted by stem cells, as the main carrier of secreted factors, are vesicles with a diameter of 40-100 nm secreted by BMSC in the extracellular environment. It contains proteins, lipids and nucleic acids. In recent years, a large number of studies have confirmed that BMSC-EV can affect the occurrence and progression of tumors, and mesenchymal stem cells can also regulate the formation of blood vessels in tumors and further promote the proliferation of cells within tumor tissues by regulating angiogenesis, stability, and maturation. However, at present, the specific mode and mechanism of BMSC-EV promoting the growth and metastasis of osteosarcoma cells in the bone marrow microenvironment are still unclear. In order to better simulate the interaction between BMSC and osteosarcoma cells in the matrix, the present invention supplemented the BMSC-EV component in the dOsEM-Fibrin hydrogel inside the organoid chip to observe the cell proliferation ability and cell-matrix interaction.

Therefore, by 3D printing a dOsEM-Fibrin-based hydrogel scaffold (structural biomimetic) combined with osteosarcoma cells and BMSC-EV (component biomimetic) on a microfluidic chip, the present invention can construct a matrix with high proliferative activity and strong strength similar to that in vivo - Cell-interacting components - Structural multilevel biomimetic osteosarcoma model. Compared with the 2D culture model, it can more accurately simulate the expression of signaling pathways related to osteosarcoma tissue in vivo, as well as the impact on downstream cell phenotypes such as proliferation, adhesion, and migration (Figure 6).

The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, They are not intended to limit the scope of the invention; in the specification and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, As those skilled in the art have mastered the prior art and the description of the present invention, any method, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used. to implement the invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology.

The methods or application scenarios described in the present invention are used for non-diagnostic and therapeutic purposes, such as basic research.

One embodiment of the present invention provides an osteosarcoma organoid model. The osteosarcoma model organoid model at least includes a biological scaffold and osteosarcoma cells and bone marrow mesenchymal stem cell-derived exosomes (BMSC- EV); the biological scaffold is a crisscross three-dimensional structure constructed from a hydrogel preparation, which includes an acellular matrix and fibrin.

Preferably, the acellular matrix is an acellular matrix of osteosarcoma tissue.

In one embodiment, the osteosarcoma tissue is of human origin.

In one embodiment, the fibrin (Fibrin) can be derived from plasma or bacterial extracts of cattle, humans, rabbits, rats, etc.

In one embodiment, the reaction of fibrinogen and thrombin can be used to generate fibrin. Thrombin can be derived from plasma or bacterial extracts of cattle, humans, rabbits, rats, etc. For example, Fibrinogen from bovine plasma (F8630-10G, sigma) and thrombin (T4648-1KU, sigma) can be used to react to generate fibrin.

The osteosarcoma cells are living cells.

In one embodiment, the osteosarcoma cells are of human origin. For example, it can be MG63 or osteosarcoma cells from osteosarcoma patients.

In one embodiment, the concentration of the acellular matrix is greater than 0 and ≤ 60 mg/mL based on the total amount of the hydrogel preparation. For example, 60 mg/mL means that the concentration of the decellularized matrix dOsEM occupying the total amount of the hydrogel preparation is 60 mg/mL. It can be greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL, 10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL-40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL, 20mg/mL-60mg/mL, 20mg/ mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL, 40mg/mL- 60mg/mL, 40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of the acellular matrix is 6 mg/mL.

In one embodiment, the concentration of fibrin is greater than 0 and ≤ 60 mg/mL based on the total amount of the hydrogel formulation. 60 mg/mL means that the concentration of the fibrin in the total amount of the hydrogel preparation is 60 mg/mL. Can be, greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL ; 10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL-40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL; 20mg/mL-60mg/mL, 20mg /mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL; 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL; 40mg/mL -60mg/mL, 40mg/mL-50mg/mL or 50mg/mL-60mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of fibrin is 20 mg/mL.

In one embodiment, based on the total amount of the hydrogel preparation, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is greater than 0 and ≤300 mg/mL. The exosome concentration was calculated using the BCA protein detection kit (23227, thermofisher) (this method is the gold standard for exosome concentration detection in the industry). For example, 300 mg/mL means that 1 mL of hydrogel preparation can load 300 mg of exosomes derived from bone marrow mesenchymal stem cells. It can be greater than 0 and ≤300mg/mL, greater than 0 and ≤200mg/mL, greater than 0 and ≤100mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤10mg/mL, greater than 0 and ≤1mg/mL, Greater than 0 and ≤500μg/mL, greater than 0 and ≤300μg/mL, greater than 0 and ≤100μg/mL; 100μg/mL-300mg/mL, 100μg/mL-200mg/mL, 100μg/mL-100mg/mL, 100μg/ mL-50mg/mL, 100μg/mL-10mg/mL, 100μg/mL-1mg/mL, 100μg/mL-500μg/mL, 100μg/mL-300μg/mL, 300μg/mL-300mg/mL, 300μg/mL- 300mg/mL, 300μg/mL-100mg/mL, 300μg/mL-50mg/mL, 300μg/mL-10mg/mL, 300μg/mL-1mg/mL, 300μg/mL-500μg/mL, 500μg/mL-300mg/ mL, 500μg/mL-300mg/mL, 500μg/mL-100mg/mL, 500μg/mL-50mg/mL, 500μg/mL-10mg/mL, 500μg/mL-1mg/mL; 1mg/mL-300mg/mL, 1mg/mL-200mg/mL, 1mg/mL-100mg/mL, 1mg/mL-50mg/mL, 1mg/mL-10mg/mL; 10mg/mL-300mg/mL, 10mg/mL-200mg/mL, 10mg/ mL-100mg/mL, 10mg/mL-50mg/mL; 100mg/mL-300mg/mL, 100mg/mL-200mg/mL or 200mg/mL-300mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is 300 μg/mL.

In one embodiment, based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is greater than 0 and ≤8*10 ⁷ /mL. For example, 8*10 ⁷ / means that 1mL hydrogel preparation can load 8*10 ⁷ osteosarcoma cells.

It can be greater than 0 and ≤8*10 ⁷ /mL, greater than 0 and ≤6*10 ⁷ /mL, greater than 0 and ≤4*10 ⁷ /mL, greater than 0 and ≤2*10 ⁷ /mL, greater than 0 and ≤ 1*10 ⁷ /mL; 1*10 ⁷ /mL-8*10 ⁷ /mL, 1*10 ⁷ /mL-6*10 ⁷ /mL, 1*10 ⁷ /mL -4*10 ⁷ /mL, 1*10 ⁷ /mL-2*10 ⁷ /mL; 2*10 ⁷ /mL-8*10 ⁷ /mL, 2*10 ⁷ /mL-6*10 ⁷ /mL, 2*10 ⁷ /mL-4*10 ⁷ /mL; 4*10 ⁷ /mL-8*10 ⁷ /mL, 4*10 ⁷ /mL-6*10 ⁷ /mL or 6*10 ⁷ /mL-8 *10 ⁷ /mL.

Preferably, based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is 1*10 ⁷ /mL.

Optionally, the bone marrow mesenchymal stem cell-derived exosomes can be derived from animals. Further, for mammals. Optionally, it can be rodents, primates, artiodactyls, humans, etc. For example, mice, rabbits, monkeys, orangutans, apes, cows, pigs, humans, etc.

In one embodiment, the hydrogel formulation also includes gelatin.

Optionally, based on the total amount of the hydrogel preparation, the concentration of the gelatin is greater than 0 and ≤ 60 mg/mL. For example, 60 mg/mL means that the gelatin occupies a concentration of 60 mg/mL in the total amount of the hydrogel preparation. It can be greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL, 10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL-40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL, 20mg/mL-60mg/mL, 20mg/ mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL, 40mg/mL- 60mg/mL, 40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of gelatin is 20 mg/mL.

In one embodiment, the osteosarcoma model organoid model further includes glycerol. Used to reduce cell damage caused by extrusion.

Optionally, based on the total amount of the hydrogel preparation, the volume fraction of the glycerin is greater than 0 and ≤30%. It can be greater than 0 and ≤30%, greater than 0 and ≤20%, greater than 0 and ≤10%, 10%-30%, 10%-20% or 20%-30%.

The present invention constructs a decellularized matrix (Decellularized Osteosarcoma Extracellular Matrix, dOsEM) of osteosarcoma tissue (Figure 4) and combines it with Fibrin material to construct a dOsEM-Fibrin hydrogel, and then uses the CCK8 method to Alginate 5 kinds of hydrogels , GelMA, HGP, Collagen, and dOsEM-Fibrin were compared for cell proliferation activities, and it was verified that the prepared dOsEM-Fibrin hydrogel can better promote the in vitro proliferation of osteosarcoma cell line (MG63 cells). And compared with traditional 3D cultured Alginate hydrogel, dOsEM-Fibrin hydrogel can significantly improve the adhesion and proliferation activity of osteosarcoma cells. RNA-seq analysis results show that compared with 2D conventional culture, dOsEM-Fibrin hydrogel can significantly improve the adhesion and proliferation activity of osteosarcoma cells. Glue can significantly promote osteosarcoma cell proliferation, adhesion, and matrix-receptor interaction. The above results preliminarily prove that dOsEM-Fibrin hydrogel can simulate the interaction between osteosarcoma cells and matrix more realistically and significantly improve the activity of osteosarcoma cells compared with 2D culture models and other 3D culture hydrogel models. and proliferative capacity.

The present invention found that some chemical preparations involved in preparing dOsEM acellular matrix may destroy bioactive proteins in the matrix microenvironment, especially exosomes (BMSC-EV) represented by bone marrow mesenchymal stem cells (BMSC) and signaling molecules, etc. Therefore, the organoid chip constructed based on dOsEM-Fibrin hydrogel cannot well simulate the interaction between BMSCs and osteosarcoma cells in the matrix. BMSC, as an important component of the bone microenvironment, are cells in the bone marrow with multi-directional differentiation potential and immune regulatory functions. Studies have shown that they are involved in the proliferation and migration of tumor tissues, the formation of the tumor microenvironment, and their interaction with tumor cells. interactions, etc., and BMSCs have also been shown to promote tumor cell growth, metastasis and immune evasion by secreting a series of cytokines such as IL-6, VEGF, TNF-a, etc. Among them, exosomes (BMSC-EV) secreted by stem cells, as the main carrier of secreted factors, are vesicles with a diameter of 40-100 nm secreted by BMSC in the extracellular environment. It contains proteins, lipids and nucleic acids. BMSC-EV can affect the occurrence and progression of tumors, and mesenchymal stem cells can also regulate the formation of blood vessels in tumors and further promote the proliferation of cells within tumor tissues by regulating angiogenesis, stability, and maturation. The specific mode and mechanism of BMSC-EV promoting the growth and metastasis of osteosarcoma cells in the bone marrow microenvironment are still unclear. In order to better simulate the interaction between BMSC and osteosarcoma cells in the matrix, the present invention supplemented the BMSC-EV component in the dOsEM-Fibrin hydrogel inside the organoid chip to observe the cell proliferation ability and cell-matrix interaction.

One embodiment of the present invention provides a method for constructing an osteosarcoma organoid model, which at least includes the following steps:

Exosomes derived from bone marrow mesenchymal stem cells and osteosarcoma cells are loaded on the biological scaffold to obtain an osteosarcoma organoid model; the biological scaffold is a crisscross three-dimensional structure constructed with a hydrogel preparation, and the water Gel formulations include acellular matrix and fibrin.

Optionally, the solvent used in the hydrogel preparation can be culture medium, water or PBS buffer.

In one embodiment, the concentration of the acellular matrix is greater than 0 and ≤ 60 mg/mL based on the total amount of the hydrogel preparation. For example, 60 mg/mL means that the concentration of the decellularized matrix dOsEM occupying the total amount of the hydrogel preparation is 60 mg/mL. It can be greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL, 10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL-40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL, 20mg/mL-60mg/mL, 20mg/ mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL, 40mg/mL- 60mg/mL, 40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of the acellular matrix is 6 mg/mL.

In one embodiment, the concentration of fibrin is greater than 0 and ≤ 60 mg/mL based on the total amount of the hydrogel formulation. 60 mg/mL means that the concentration of the fibrin in the total amount of the hydrogel preparation is 60 mg/mL. Can be, greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL ;10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL -40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL; 20mg/mL-60mg/mL, 20mg/mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg /mL; 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL; 40mg/mL-60mg/mL, 40mg/mL-50mg/mL or 50mg/mL-60mg/mL .

Preferably, based on the total amount of the hydrogel preparation, the concentration of fibrin is 20 mg/mL.

In one embodiment, based on the total amount of the hydrogel preparation, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is greater than 0 and ≤300 mg/mL. The exosome concentration was calculated using the BCA protein detection kit (23227, thermofisher) (this method is the gold standard for exosome concentration detection in the industry). For example, 300 mg/mL means that 1 mL of hydrogel preparation can load 300 mg of exosomes derived from bone marrow mesenchymal stem cells. It can be greater than 0 and ≤300mg/mL, greater than 0 and ≤200mg/mL, greater than 0 and ≤100mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤10mg/mL, greater than 0 and ≤1mg/mL, Greater than 0 and ≤500μg/mL, greater than 0 and ≤300μg/mL, greater than 0 and ≤100μg/mL; 100μg/mL-300mg/mL, 100μg/mL-200mg/mL, 100μg/mL-100mg/mL, 100μg/ mL-50mg/mL, 100μg/mL-10mg/mL, 100μg/mL-1mg/mL, 100μg/mL-500μg/mL, 100μg/mL-300μg/mL, 300μg/mL-300mg/mL, 300μg/mL- 300mg/mL, 300μg/mL-100mg/mL, 300μg/mL-50mg/mL, 300μg/mL-10mg/mL, 300μg/mL-1mg/mL, 300μg/mL-500μg/mL, 500μg/mL-300mg/ mL, 500μg/mL-300mg/mL, 500μg/mL-100mg/mL, 500μg/mL-50mg/mL, 500μg/mL-10mg/mL, 500μg/mL-1mg/mL; 1mg/mL-300mg/mL, 1mg/mL-200mg/mL, 1mg/mL-100mg/mL, 1mg/mL-50mg/mL, 1mg/mL-10mg/mL; 10mg/mL-300mg/mL, 10mg/mL-200mg/mL, 10mg/ mL-100mg/mL, 10mg/mL-50mg/mL; 100mg/mL-300mg/mL, 100mg/mL-200mg/mL or 200mg/mL-300mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is 300 μg/mL.

In one embodiment, based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is greater than 0 and ≤8*10 ⁷ /mL. For example, 8*10 ⁷ / means that 1mL hydrogel preparation can load 8*10 ⁷ osteosarcoma cells.

It can be greater than 0 and ≤8*10 ⁷ /mL, greater than 0 and ≤6*10 ⁷ /mL, greater than 0 and ≤4*10 ⁷ /mL, greater than 0 and ≤2*10 ⁷ /mL, greater than 0 and ≤ 1*10 ⁷ /mL; 1*10 ⁷ /mL-8*10 ⁷ /mL, 1*10 ⁷ /mL-6*10 ⁷ /mL, 1*10 ⁷ /mL-4*10 ⁷ /mL, 1 *10 ⁷ /mL-2*10 ⁷ /mL; 2*10 ⁷ /mL-8*10 ⁷ /mL, 2*10 ⁷ /mL-6*10 ⁷ /mL, 2*10 ⁷ /mL-4* 10 ⁷ /mL; 4*10 ⁷ /mL-8*10 ⁷ /mL, 4*10 ⁷ /mL-6*10 ⁷ /mL or 6*10 ⁷ /mL-8*10 ⁷ /mL.

Preferably, based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is 1*10 ⁷ /mL.

Optionally, the decellularized matrix is obtained by the following method: decellularizing the biological tissue to obtain the decellularized matrix. cell matrix.

Optionally, the biological tissue is osteosarcoma tissue.

In one embodiment, the osteosarcoma tissue is of human origin.

Decellularization refers to the removal of cells from biological tissues and the remaining supporting tissue.

In one embodiment, the hydrogel formulation also includes gelatin.

Optionally, based on the total amount of the hydrogel preparation, the concentration of the gelatin is greater than 0 and ≤ 60 mg/mL. For example, 60 mg/mL means that the gelatin occupies a concentration of 60 mg/mL in the total amount of the hydrogel preparation. It can be greater than 0 and ≤60mg/mL, greater than 0 and ≤50mg/mL, greater than 0 and ≤40mg/mL, greater than 0 and ≤30mg/mL, greater than 0 and ≤20mg/mL, greater than 0 and ≤10mg/mL, 10mg/mL-60mg/mL, 10mg/mL-50mg/mL, 10mg/mL-40mg/mL, 10mg/mL-30mg/mL, 10mg/mL-20mg/mL, 20mg/mL-60mg/mL, 20mg/ mL-50mg/mL, 20mg/mL-40mg/mL, 20mg/mL-30mg/mL, 30mg/mL-60mg/mL, 30mg/mL-50mg/mL, 30mg/mL-40mg/mL, 40mg/mL- 60mg/mL, 40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.

Preferably, based on the total amount of the hydrogel preparation, the concentration of gelatin is 20 mg/mL.

In one embodiment, the osteosarcoma model organoid model further includes glycerol. Used to reduce cell damage caused by extrusion.

Optionally, based on the total amount of the hydrogel preparation, the volume fraction of the glycerin is greater than 0 and ≤30%. It can be greater than 0 and ≤30%, greater than 0 and ≤20%, greater than 0 and ≤10%, 10%-30%, 10%-20% or 20%-30%.

In one embodiment, the osteosarcoma organoid model is made by 3D printing.

As shown in Figures 1-1 and 1-2, one embodiment of the present invention provides a microfluidic chip, which contains:

Organoid culture area 1, the organoid culture area 1 contains the aforementioned osteosarcoma organoid model;

The liquid inlet 2 is connected to the organoid culture area 1 and is used to pump in fluid.

The liquid outlet 3 is connected to the organoid culture area 1 and is used to discharge fluid.

Microfluidic channel 4 is used to connect the organoid culture area 1, the liquid inlet 2 and the liquid outlet 3.

Fluid can be injected into the microfluidic chip. The fluid may be culture medium. The culture medium generally uses a-MEM culture medium (gibco).

The organoid culture medium contains several interconnected chambers, and the aforementioned osteosarcoma organ model can be injected into each chamber.

Optionally, the microfluidic chip includes a cover plate 5 and a bottom plate 6 that can fit together, and the organoid culture area 1 and the microfluidic channel 4 are carved on the bottom plate 6 . The liquid outlet 3 and/or the liquid inlet 2 can be engraved on the bottom plate or the cover plate. The cover plate and base plate can be connected by adhesive. For example, the connection is via an adhesive film 7 (DSF).

Preferably, the material of the cover plate and the bottom plate are different.

Optionally, the bottom plate can also be divided into an upper bottom plate and a lower bottom plate that can fit together. The organoid culture area 1 and the microfluidic channel 4 are carved on the upper bottom plate. The upper and lower base plates can be connected by an adhesive film 7 .

Optionally, the bottom plate may be made of PMMA (polymethylmethacrylate) and/or glass.

Optionally, the cover plate is made of PDMS (polydimethylsiloxane), which is beneficial to gas exchange.

The inner diameter of the microfluidic channel is 50 μm-1cm.

As shown in Figures 1-3, one embodiment of the present invention provides a method for preparing the aforementioned microfluidic chip, which at least includes the following steps:

The aforementioned osteosarcoma organoid model is placed in the organoid culture area; fluid is injected into the microfluidic channel and the organoid culture area through the liquid inlet to obtain the microfluidic chip.

Further, the method also includes the following steps: after placing the aforementioned osteosarcoma organoid model in the organoid culture area, the cover plate and the bottom plate are closed.

The aforementioned osteosarcoma organoid model can be injected into the organoid culture area in a non-gelled state.

One embodiment of the present invention provides a dynamic culture model of osteosarcoma model organoids. The dynamic culture model includes the aforementioned microfluidic chip and a microfluidic pump. The microfluidic pump is connected to the liquid inlet and outlet of the aforementioned microfluidic chip. The port is connected to provide power for fluid flow within the microfluidic chip.

The microfluidic pump is a microperistaltic pump.

Optionally, the osteosarcoma model organoid dynamic culture model also includes a liquid reservoir. The liquid reservoir is used to store the fluid delivered to the liquid inlet and the fluid discharged from the liquid outlet. The dynamic culture model of osteosarcoma model organoids forms a circular closed tube.

As shown in Figures 1 and 11, the present invention provides the application of the aforementioned osteosarcoma organoid model or microfluidic chip or osteosarcoma model organoid dynamic culture model in anti-osteosarcoma drug screening.

The anti-osteosarcoma drug screening according to the present invention can be a single drug screening or a pharmaceutical composition screening.

The drug screening may be a general drug screening for osteosarcoma or a drug screening for specific osteosarcoma tissues.

In one embodiment, as shown in Figure 12, first, the patient biopsy samples taken clinically are used to pathologically confirm the cells therein, and then the remaining tissue is used to construct the 3D bioprinting ink.

Secondly, a multi-organ microfluidic drug screening platform is constructed through 3D bioprinting technology to accurately simulate and observe tumor response to drugs in real time.

Example 1

a) Extraction and identification of BMSC-EV

New Zealand male young rabbits aged 1-2 weeks were selected and sterilized with 75% alcohol to remove the femur, tibia and humerus. After washing with PBS, use scissors to cut off both ends of the bone to expose the central medullary cavity, and rinse with Alpha-MEM medium containing 5000 U/mL heparin. Bone marrow cavity. Resuspend the cells in the collected culture medium, mix and centrifuge at 1800 rpm for 10 minutes. The cells were then resuspended in Alpha-MEM complete medium containing 20% FBS and 1% double antibody, and seeded in a 75cm ² cell culture flask. Incubate in an incubator at 37°C, 5% CO ₂ and 95% humidity until the cells are fused and then passaged. P2-P3 BMSCs were used to extract stem cell exosomes. When the cell confluence was about 70%, the cell culture medium was replaced with serum-free medium, and the cell culture supernatant was collected after continuing to culture for 72 hours. Centrifuge at 1,200 rpm to remove cellular impurities, and then ultracentrifuge at 100,000 g for 90 min at 4°C. After centrifugation, discard the supernatant and resuspend it in 0.5 mL DPBS to obtain BMSC-EV. Later, TEM was used to observe the morphology of exosomes, nanoparticle tracking analysis technology NTA was used to observe the particle size distribution of exosomes, Western Blot was used to verify the characteristics of exosomes Marker CD9, CD63, and Diol-labeled exosomes were used to observe the response of cells to exosomes. Uptake, and the impact of BMSC-EV on the activity, proliferation and migration behavior of osteosarcoma cell line MG63 was determined by detecting cell activity, cell proliferation and cell migration.

The results are shown in Figures 2 and 3, BMSC-EV promoted the proliferation and migration of MG63. BMSC-EV enhanced the bone-related gene expression of MG63 (Figure 3).

b) Extraction and identification of dOsEM acellular matrix

Fresh human osteosarcoma tissue (transported in culture medium) obtained clinically was placed in 0.25% trypsin and stirred at 250 rpm for 6 hours. After washing three times with water, 100 mL of 70% ethanol was stirred at 250 rpm for 10 hours and washed with 3% H ₂ O ₂ for 15 minutes. After washing with water three times, it was treated with 1% TritonX-100 and 0.26% EDTA/Tris for 6 hours. Thereafter, it was washed three times with water for 15 min, treated with 0.1% peracetic acid/4% ethanol for 2 h, washed several times with double-distilled water for 15 min and then freeze-dried. NanoDrop was then used to quantify the DNA content in the acellular matrix, and GAG and collagen Elisa quantitative kits were used to quantitatively detect the above components. At the same time, protein spectroscopy is used to conduct qualitative and quantitative analysis of the matrix components inside the matrix.

b-1) Protein profiling

Use biological mass spectrometry technology to analyze the bioactive proteins in dOsEM and combine it with bioinformatics molecular screening to determine that the main extracellular matrix proteins that can play a key regulatory role in dOsEM (such as Collagen I, Fibrinogen, Laminin, Collagen III, Collagen XV, Periostin, Collagen XI, ALB, etc.). The specific operation is that after the protein cleavage sample of the sample is initially separated by SDS-PAGE electrophoresis, the Kaodian destaining solution is destained and a sequencing-grade trypsin solution is added to react overnight at 37°C. After ultrasonic lyophilization, the sample is analyzed using capillary high-performance liquid chromatography combined with ESI mass spectrometry. Analysis: Maxquant software was used to analyze the data and combined with the STRING database to identify proteins and their mutual regulatory relationships.

The results are shown in Figure 3-1. The results show that the active ingredients are similar in composition and proportion to osteosarcoma (Os).

c) Preparation and characterization of dOsEM-Fibrin hydrogel for printing

The freeze-dried dOsEM decellularized matrix and pepsin were stirred and dissolved at a ratio of 10:1 for 48 hours, and the pH was adjusted to about 7.4 with NaOH to a final concentration of 6 mg/mL. The dOsEM-Fibrin bio-ink was formed according to the ingredients and proportions listed in Table 1, and was mixed with 20 IU/mL Thrombin solution and cross-linked for 30 minutes to achieve the structural formation of the hydrogel. and to it Rheological properties and mechanical properties were characterized. The characterization results of osteosarcoma acellular matrix dOsEM and dOsEM-Fibrin hydrogel are shown in Figure 5. It shows that the process does not destroy the Collagen content and removes DNA and GAG components.

Table 1. Ingredients for preparation of BMSC-EV-loaded dECM hydrogel

Table 2 BMSC-EV mass spectrometry analysis results of active ingredients in dOsEM-Fibrin hydrogel

The BMSC-EV mass spectrometry analysis results of the active ingredients in dOsEM-Fibrin hydrogel are shown in Table 2, showing that the active ingredients are mainly COL-XII, Vimentin, COL-I, FN1, COL-III, COL-V, etc.

Rheological testing: HR-2Discovery rheometer (TA Instruments, Newcastle, DE, USA) and 25mm parallel plate fixture were used. Strain was measured from 0.01 to 100 at an angular frequency of 6.28 rad/s. The rheometer was run using TA Instrument's programmable software and data were collected from triplicate samples with the solution temperature maintained at 20°C. The results are shown in Figure 6, indicating that the osteosarcoma acellular matrix dOsEM and dOsEM-Fibrin hydrogel have good performance.

d) Optimization of conditions for 3D bioprinting of dOsEM-Fibrin bioscaffold loaded with BMSC-EV

Using the Novaprint 3D printer, the dOsEM bioscaffold was constructed by extruding the hydrogel and integrating it in a layer-by-layer manner. Specific steps are as follows:

(a) Turn on the power supply, pneumatic valves, and condensing devices of the equipment, and calibrate the equipment.

(b) Before printing, use Magic software to construct a 6.5×6.5×1mm 3D printing bracket model "Box 6.5×6.5×1.st2", and use the layering software provided by the 3D printer to cut the above model to obtain the printing path file.

(c) Thereafter, the cells were divided into BMSC-EV-dOsEM-Fibrin group and dOsEM-Fibrin group according to Table 1 to obtain bioink for 3D printing. The bioink is loaded into the printing barrel, and three different caliber straight needles (100, 200, 250 μm) are used as the print head for printing. The printing unit is placed on the barrel sleeve of the 3D bioprinter.

(d) After installing the printing barrel and the printing needle into the bioprinting head of the bioprinter, set the printing speed to 3mm/s, the printing pressure to 1.7bar, and the printed pattern to be a cuboid (6.5×6.5×1mm). In addition, according to the relevant research results of oxygen diffusion (different printing spacing will change the oxygen and nutrient transport inside the stent, the results come from Lu, Zuyan, et al. "An oxygen-releasing device to improve the survival of mesenchymal stem cells inissue engineering ."Biofabrication 11.4(2019):045012.), set different printing spacings (0.6mm, 1.0mm and 2.0mm) respectively, and in order to eliminate the effect of temperature on the viscosity of the hydrogel and ensure the survival of cells inside the hydrogel, Set the temperature of the print extrusion head to 37°C.

(e) The osteosarcoma chip is composed of two different chip structures (PMMA and PDMS). The bottom layer is used for direct imaging of microtissues/cells, while the top PDMS layer facilitates gas exchange. To avoid particle contamination, sterilize under UV light for 30 minutes. Then, place the microfluidic chip with prefabricated holes and flow channels in the central area of the printer, and use air pressure to drive the extruded bio-ink to print onto the organoid culture area on the bottom plate of the microfluidic chip. After printing, the 3D printed scaffold is The sample was added to Throbmin (200IU/mL) solution and continued cross-linking at 37°C for 30 minutes.

(f) After printing is completed, place the cover on the bottom plate. The cover plate consists of a plasma-bonded adhesive and a PDMS cover. The port on the cover plate houses polytetrafluoroethylene (PTFE) tubing and connects the chip to the microperistaltic pump circuit. Then, connect the pipe from the liquid inlet to the liquid reservoir through the PVC pipe, and connect the liquid outlet to drain into the liquid reservoir, thus establishing a closed-loop system. To initiate media recirculation in the system, a PVC pipe is installed on the microperistaltic pump. Each medium storage tank was filled with 1.6 mL of medium at a very low flow rate of 4 μL/min to simulate the culture of organoids under the action of a circulatory system. The effectiveness of the culture system was verified through in vitro cell activity, cell migration, and cell apoptosis inside the chip.

Cell proliferation activity in vitro

The CCK8 kit and CaAM/PI fluorescence kit were used to detect the proliferation of cells inside the hydrogel. The specific operation is to use the CCK8 kit to dilute the culture medium at a ratio of 1:10, then aspirate the culture medium inside the well plate, add 300 μL of CCK8 dilute solution, incubate at room temperature for 2 hours, and record for 1 day and 3 days respectively. The absorbance at 450nm inside the well plate at 5 days and 7 days, 4 parallel samples in each group. CaAM/PI is to add CaAM working solution (1:1000) to the bioprinting scaffold containing cells, incubate at 37°C for 45 minutes, then add PI working solution (1:3000), incubate at 37°C for 15 minutes, and then wash with PBS, and Use laser confocal microscopy to observe the ratio of live and dead cells. Alginate hydrogel (Sigma-Aldrich, A0682) was used as a routine control group.

cell adhesion activity

The cytoskeleton was labeled using a phalloidin detection kit, and the adhesion spreading of the internal cells was observed by immunofluorescence microscopy. The specific operation is that after the scaffold is cultured for 48 hours, the culture medium is removed, washed three times with PBS, and then 2.5% paraformaldehyde is added and fixed at room temperature for 60 minutes. Then use PBS containing 0.1% TritonX-100 to wash 3 times, dilute the Actin-Tracker Red dye in PBS containing 1% BSA and 0.1% TritonX-100 at a ratio of 1:100, incubate at room temperature in the dark for 60 minutes, and then use The cells were washed three times with PBS containing 0.1% TritonX-100, and finally the nuclear stain DAPI was added and the cytoskeleton morphology was observed under a laser confocal microscope.

The results are shown in Figure 8, indicating that compared with the control alginate hydrogel, the 3D bioprinted dOsEM-Fibrin hydrogel scaffold can significantly enhance the in vitro proliferation and adhesion activity of osteosarcoma cells.

e) Use 3D osteosarcoma organoid chips to explore the therapeutic effect of the CXCR4-specific inhibitor Plerixafor combined with osteosarcoma chemotherapy drugs on osteosarcoma with high proliferative activity

First, by combining different concentrations of doxorubicin (0.01μM-100μM), cisplatin (0.03μM-300μM), methotrexate (0.03μM-10μM), ifosfamide (0.3mM-10mM), Plerixafor ( 1nM-100μM) was added to the culture medium in the microfluidic chip and circulated for 3 days. Afterwards, the cell viability was detected using the CCK8 method to obtain the IC50 concentration of the chemotherapy drug. And by combining different concentrations of doxorubicin, cisplatin, methotrexate, ifosfamide and other drugs with the specific inhibitor of CXCR4 Plerixafor, the culture medium mixed with different drugs was added to the microorganism in sequence. The cells were circulated and cultured in the fluidic chip for 3 days, and then the internal osteosarcoma cell activity was detected using the CaAM/PI method, and ImageJ was used for semi-quantitative analysis of the cell activity.

The results are shown in Figures 9 and 10. By constructing a drug screening platform using the osteosarcoma organoid chip of the present invention, it is clear that the combination of Plerixafor and Dox can significantly increase the killing effect of Dox on osteosarcoma cells with high proliferative activity. Compared with the conventional 2D single cell culture (2D Mono) group, the metabolic function of the 3D bioprinted dOsEM-Fibrin hydrogel scaffold of the present invention is improved.

The above examples are for illustrating the disclosed embodiments of the present invention and are not to be construed as limitations of the present invention. In addition, various modifications listed herein, as well as variations in methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (10)

An osteosarcoma organoid model, characterized in that the osteosarcoma model organoid model at least includes a biological scaffold and exosomes derived from osteosarcoma cells and bone marrow mesenchymal stem cells loaded on the biological scaffold; the biological scaffold It is a crisscross three-dimensional structure constructed from a hydrogel preparation that includes acellular matrix and fibrin. The osteosarcoma organoid model according to claim 1, further comprising one or more of the following features: a. The acellular matrix is the acellular matrix of osteosarcoma tissue; b. The osteosarcoma cells are living cells; c. The hydrogel preparation also includes gelatin; d. The hydrogel preparation also includes glycerin; e. Based on the total amount of the hydrogel preparation, the concentration of the acellular matrix is greater than 0 and ≤ 60 mg/mL; f. Based on the total amount of the hydrogel preparation, the concentration of fibrin is greater than 0 and ≤ 60 mg/mL; g. Based on the total amount of the hydrogel preparation, the concentration of exosomes derived from bone marrow mesenchymal stem cells is greater than 0 and ≤300 mg/mL; h. Based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is greater than 0 and ≤8*10 ⁷ /mL. The osteosarcoma organoid model of claim 2, wherein when feature c is included, the gelatin concentration is greater than 0 and ≤ 60 mg/mL based on the total amount of the hydrogel preparation; and/ or, When feature d is included, the volume fraction of glycerol is greater than 0 and ≤30% based on the total amount of the hydrogel preparation. A method for constructing an osteosarcoma organoid model includes at least the following steps: Exosomes derived from bone marrow mesenchymal stem cells and osteosarcoma cells are loaded on the biological scaffold to obtain an osteosarcoma organoid model; the biological scaffold is a crisscross three-dimensional structure constructed with a hydrogel preparation, and the water Gel formulations include acellular matrix and fibrin. The method for constructing an osteosarcoma organoid model according to claim 4, further comprising one or more of the following features: a. The acellular matrix is the acellular matrix of osteosarcoma tissue; b. The osteosarcoma cells are living cells; c. The hydrogel preparation also includes gelatin; d. The hydrogel preparation also includes glycerin; e. Based on the total amount of the hydrogel preparation, the concentration of the acellular matrix is greater than 0 and ≤ 60 mg/mL; f. Based on the total amount of the hydrogel preparation, the concentration of fibrin is greater than 0 and ≤ 60 mg/mL; g. Based on the total amount of the hydrogel preparation, the concentration of exosomes derived from bone marrow mesenchymal stem cells is greater than 0 And ≤300mg/mL; h. Based on the total amount of the hydrogel preparation, the content of the osteosarcoma cells is greater than 0 and ≤8*10 ⁷ /mL. The method for preparing an osteosarcoma organoid model according to claim 5, wherein when feature c is included, the concentration of the gelatin is greater than 0 and ≤ 60 mg/kg based on the total amount of the hydrogel preparation. mL; and/or, When feature d is included, the volume fraction of glycerin is greater than 0 and ≤30% based on the total amount of the hydrogel preparation. A microfluidic chip, which contains: Organoid culture area (1), the organoid culture area contains the osteosarcoma organoid model according to any one of claims 1-3; The liquid inlet (2) is connected to the organoid culture area (1) and is used to pump fluid; The liquid outlet (3) is connected with the organoid culture area (1) and is used to discharge fluid; The microfluidic channel (4) is used to connect the organoid culture area (1), the liquid inlet (2) and the liquid outlet (3). A method for preparing a microfluidic chip, at least including the following steps: placing the osteosarcoma organoids described in any one of claims 1-3 in the organoid culture area; injecting fluid through the liquid inlet into microfluidic channels and organoids. Organ culture area to obtain the microfluidic chip. An osteosarcoma model organoid dynamic culture model, the dynamic culture model includes the microfluidic chip and a microfluidic pump according to claim 7, the microfluidic pump is connected to the liquid inlet and the liquid inlet in the microfluidic chip. The liquid outlet is connected and used to provide power for fluid flow in the microfluidic chip. The osteosarcoma organoid model according to any one of claims 1 to 3 or the microfluidic chip according to claim 7 or the osteosarcoma model organoid dynamic culture model according to claim 9 in anti-osteosarcoma drug screening application.