CN115651909B - Osteosarcoma organoid model, construction method and application - Google Patents
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Abstract
The invention provides a osteosarcoma organoid model, which at least comprises a biological bracket, and osteosarcoma cells and exosomes derived from bone marrow mesenchymal stem cells loaded on the biological bracket; the biological scaffold is a crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin. According to the invention, a dOsEM-fiber hydrogel loading system is combined with BMSC-EV for the first time, a component-structure multi-level bionic optimized osteosarcoma organoid chip is integrally constructed on a microfluidic chip by using a 3D biological printing technology, an organoid dynamic culture model capable of simulating an in-vivo osteosarcoma matrix microenvironment is constructed, and an ideal model foundation is laid for in-vitro 3D culture of subsequent physiological and pathological bone tissues.
Description
Technical Field
The invention relates to the field of medicines, in particular to an osteosarcoma organoid model, a construction method and application thereof.
Background
Currently, biomedical research related to tumors relies mainly on in vitro cultured human cells or rodent models. Although studies using cultured cells in vitro can directly reflect the effects of drugs, the responses in physiological function are still significantly different from the actual physiological function of the human body. In contrast, animal models can mimic physiological functions at an organ or organs level, but are limited in that inherent differences in physiological functions between animals and humans can expose part of the tumor-effective drug candidates to the risk of failure in human trials. Biomimetic systems, in particular organ-chips, are important models for in vitro studies of tissue physiology and pathological functions that have appeared in recent years. The micro engineering bionic organism tissue system built on the micro fluid chip can combine the advantages of the human cell in-vitro simulation model and the rodent model, and culture human tissues by simulating the action of the human blood circulation system to construct a tissue structure similar to the human tissues. By developing and utilizing the microcirculation systems, in-vitro tissue physiological and pathological disease models can be established to understand biological mechanisms and screen potential drugs. The above studies indicate that these microfluidic systems have great potential in biomedical, pharmaceutical and toxicological applications in the medical field.
Although organ chips have great application prospects in clinical applications. The existing 3D osteosarcoma organ chip still cannot regulate and control the spatial structure, cell distribution and the like of osteosarcoma matrix microscopically due to the influence of the processing technology, and also cannot accurately regulate and control the exchange of oxygen and nutrient substances in the matrix, so that the characteristics of high proliferation activity, high cell density and strong matrix-cell interaction of osteosarcoma tissues cannot be simulated accurately at a microscopic level, and a certain gap remains between the chip and the active osteosarcoma tissues in vivo. Moreover, at present, a single organ chip only singly researches the influence of mechanical stimulation of a circulatory system on physiological and pathological behaviors, and the influence of biological functions and metabolic behaviors of other solid organs on tumorigenesis and development and treatment is greatly ignored, because the biological processes in the body cannot be well simulated.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an osteosarcoma organoid model, a construction method and an application.
The invention constructs a component-structure multi-stage bionic organoid dynamic culture model capable of simulating in-vivo osteosarcoma matrix microenvironment by combining the 3D biological printing technology and the organoid chip model and (1) 3D printing on a microfluidic chip to load osteosarcoma cells and BMSC-EV of a patient by using a dOsEM-fiber hydrogel bracket. And the drug combination scheme is screened by a 3D multi-level bionic organoid chip to be used for the treatment potential of the high proliferation activity osteosarcoma.
According to the invention, a multi-stage bionic osteosarcoma organoid dynamic culture model capable of simulating high proliferation and transfer activity of in-vivo osteosarcoma is constructed by 3D printing of a hydrogel bracket of bone tissue acellular matrix (dOsEM-fibromin) on a microfluidic chip and combining osteosarcoma cells and BMSC-EV. And the treatment effect of various inhibitors combined with osteosarcoma first-line chemotherapeutics on the osteosarcoma with high proliferation activity is explored, so that the application value of the model in personalized diagnosis and treatment of osteosarcoma is clear.
The invention is helpful for in-depth understanding of physical and chemical components and physical structures in physiological and pathological microenvironments on the specific actions and mechanisms of the osteosarcoma cell proliferation and adhesion behaviors, and defines the significance of the multistage bionic tumor organ chip detection platform constructed based on 3D biological printing for in-vitro drug screening of osteosarcoma, and provides a pre-clinical drug evaluation platform which is more similar to a human body for the current brand-new biological treatment, immunotherapy and targeted treatment.
The first aspect of the invention provides an osteosarcoma organoid model, which at least comprises 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 crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin.
The second aspect of the invention provides a method for constructing an osteosarcoma organoid model, which at least comprises the following steps:
loading exosomes derived from mesenchymal stem cells and osteosarcoma cells on the biological scaffold to obtain an osteosarcoma organoid model; the biological scaffold is a crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin.
A third aspect of the present invention provides a microfluidic chip, comprising:
an organoid culture zone containing the osteosarcoma organoid model described above;
a liquid inlet communicated with the organoid culture area and used for pumping fluid;
a liquid outlet communicated with the organoid culture zone for discharging fluid;
and the micro-flow channel is used for communicating the organoid culture area, the liquid inlet and the liquid outlet.
The fourth aspect of the present invention provides a method for manufacturing a microfluidic chip, at least comprising the steps of: placing the osteosarcoma organoid in an organoid culture zone; and injecting fluid into the micro-flow channel and the organoid culture area through the liquid inlet to obtain the micro-flow control chip.
The fifth aspect of the invention provides a dynamic culture model of osteosarcoma model organoid, which comprises the microfluidic chip and a microfluidic pump, wherein the microfluidic pump is communicated with a liquid inlet and a liquid outlet in the microfluidic chip and is used for providing power for fluid flow in the microfluidic chip.
The sixth aspect of the invention provides the use of the osteosarcoma organoid model described above or the microfluidic chip described above or the osteosarcoma organoid dynamic culture model described above in the screening of anti-osteosarcoma drugs.
As described above, the osteosarcoma organoid model, the construction method and the application of the osteosarcoma organoid model have the following beneficial effects:
(1) The invention combines the advantages of the 3D biological printing technology and the organoid chip screening platform which are the forefront in the world at present, further deepens and innovates the technology, combines a dOsEM-fiber hydrogel loading system with BMSC-EV for the first time, integrally builds the osteosarcoma organoid chip which is 'component-structure' multi-level bionic optimization on a microfluidic chip by using the 3D biological printing technology, builds an organoid dynamic culture model which can simulate the in-vivo osteosarcoma matrix microenvironment, and lays an ideal model foundation for the in-vitro 3D culture of the subsequent physiological and pathological bone tissues.
(2) The invention takes bone matrix mesenchymal stem cell exosomes (BMSC-EV) as a research object, and creatively explores a specific action mechanism of BMSC-EV activated CXCL12/CXCR4 signal axes to regulate and control proliferation and invasion of osteosarcoma cells under the bone sarcoma matrix microenvironment. The 3D osteosarcoma organoid chip is systematically clarified to better simulate the CXCL12/CXCR4 regulation and control effect under in-vivo osteosarcoma matrix-cell interaction compared with a 2D culture model, and the potential application value of the model is revealed. In addition, the project innovatively and deeply reveals that BMSC influences the osteosarcoma matrix microenvironment through secretion of exosomes and matrix components together, and regulates proliferation and metastasis behaviors of osteosarcoma cells.
(3) The invention creatively researches the curative effect of the CXCR4 specific inhibitor Plerixafor combined osteosarcoma first-line chemotherapy drugs on the osteosarcoma with high proliferation activity through the multi-stage bionic organoid chip, further verifies the advantage of the 3D printing multi-stage bionic osteosarcoma organoid chip in simulating the in-vivo drug effect, and establishes an effective in-vitro evaluation platform for screening and optimizing the personalized multi-drug treatment scheme of osteosarcoma.
The organ chip has strong clinical application value for personalized medicine evaluation, and can accurately and rapidly observe the toxicity of the medicine to target organs and other multiple organs of a patient in real time. The invention provides a brand-new diagnosis and treatment strategy and treatment evaluation means for the diagnosis and treatment of osteosarcoma diseases by using brand-new technical means. The invention provides a powerful tool for clinical research and treatment of bone tumor, fills the blank of in vitro model of bone and meat tumor, and has good economic benefit. On the other hand, a new research idea can be provided for searching relevant therapeutic targets of osteosarcoma, thereby having important value.
Drawings
Fig. 1: the flow chart of the invention is shown in the schematic diagram.
Fig. 1-1: an exploded view of a microfluidic chip according to an embodiment of the present invention.
Fig. 1-2: structure of microfluidic chip according to an embodiment of the present invention.
Fig. 1-3: the preparation flow of the microfluidic chip of the embodiment of the invention.
Fig. 2: characterization of BMSC-EV. (A) cell morphology map of BMSC. (B) flow cell analysis of surface markers of BMSCs. (C) TEM micro morphology map of BMSC-EV. (D) particle size distribution plot of BMSC-EV. (E) cell uptake map of BMSC-EV. (F) Results of BMSC-EV specific marker West Blot analysis.
Fig. 3: BMSC-EV promotes proliferation and migration of MG 63. (A) MTT assay to detect proliferation of BMSC-EV at various concentrations. (B, C) scratch test detects migration of BMSC-EV at different concentrations. (D) BMSC-EV enhanced bone-related gene expression of MG 63.
Fig. 3-1: the active ingredients of the dOsEM were analyzed using protein biological mass spectrometry.
Fig. 4: preparation of an osteosarcoma acellular matrix dOsEM and dOsEM-Fibrin hydrogel.
Fig. 5: characterization of osteosarcoma decellularized matrix, dOsEM, and dOsEM-Fibrin hydrogel. And (A) identifying the effective components in the dOsEM by protein mass spectrometry. And (B) identifying the microcosmic morphology in the dOsEM by TEM. (C) Quantitative analysis of DNA, collagen and GAG components in the dsem indicated the effect of the osteosarcoma on the respective components before and after decellularized matrix treatment.
Fig. 6: rheological Properties of the dOsEM-Fibrin hydrogels. (A) The dependence of storage modulus (G ') and loss modulus (G') on shear strain for various concentrations of the dOsEM-fiber hydrogel material. (B) Viscosity dependence of different concentrations of the dOsEM-fiber hydrogel material on shear strain.
Fig. 7: (A) Schematic of the manufacturing process of the 3D printed on-chip osteosarcoma model. (B) Optical micrographs and macroscopic views of osteosarcoma on-chip models were 3D printed. (C) A 3D printed osteosarcoma chip model for drug screening applications.
Fig. 8: (A) Compared with Alginate hydrogel, the 3D bioprinting of the organoid chip constructed by the dOsEM-fiber hydrogel can remarkably improve the in-vitro cell adhesion activity (A upper: phalloidin immunofluorescence staining) and proliferation activity (A lower: living and dead cell immunofluorescence staining) of osteosarcoma cells. (B) cck8 assay for bone tissue cell proliferation. The dOsEM-Fibrin hydrogel can remarkably promote proliferation of cells related to physiological bone tissues. (C) cck8 assay for osteosarcoma cell proliferation. The dOsEM-Fibrin hydrogel can obviously promote proliferation of pathological osteosarcoma cells.
Fig. 9: (A) Constructing a drug screening platform by using a 3D printed osteosarcoma organoid chip; b is the effect of different drug treatments on the activity of cells inside osteosarcoma tissue (B is the result of live-dead fluorescent staining); c is a quantitative statistical result of the cell activity in B. (B, C) it was determined that the combination of Plerixafor with Dox significantly increased the killing effect of Dox on high proliferative osteosarcoma cells.
Fig. 10: metabolic function of 3D bioprinted dsem-Fibrin hydrogel scaffolds. For comparison of the 3D hydrogel chip (3D chip) group with the conventional 2D single cell culture (2D Mono) group, half-mortem (IC 50) results of cells for different drugs were obtained by detecting cell activity using MTT.
Fig. 11: the technical roadmap of the invention.
Fig. 12: the application mode of the invention is a flow chart.
1-organoid culture zone
2-liquid inlet
3-liquid outlet
4-micro flow channel
5-cover plate
6-baseboard
7-adhesive film
Detailed Description
Construction of micro tumor tissue by 3D biological printing technology
First, 3D printing technology is an effective way to construct tissue biological scaffolds. Compared with other 3D culture models, the three-dimensional culture model can accurately arrange cells and extracellular matrixes, control local tissue microenvironment, further construct in-vivo tissue-like forms and mechanical environments, and simulate in-vivo cell-cell interaction and cell-extracellular matrix interaction. In addition, the porosity inside the stent can be precisely controlled by blending materials and cells in advance and regularly arranging the materials and the cells after extrusion, so that oxygen and nutrient substance transmission and metabolic waste exchange are facilitated, and the survival of cells inside the stent is further maintained. In recent years, the construction of a 3D biological printing active scaffold with biological hydrogel loaded stem cells as printing ink has high potential in the tissue regeneration and repair directions. It mainly comprises three organic components: functional cells, biological scaffold material, and loaded bioactive components. The choice of the biological scaffold material and the supported bioactive components is the core of the technology. The loaded bioactive component. In addition, the micro tissue construction can be better realized by means of a high-precision processing technology special for a 3D biological printing technology, so that microscopic components and structures of the tissue can be better simulated, and the defects of the traditional tissue chip in the field are relieved.
In addition, 3D bioprinting technology also exhibits its unique advantages in constructing solid tumor models. Traditional 2D approaches are severely limited in the construction of tumor tissue due to the lack of natural microenvironment features of tumor tissue in vivo in two-dimensional monolayer cell models. On the other hand, rodent tumor-bearing models established in immunocompromised mice also do not mimic well the development and progression of tumors in humans. To overcome the above obstacles, more and more researches begin to use in vitro 3D tumor models based on human cancer cells to accurately simulate human cancer tissue characteristics, thereby researching the influence of the model on tumor cell morphology, proliferation, drug metabolism, gene expression and protein synthesis. Related technologies such as multicellular spheroid technology, 3D scaffold seeding technology, hydrogel embedding technology, microfluidic chips, cell patterns, etc. have also been developed for the construction of 3D in vitro tumor models. Although these studies reveal great potential for application of in vitro 3D tumor models, it is still difficult to simulate complex 3D tumor microenvironments in most of the above models due to manufacturing technology limitations. Whereas 3D biotechnology offers more possibilities for research in studying disease pathogenesis and discovery of new drugs by constructing tumor cells, interactions of extracellular matrix (ECM) materials. Compared with the traditional 2D plane culture model, the 3D printing bracket has higher tumor proliferation activity and colony forming capacity. The 3D biological printing technology can better simulate the interaction of tumor cells and extracellular matrix (ECM), accurately control the transmission of nutrient substances in the stent, and more accurately simulate the pathological microenvironment of the tumor tissue with high proliferation activity in vivo.
3D biological printing technology to construct a component-structure multilevel bionic organoid dynamic culture model capable of simulating in-vivo osteosarcoma matrix microenvironment
However, some of the chemical treatments involved in preparing the dOsEM decellularized matrix may destroy bioactive proteins in the matrix microenvironment, especially exosomes (BMSC-EV) represented by bone marrow mesenchymal stem cells (BMSCs), signaling molecules, etc., and thus organoid chips constructed based on the dOsEM-fiber hydrogels do not mimic the interaction of BMSCs with osteosarcoma cells in the matrix well. BMSCs, which are important components in bone microenvironments, are cells in bone marrow with multidirectional differentiation potential and immune regulation function, have been studied to be involved in proliferation and migration of tumor tissues, formation of tumor microenvironments, interaction with tumor cells, and the like, and have also been studied to confirm that BMSCs can promote tumor cell growth, metastasis, and immune escape by secreting a series of cytokines such as IL-6, VEGF, TNF-a, and the like. Wherein exosomes secreted by stem cells (BMSC-EV) are vesicles with diameters of 40-100nm secreted by BMSCs in the extracellular environment as the main load medium for secretion factors. Which contains proteins, lipids, nucleic acids, etc. In recent years, a great deal of research has demonstrated that BMSC-EV can affect the development and progression of tumors, and that mesenchymal stem cells can also regulate the formation of blood vessels in tumors and further promote proliferation of cells inside tumor tissues by regulating angiogenesis, stabilization and maturation. However, the specific mode of action and mechanism of BMSC-EV in bone marrow microenvironment to promote osteosarcoma cell growth and metastasis is currently unknown. To better mimic the interaction of BMSC with osteosarcoma cells in the matrix, the present invention complements the dsem-Fibrin hydrogel inside the organoid chip with BMSC-EV components to observe cell proliferation capacity and cell-matrix interactions.
Therefore, the invention can construct the component-structure multilevel bionic osteosarcoma model with high proliferation activity and strong matrix-cell interaction in vivo by 3D printing of the hydrogel bracket (structure bionic) composite osteosarcoma cells and BMSC-EV (component bionic) based on the dOsEM-Fibrin on the microfluidic chip. It more accurately mimics the expression of osteosarcoma tissue associated signaling pathways in vivo, and effects on cell phenotypes such as downstream proliferation, adhesion, migration, etc., compared to 2D culture models (fig. 6).
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention is capable of other and different embodiments and its several details are capable of modification and/or various other uses and applications in various respects, all without departing from the spirit of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention as would be apparent to one of skill in the art having possession of the prior art and having possession of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
The methods or application scenarios described herein are for non-diagnostic and therapeutic uses, such as may be used in basic research.
An embodiment of the present invention provides an osteosarcoma model organoid model, which at least includes a biological scaffold and osteosarcoma cells and bone marrow mesenchymal stem cell-derived exosomes (BMSC-EV) loaded on the biological scaffold; the biological scaffold is a crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin.
Preferably, the acellular matrix is of osteosarcoma tissue.
In one embodiment, the osteosarcoma tissue is of human origin.
In one embodiment, the Fibrin (Fibrin) may be derived from bovine, human, rabbit, rat, etc. plasma or bacterial extracts.
In one embodiment, fibrinogen and thrombin may be used to react to form fibrin. Thrombin can be derived from bovine, human, rabbit, rat, etc. plasma or bacterial extract. For example, fibrinogen Fibrinogen from bovine plasma (F8630-10G, sigma) and Thrombin Thrombin (T4648-1 KU, sigma) may be used to react to form fibrin.
The osteosarcoma cell is a living cell.
In one embodiment, the osteosarcoma cells are derived from a human. For example, it may be MG63 or osteosarcoma cells of a osteosarcoma patient.
In one embodiment, the concentration of decellularized matrix is greater than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel formulation. For example, 60mg/mL indicates that the concentration of the decellularized matrix dOsEM occupying the total amount of the hydrogel formulation is 60mg/mL. Can be greater than 0 and less than or equal to 60mg/mL, greater than 0 and less than or equal to 50mg/mL, greater than 0 and less than or equal to 40mg/mL, greater than 0 and less than or equal to 30mg/mL, greater than 0 and less than or equal to 20mg/mL, greater than 0 and less than or equal to 10mg/mL,10mg/mL-60mg/mL,10 mg/mL-50mg/mL, 10mg/mL-40mg/mL,10mg/mL-30mg/mL,10mg/mL-20mg/mL,20 mg/mL-60mg/mL, 20mg/mL-50mg/mL,20 mg/mL-30mg/mL,30 mg/mL-60mg/mL, 30mg/mL-50mg/mL,30mg/mL-40mg/mL,40mg/mL-60mg/mL,40 mg/mL-50mg/mL, or 50mg/mL-60mg/mL.
Preferably, the concentration of the acellular matrix is 6mg/mL based on the total amount of the hydrogel formulation.
In one embodiment, the concentration of fibrin is greater than 0 and less than or equal to 60mg/mL based on the total amount of hydrogel formulation. 60mg/mL indicates that the concentration of fibrin in the total hydrogel formulation is 60mg/mL. Can be more than 0 and less than or equal to 60mg/mL, more than 0 and less than or equal to 50mg/mL, more than 0 and less than or equal to 40mg/mL, more than 0 and less than or equal to 30mg/mL, more than 0 and less than or equal to 20mg/mL, more than 0 and less than or equal to 10mg/mL;10mg/mL-60mg/mL,10mg/mL-50mg/mL,10 mg/mL-40mg/mL, 10mg/mL-30mg/mL,10mg/mL-20mg/mL;20mg/mL-60mg/mL,20 mg/mL-50mg/mL, 20mg/mL-40mg/mL,20mg/mL-30mg/mL;30mg/mL-60mg/mL,30 mg/mL-50mg/mL, 30mg/mL-40mg/mL;40mg/mL-60mg/mL,40mg/mL-50mg/mL or 50 mg/mL-60 mg/mL.
Preferably, the concentration of fibrin is 20mg/mL based on the total amount of hydrogel formulation.
In one embodiment, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is greater than 0 and less than or equal to 300mg/mL based on the total amount of the hydrogel formulation. Exosome concentration was calculated by BCA protein assay kit (23227, theromofeis) (this method is a gold standard for exosome concentration assay in the industry). For example, 300mg/mL indicates that 1mL of the hydrogel formulation can be loaded with 300mg of bone marrow mesenchymal stem cell-derived exosomes. Can be more than 0 and less than or equal to 300mg/mL, more than 0 and less than or equal to 200mg/mL, more than 0 and less than or equal to 100mg/mL, more than 0 and less than or equal to 50mg/mL, more than 0 and less than or equal to 10mg/mL, more than 0 and less than or equal to 1mg/mL, more than 0 and less than or equal to 500 mug/mL, more than 0 and less than or equal to 300 mug/mL, more than 0 and less than or equal to 100 mug/mL; 100 μg/mL-300mg/mL,100 μg/mL-200mg/mL,100 μg/mL-100mg/mL,100 μg/mL-50mg/mL, 100 μg/mL-10mg/mL,100 μg/mL-1mg/mL,100 μg/mL-500 μg/mL,100 μg/mL-300 μg/mL,300 μg/mL-300mg/mL,300 μg/mL-100mg/mL,300 μg/mL-50mg/mL,300 μg/mL-10mg/mL,300 μg/mL-1mg/mL,300 μg/mL-500 μg/mL,500 μg/mL-300mg/mL,500 μg/mL-100mg/mL,500 μg/mL-50mg/mL, 500 μg/mL-10mg/mL,500 μg/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,10 mg/mL-200mg/mL, 10mg/mL-100mg/mL,10mg/mL-50mg/mL;100mg/mL-300mg/mL,100 mg/mL-200mg/mL or 200mg/mL-300mg/mL.
Preferably, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is 300 μg/mL based on the total amount of the hydrogel formulation.
In one embodiment, the osteosarcoma cells are present in an amount of greater than 0 and less than or equal to 8 x 10 based on the total amount of the hydrogel formulation 7 /mL. For example 8 x 10 7 1mL hydrogel formulation loadable 8 x 10 7 And osteosarcoma cells.
Can be more than 0 and less than or equal to 8 x 10 7 Per mL, greater than 0 and less than or equal to 6 x 10 7 Per mL, greater than 0 and less than or equal to 4 x 10 7 Per mL, greater than 0 and less than or equal to 2 x 10 7 Per mL, greater than 0 and less than or equal to 1 x 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 Per mL or 6 x 10 7 /mL-8*10 7 /mL。
Preferably, the content of the osteosarcoma cells is 1×10 based on the total amount of the hydrogel preparation 7 /mL。
Alternatively, the bone marrow mesenchymal stem cell-derived exosomes may be derived from an animal. Further, it is mammalian. Alternatively, rodents, primates, artiodactyls, humans, and the like may be used. Such as mice, rabbits, monkeys, chimpanzees, apes, cattle, pigs, humans, etc.
In one embodiment, gelatin is also included in the hydrogel formulation.
Alternatively, the gelatin concentration is greater than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel formulation. For example, 60mg/mL indicates that the gelatin occupies a concentration of 60mg/mL of the total hydrogel formulation. Can be greater than 0 and less than or equal to 60mg/mL, greater than 0 and less than or equal to 50mg/mL, greater than 0 and less than or equal to 40mg/mL, greater than 0 and less than or equal to 30mg/mL, greater than 0 and less than or equal to 20mg/mL, greater than 0 and less than or equal to 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,20 mg/mL-30mg/mL,30mg/mL-60mg/mL,30mg/mL-50mg/mL,30 mg/mL-40mg/mL, 40mg/mL-60mg/mL,40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.
Preferably, the gelatin concentration is 20mg/mL based on the total hydrogel formulation.
In one embodiment, glycerol is also included in the osteosarcoma model organoid model. For reducing cell damage caused by extrusion.
Alternatively, the volume fraction of glycerol is greater than 0 and less than or equal to 30% based on the total amount of the hydrogel formulation. May be greater than 0 and less than or equal to 30%, greater than 0 and less than or equal to 20%, greater than 0 and less than or equal to 10%,10% -30%,10% -20% or 20% -30%.
The invention constructs the deosEM-Fibrin hydrogel by constructing the decellularized matrix (Decellularized Osteosarcoma Extracellular Matrix, dOsEM) of the osteosarcoma tissue (figure 4) and combining with a Fibrin material, and then compares the cell proliferation activity of 5 hydrogels Alginate, gelMA, HGP, collagen and dOsEM-Fibrin by a CCK8 method, so that the prepared dOsEM-Fibrin hydrogel can better promote the proliferation of the osteosarcoma cell line (MG 63 cells) in vitro. And compared with the traditional 3D culture Alginate hydrogel, the dOsEM-Fibrin hydrogel can remarkably improve the adhesion of osteosarcoma cells, and the proliferation activity RNA-seq analysis result shows that compared with the 2D conventional culture, the dOsEM-Fibrin hydrogel can remarkably promote the proliferation, adhesion and matrix-receptor interaction of osteosarcoma cells. The above results initially demonstrate that the dOsEM-Fibrin hydrogel can more truly mimic osteosarcoma cell-matrix interactions than 2D culture models and other 3D culture hydrogel models, and more significantly enhance osteosarcoma cell activity and proliferation capacity.
The present invention has found that some of the chemical treatments involved in preparing the dOsEM acellular matrix may destroy bioactive proteins in the matrix microenvironment, especially exosomes (BMSC-EV) represented by bone marrow mesenchymal stem cells (BMSCs), signal molecules, etc., and thus the organoid chip constructed based on the dOsEM-Fibrin hydrogel does not mimic the interaction of BMSCs with osteosarcoma cells in the matrix well. BMSCs, which are important components in bone microenvironments, are cells in bone marrow with multidirectional differentiation potential and immune regulation function, have been studied to be involved in proliferation and migration of tumor tissues, formation of tumor microenvironments, interaction with tumor cells, and the like, and have also been studied to confirm that BMSCs can promote tumor cell growth, metastasis, and immune escape by secreting a series of cytokines such as IL-6, VEGF, TNF-a, and the like. Wherein exosomes secreted by stem cells (BMSC-EV) are vesicles with diameters of 40-100nm secreted by BMSCs in the extracellular environment as the main load medium for secretion factors. Which 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 within tumors and further promote proliferation of cells within tumor tissues by regulating angiogenesis, stabilization and maturation. The specific mode of action and mechanism of BMSC-EV in bone marrow microenvironment to promote osteosarcoma cell growth and metastasis are not known. To better mimic the interaction of BMSC with osteosarcoma cells in the matrix, the present invention complements BMSC-EV components in the dsem-Fibrin hydrogels inside the organoid chip to observe cell proliferation capacity and cell-matrix interactions.
An embodiment of the present invention provides a method for constructing osteosarcoma organoid model, at least comprising the steps of:
loading exosomes derived from mesenchymal stem cells and osteosarcoma cells on the biological scaffold to obtain an osteosarcoma organoid model; the biological scaffold is a crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin.
Alternatively, the solvent used for the hydrogel formulation may be a culture medium, water or PBS buffer.
In one embodiment, the concentration of decellularized matrix is greater than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel formulation. For example, 60mg/mL indicates that the concentration of the decellularized matrix dOsEM occupying the total amount of the hydrogel formulation is 60mg/mL. Can be greater than 0 and less than or equal to 60mg/mL, greater than 0 and less than or equal to 50mg/mL, greater than 0 and less than or equal to 40mg/mL, greater than 0 and less than or equal to 30mg/mL, greater than 0 and less than or equal to 20mg/mL, greater than 0 and less than or equal to 10mg/mL,10mg/mL-60mg/mL,10 mg/mL-50mg/mL, 10mg/mL-40mg/mL,10mg/mL-30mg/mL,10mg/mL-20mg/mL,20 mg/mL-60mg/mL, 20mg/mL-50mg/mL,20 mg/mL-30mg/mL,30 mg/mL-60mg/mL, 30mg/mL-50mg/mL,30mg/mL-40mg/mL,40mg/mL-60mg/mL,40 mg/mL-50mg/mL, or 50mg/mL-60mg/mL.
Preferably, the concentration of the acellular matrix is 6mg/mL based on the total amount of the hydrogel formulation.
In one embodiment, the concentration of fibrin is greater than 0 and less than or equal to 60mg/mL based on the total amount of hydrogel formulation. 60mg/mL indicates that the concentration of fibrin in the total hydrogel formulation is 60mg/mL. Can be more than 0 and less than or equal to 60mg/mL, more than 0 and less than or equal to 50mg/mL, more than 0 and less than or equal to 40mg/mL, more than 0 and less than or equal to 30mg/mL, more than 0 and less than or equal to 20mg/mL, more than 0 and less than or equal to 10mg/mL;10mg/mL-60mg/mL,10mg/mL-50mg/mL,10 mg/mL-40mg/mL, 10mg/mL-30mg/mL,10mg/mL-20mg/mL;20mg/mL-60mg/mL,20 mg/mL-50mg/mL, 20mg/mL-40mg/mL,20mg/mL-30mg/mL;30mg/mL-60mg/mL,30 mg/mL-50mg/mL, 30mg/mL-40mg/mL;40mg/mL-60mg/mL,40mg/mL-50mg/mL or 50 mg/mL-60 mg/mL.
Preferably, the concentration of fibrin is 20mg/mL based on the total amount of hydrogel formulation.
In one embodiment, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is greater than 0 and less than or equal to 300mg/mL based on the total amount of the hydrogel formulation. Exosome concentration was calculated by BCA protein assay kit (23227, theromofeis) (this method is a gold standard for exosome concentration assay in the industry). For example, 300mg/mL indicates that 1mL of the hydrogel formulation can be loaded with 300mg of bone marrow mesenchymal stem cell-derived exosomes. Can be more than 0 and less than or equal to 300mg/mL, more than 0 and less than or equal to 200mg/mL, more than 0 and less than or equal to 100mg/mL, more than 0 and less than or equal to 50mg/mL, more than 0 and less than or equal to 10mg/mL, more than 0 and less than or equal to 1mg/mL, more than 0 and less than or equal to 500 mug/mL, more than 0 and less than or equal to 300 mug/mL, more than 0 and less than or equal to 100 mug/mL; 100 μg/mL-300mg/mL,100 μg/mL-200mg/mL,100 μg/mL-100mg/mL,100 μg/mL-50mg/mL, 100 μg/mL-10mg/mL,100 μg/mL-1mg/mL,100 μg/mL-500 μg/mL,100 μg/mL-300 μg/mL,300 μg/mL-300mg/mL,300 μg/mL-100mg/mL,300 μg/mL-50mg/mL,300 μg/mL-10mg/mL,300 μg/mL-1mg/mL,300 μg/mL-500 μg/mL,500 μg/mL-300mg/mL,500 μg/mL-100mg/mL,500 μg/mL-50mg/mL, 500 μg/mL-10mg/mL,500 μg/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,10 mg/mL-200mg/mL, 10mg/mL-100mg/mL,10mg/mL-50mg/mL;100mg/mL-300mg/mL,100 mg/mL-200mg/mL or 200mg/mL-300mg/mL.
Preferably, the concentration of the bone marrow mesenchymal stem cell-derived exosomes is 300 μg/mL based on the total amount of the hydrogel formulation.
In one embodiment, the osteosarcoma cells are present in an amount of greater than 0 and less than or equal to 8 x 10 based on the total amount of the hydrogel formulation 7 /mL. For example 8 x 10 7 1mL hydrogel formulation loadable 8 x 10 7 And osteosarcoma cells.
Can be more than 0 and less than or equal to 8 x 10 7 Per mL, greater than 0 and less than or equal to 6 x 10 7 Per mL, greater than 0 and less than or equal to 4 x 10 7 Per mL, greater than 0 and less than or equal to 2 x 10 7 Per mL, greater than 0 and less than or equal to 1 x 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 Per mL or 6 x 10 7 /mL-8*10 7 /mL。
Preferably, the content of the osteosarcoma cells is 1×10 based on the total amount of the hydrogel preparation 7 /mL。
Alternatively, the decellularized matrix is obtained using the following method: and obtaining biological tissues, and then performing decellularization to obtain a decellularized matrix.
Optionally, the biological tissue is osteosarcoma tissue.
In one embodiment, the osteosarcoma tissue is of human origin.
Decellularization refers to a supporting tissue that remains after removal of cells from a living tissue.
In one embodiment, gelatin is also included in the hydrogel formulation.
Alternatively, the gelatin concentration is greater than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel formulation. For example, 60mg/mL indicates that the gelatin occupies a concentration of 60mg/mL of the total hydrogel formulation. Can be greater than 0 and less than or equal to 60mg/mL, greater than 0 and less than or equal to 50mg/mL, greater than 0 and less than or equal to 40mg/mL, greater than 0 and less than or equal to 30mg/mL, greater than 0 and less than or equal to 20mg/mL, greater than 0 and less than or equal to 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,20 mg/mL-30mg/mL,30mg/mL-60mg/mL,30mg/mL-50mg/mL,30 mg/mL-40mg/mL, 40mg/mL-60mg/mL,40mg/mL-50mg/mL, or 50mg/mL-60mg/mL.
Preferably, the gelatin concentration is 20mg/mL based on the total hydrogel formulation.
In one embodiment, glycerol is also included in the osteosarcoma model organoid model. For reducing cell damage caused by extrusion.
Alternatively, the volume fraction of glycerol is greater than 0 and less than or equal to 30% based on the total amount of the hydrogel formulation. May be greater than 0 and less than or equal to 30%, greater than 0 and less than or equal to 20%, greater than 0 and less than or equal to 10%,10% -30%,10% -20% or 20% -30%.
In one embodiment, the osteosarcoma organoid model is made using 3d printing.
As shown in fig. 1-1 and fig. 1-2, an embodiment of the present invention provides a microfluidic chip, where the microfluidic chip includes:
an organoid culture zone 1, wherein the organoid culture zone 1 contains the osteosarcoma organoid model;
and a liquid inlet 2 communicated with the organoid culture zone 1 and used for pumping fluid.
And a liquid outlet 3 communicated with the organoid culture zone 1 and used for discharging fluid.
And the micro-flow channel 4 is used for communicating 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 a culture medium. The medium is generally selected from a-MEM medium (gibco).
The organoid culture comprises a plurality of chambers which are communicated with each other, and the 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 be attached to each other, and the organoid culture area 1 and the micro flow channel 4 are engraved on the bottom plate 6. The liquid outlet 3 and/or the liquid inlet 2 can be carved on the bottom plate or the cover plate. The cover plate and the base plate may be connected by an adhesive. For example by means of an adhesive film 7 (DSF).
Preferably, the material of the cover plate and the material of the base plate are different.
Alternatively, the bottom plate may be divided into an upper bottom plate and a lower bottom plate that can be attached, and the organoid culture area 1 and the microfluidic channel 4 are engraved on the upper bottom plate. The upper and lower plates may be connected by an adhesive film 7.
Alternatively, the material of the bottom plate may be PMMA (polymethyl methacrylate) and/or glass.
Optionally, the material of the cover plate is PDMS (polydimethylsiloxane), which is beneficial to gas exchange.
The inner diameter of the micro flow channel is 50 mu m-1cm.
As shown in fig. 1 to 3, an embodiment of the present invention provides a method for preparing the microfluidic chip, which at least includes the following steps:
placing the osteosarcoma organoid model in an organoid culture zone; and injecting fluid into the micro-flow channel and the organoid culture area through the liquid inlet to obtain the micro-fluidic chip.
Further, the method also comprises the following steps: after the osteosarcoma organoid model is placed in the organoid culture zone, the cover plate and the bottom plate are covered.
The osteosarcoma organoid model described above may be injected into the organoid culture zone in a non-gel state.
An embodiment of the invention provides an osteosarcoma model organoid dynamic culture model, which comprises the microfluidic control chip and a microfluidic pump, wherein the microfluidic pump is communicated with a liquid inlet and a liquid outlet in the microfluidic chip and is used for providing power for fluid flow in the microfluidic chip.
The micro-flow pump is a micro peristaltic pump.
Optionally, the osteosarcoma model organoid dynamic culture model further comprises a liquid reservoir. The liquid storage device is used for storing the fluid conveyed to the liquid inlet and storing the fluid discharged from the liquid outlet. So that the osteosarcoma model organoid dynamic culture model forms an annular closed pipeline.
As shown in fig. 1 and 11, the invention provides the application of the osteosarcoma organoid model or the microfluidic chip or the osteosarcoma model organoid dynamic culture model in the screening of osteosarcoma resistant drugs.
The anti-osteosarcoma drug screening of the invention can be single drug screening or drug composition screening.
The drug screening can be a general drug screening of osteosarcoma or a drug screening aiming at specific osteosarcoma tissue.
In one embodiment, as shown in FIG. 12, first, after pathological confirmation of cells therein by a clinically taken patient biopsy sample, the remaining tissue is used for construction of the combined 3D bioprinting ink.
Secondly, a multi-organ microfluidic drug screening platform is constructed by a 3D biological printing technology so as to accurately simulate and observe the response of tumors to drugs in real time.
Example 1
a) Extraction and identification of BMSC-EV
Selecting 1-2 weeks old XinxiSterilizing young rabbits with 75% alcohol, and taking femur, tibia and humerus. After PBS washing, the bone ends were cut off using scissors, the central bone marrow cavity was exposed, and the bone marrow cavity was flushed with Alpha-MEM medium containing 5000U/mL heparin. The collected medium was resuspended in cells and centrifuged at 1800rpm for 10 minutes after mixing. Cells were then resuspended in Alpha-MEM complete medium containing 20% FBS,1% diabody and inoculated at 75cm 2 Cell culture flasks. 37 ℃, 5% CO 2 Incubating in a 95% humidity incubator, and passaging after cell fusion. BMSCs using P2-P3 were used to extract stem cell exosomes, and after the cell fusion degree was about 70%, the cell culture medium was replaced with serum-free medium, and the culture was continued for 72 hours, the cell culture supernatant was collected. Cell impurities were removed by centrifugation at 1200rpm, followed by ultracentrifugation at 4℃for 90min using 100000g, supernatant was discarded after the end of centrifugation, and 0.5mL DPBS was resuspended to give BMSC-EV. And then observing the exosome shape by using a TEM, observing the exosome particle size distribution by using a nanoparticle tracking analysis technology NTA, verifying the characteristics Marker CD9 and CD63 of the exosome by using Western Blot, observing the uptake of the exosome by using a Diol-marked exosome, and determining the influence of BMSC-EV on the activity, proliferation and migration behaviors of the osteosarcoma cell line MG63 by detecting the cell activity, cell proliferation and cell migration.
The results are shown in FIGS. 2 and 3, in which BMSC-EV promotes proliferation and migration of MG 63. BMSC-EV enhanced bone-related gene expression of MG63 (FIG. 3).
b) Extraction and identification of dOsEM acellular matrix
Clinically obtained fresh human osteosarcoma tissue (transferred in culture medium) is placed in 0.25% trypsin at 250rpm for stirring treatment for 6h, washed three times, 100mL 70% ethanol at 250rpm for stirring treatment for 10h,3% H 2 O 2 After washing for 15min, three times, 1% Triton X-100 and 0.26% EDTA/Tris for 6h. Thereafter, the mixture was washed 3 times with water for 15min, treated with 0.1% peroxyacetic acid/4% ethanol for 2h, washed with double distilled water for 15min for a plurality of times, and lyophilized. The DNA content in the decellularized matrix was then quantified using a NanoDrop, and the above components were quantitatively detected using a GAG and collagen Elisa quantification kit. At the same time, protein mass spectrometry is used to perform matrix composition inside the matrixQualitative and quantitative analysis.
b-1) Mass Spectrometry of proteins
Bioactive proteins in the dOsEM are resolved by using a biological mass spectrometry technology, and a bioinformatic molecular screening is combined and a major extracellular matrix protein which can play a key regulation role in the dOsEM is determined (such as Collagen I, fibrinogen, laimin, collagen III, collagen XV, periostin, collagen XI, ALB and the like). The specific operation is that after the protein cracking sample of the sample is primarily separated through SDS-PAGE electrophoresis, the dyeing decolorization liquid is used for decolorization, the sample is added into a sequencing-grade pancreatin solution for reaction at 37 ℃ for overnight, after ultrasonic freeze-drying, the sample is analyzed by combining capillary high performance liquid chromatography with ESI mass spectrum, and the protein and the mutual regulation relation thereof are identified by analyzing data through Maxquat soft parts and combining with STRING database.
The results are shown in FIG. 3-1, and the results show that the effective components are similar to osteosarcoma (Os) in composition and proportion.
c) Preparation and characterization of printable dOsEM-Fibrin hydrogels
The lyophilized dOsEM acellular matrix and pepsin are stirred for 48 hours for dissolution, and the PH is regulated to about 7.4 by NaOH so that the final concentration is 6mg/mL. The components and the proportions shown in the table 1 are sequentially configured to form the dOsEM-Fibrin biological ink, and the dOsEM-Fibrin biological ink is mixed with a Thrombin solution of 20IU/mL and crosslinked for 30 minutes, so that the structural molding of the hydrogel is realized. And the rheological property and the mechanical property of the material are characterized. The characterization of the osteosarcoma decellularized matrix, dOsEM, and dOsEM-Fibrin hydrogel is shown in FIG. 5. Indicating that the process did not destroy the content of Collagen and that this component of DNA and GAGs was removed.
TABLE 1 preparation composition Table of BMSC-EV loaded dECM hydrogels
TABLE 2 results of BMSC-EV mass spectrometry analysis of the active ingredient in the dOsEM-Fibrin hydrogel
| Fasta headers | Intensity Exo.raw |
| COL12A1 | 538270000 |
| VIM | 347170000 |
| COL1A2 | 169350000 |
| FN1 | 131410000 |
| COL3A1 | 27270000 |
| COL5A2 | 26782000 |
| COL11A1 | 8505800 |
The result of BMSC-EV mass spectrometry analysis of the effective component in the dOsEM-Fibrin hydrogel is shown in Table 2, which shows that the effective component is mainly COL-XII, vimentin, COL-I, FN1, COL-III, COL-V and the like.
Rheological detection: HR-2Discovery rheometer (TA Instruments, newcastle, DE, USA) and 25mm parallel plate clamps were used. The strain is measured from 0.01 to 100 at an angular frequency of 6.28 rad/s. The rheometer was run using programmable software from TA Instrument, data was collected from triplicate samples, and the solution temperature was maintained at 20 ℃. The results are shown in FIG. 6, which demonstrate that the osteosarcoma decellularized matrix dOsEM and dOsEM-Fibrin hydrogel has good performance.
d) Condition optimization of 3D bioprinting BMSC-EV loaded dOsEM-Fibrin bioscaffold
The mosem bioscaffold was constructed by extruding the hydrogel and integrating it in a layer-by-layer stack using a Novaprint 3D printer. The method comprises the following specific steps:
(a) Turning on the power supply, pneumatic valve and condensing unit of the equipment and calibrating the equipment.
(b) Before printing, a 3D printing bracket model 'Box 6.5X6.5X1.st2' of 6.5X6.5X1 mm is constructed by using Magic software, and the model is cut by using layering software matched with a 3D printer to obtain a printing path file.
(c) Thereafter, the bio-ink for 3d printing was obtained by dividing into BMSC-EV-dOsEM-Fibrin group and dOsEM-Fibrin group according to Table 1. The bio-ink is loaded into a printing cartridge and printed using three different bore straight pins (100,200,250 μm) as the printhead. The printing unit is placed on the cartridge sleeve of the 3D bio-printer.
(d) After the printing cartridge was mounted to the bio-printer bio-print head in cooperation with the print head, the printing speed was set to 3mm/s, the printing pressure was 1.7bar, and the print pattern was rectangular parallelepiped (6.5x6.5x1 mm). In addition, according to the results of the related studies on oxygen diffusion (printing intervals are different, oxygen and nutrient transport inside the stent are changed, which results are obtained from Lu, zuyan, et al, "An oxygen-releasing device to improve the survival of mesenchymal stem cells in tissue engineering," Biofabrication 11.4 (2019): 045012.), different printing intervals (0.6 mm,1.0mm and 2.0 mm) are set, respectively, and in order to eliminate the influence of temperature on the hydrogel adhesiveness and to ensure survival of cells inside the hydrogel, the temperature of the printing extrusion head is set to 37 ℃.
(e) Osteosarcoma chips are composed of two different chip structures (PMMA and PDMS), the bottom layer for direct imaging of the micro-tissue/cells, while the top PDMS layer aids in gas exchange. To avoid particle contamination, sterilization was performed under ultraviolet light for 30 minutes. And then placing the microfluidic chip with the prefabricated pore channels and flow channels in the central area of a printer, printing the biological ink to a organoid culture area on a bottom plate of the microfluidic chip by using air pressure drive, adding the 3D printing bracket sample into Throbmin (200 IU/mL) solution after printing, and continuously crosslinking for 30 minutes at 37 ℃.
(f) And after printing, covering the cover plate on the bottom plate. The cover plate consists of a plasma-bonded adhesive and a PDMS cover. Ports on the cover plate were fitted with Polytetrafluoroethylene (PTFE) tubing and the chip was connected to a micro peristaltic pump loop. The tubing from the inlet is then connected to the reservoir via PVC tubing and connected to the outlet for draining into the reservoir, thereby creating a closed loop system. To initiate medium recirculation in the system, PVC tubing was mounted on a micro peristaltic pump. Each medium reservoir was filled with 1.6mL of medium at an extremely low flow rate of 4. Mu.L/min, thereby simulating the circulation system for organoids. And the effectiveness of the culture system is verified by cell activity, cell migration and apoptosis in the chip in vitro.
Cell proliferation Activity in vitro
The proliferation of cells inside the hydrogel was detected separately using CCK8 kit and CaAM/PI fluorescent kit. The specific procedure was to dilute with medium at a ratio of 1:10 using CCK8 kit, afterwards to blot out the medium inside the well plate and to add 300 μl of CCK8 diluted solution, incubate at room temperature for 2h, record absorbance at 450nm inside the well plate for 1 day, 3 days, 5 days, 7 days, 4 replicates each. CaAM/PI is prepared by adding CaAM working solution (1:1000) to a bioprinting scaffold containing cells, incubating at 37℃for 45min, then adding PI working solution (1:3000), incubating at 37℃for 15min, then washing with PBS, and observing the proportion of living and dead cells using a laser confocal microscope. Alginate hydrogel (Sigma-Aldrich, A0682) was used as a conventional control.
Cell adhesion Activity
The cytoskeleton was labeled using a phalloidin detection kit, and the adhesion and spreading of the internal cells were observed by immunofluorescence microscopy. The specific operation is that after 48 hours of bracket culture, the culture medium is removed, PBS is used for washing three times, 2.5 percent polymaleic aldehyde is added, and the mixture is fixed for 60 minutes at room temperature. Then, PBS containing 0.1% Triton X-100 was used to wash 3 times in sequence, and PBS containing 1% BSA and 0.1% Triton X-100 was used to dilute the Actin-Tracker Red dye at a ratio of 1:100, incubated at room temperature for 60min in the dark, and PBS containing 0.1% Triton X-100 was used to wash 3 times, and finally, the nuclear stain DAPI was added and the morphology of the cytoskeleton was observed under a confocal laser microscope.
The results are shown in FIG. 8, which demonstrate that 3D bioprinted dOsEM-Fibrin hydrogel scaffolds significantly enhance the proliferation and adhesive activity of osteosarcoma cells in vitro, as compared to control alginate hydrogels.
e) Exploration of the therapeutic Effect of CXCR4 specific inhibitor Plerixafor in combination with osteosarcoma chemotherapeutic agent on highly proliferative active osteosarcomas Using 3D osteosarcoma organoid chip
The IC50 concentration of the chemotherapeutic agent was obtained by first adding different concentrations of doxorubicin (0.01. Mu.M-100. Mu.M), cisplatin (0.03. Mu.M-300. Mu.M), methotrexate (0.03. Mu.M-10. Mu.M), ifosfamide (0.3 mM-10 mM), plerixafor (1 nM-100. Mu.M) to the medium in the microfluidic chip for 3 days by cycling, and then detecting the cell activity by CCK8 method. And the medicines such as doxorubicin, cisplatin, methotrexate and ifosfamide with different concentrations are sequentially combined with a specific inhibitor Plerixafor of CXCR4, a culture medium mixed with the different medicines is sequentially added into a microfluidic chip for circulating culture for 3 days, and then the activity of the internal osteosarcoma cells is detected by using a CaAM/PI method and semi-quantitative analysis is carried out on the cell activity by using an ImageJ.
As shown in fig. 9 and 10, the drug screening platform is constructed by using the osteosarcoma organoid chip of the present invention, and it is clear that the combination of plaixafor and Dox can significantly increase the killing effect of Dox on osteosarcoma cells with high proliferation activity. Compared with the conventional 2D single cell culture (2D Mono) group, the 3D bioprinting dOsEM-Fibrin hydrogel scaffold has improved metabolic function.
The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.
Claims (9)
1. The 3D printed osteosarcoma model organoid model is characterized by at least comprising a biological scaffold, osteosarcoma cells loaded on the biological scaffold and exosomes derived from bone marrow mesenchymal stem cells; the biological scaffold is a crisscrossed three-dimensional structure constructed by a hydrogel preparation, and the hydrogel preparation comprises acellular matrix and fibrin; the 3D printed osteosarcoma organoid model further includes the following features:
a. The acellular matrix is of osteosarcoma tissue;
b. the osteosarcoma cell is a living cell;
c. the hydrogel preparation also comprises gelatin;
d. the hydrogel preparation also comprises glycerol;
e. the concentration of the acellular matrix is more than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel preparation;
f. the concentration of the fibrin is more than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel preparation;
g. the concentration of the exosomes derived from the bone marrow mesenchymal stem cells is more than 0 and less than or equal to 300mg/mL based on the total amount of the hydrogel preparation;
h. the content of the osteosarcoma cells is more than 0 and less than or equal to 8 multiplied by 10 based on the total amount of the hydrogel preparation 7 /mL。
2. The 3D printed osteosarcoma organoid model of claim 1, wherein the gelatin concentration is greater than 0 and less than or equal to 60mg/mL based on the total amount of hydrogel formulation; and/or the number of the groups of groups,
the volume fraction of the glycerol is more than 0 and less than or equal to 30 percent based on the total amount of the hydrogel preparation.
3. The method for constructing a 3D printed osteosarcoma organoid model according to claim 1 or 2, comprising at least the steps of:
carrying exosomes and osteosarcoma cells derived from bone marrow mesenchymal stem cells on the biological scaffold to obtain a 3D printed osteosarcoma organoid model; the biological scaffold is a crisscrossed three-dimensional structure constructed with a hydrogel formulation comprising decellularized matrix and fibrin.
4. A method of constructing a 3D printed osteosarcoma organoid model according to claim 3, further comprising a plurality of the following features:
a. the acellular matrix is of osteosarcoma tissue;
b. the osteosarcoma cell is a living cell;
c. the hydrogel preparation also comprises gelatin;
d. the hydrogel preparation also comprises glycerol;
e. the concentration of the acellular matrix is more than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel preparation;
f. the concentration of the fibrin is more than 0 and less than or equal to 60mg/mL based on the total amount of the hydrogel preparation;
g. the concentration of the exosomes derived from the bone marrow mesenchymal stem cells is more than 0 and less than or equal to 300mg/mL based on the total amount of the hydrogel preparation;
h. the content of the osteosarcoma cells is more than 0 and less than or equal to 8 multiplied by 10 based on the total amount of the hydrogel preparation 7 /mL。
5. The method of preparing a 3D printed osteosarcoma organoid model of claim 4, wherein the gelatin concentration is greater than 0 and less than or equal to 60mg/mL based on the total amount of hydrogel formulation; and/or the number of the groups of groups,
the volume fraction of the glycerol is more than 0 and less than or equal to 30 percent based on the total amount of the hydrogel preparation.
6. A microfluidic chip comprising thereon:
a organoid culture zone (1) containing a 3D printed osteosarcoma organoid model according to any of claims 1-2;
a liquid inlet (2) communicated with the organoid culture area (1) and used for pumping fluid;
a liquid outlet (3) communicated with the organoid culture zone (1) for discharging fluid;
the micro-flow channel (4) is used for communicating the organoid culture area (1), the liquid inlet (2) and the liquid outlet (3);
the organoid culture zone contains a 3D printed osteosarcoma organoid model according to any of claims 1-2 in 3D printing.
7. The preparation method of the microfluidic chip at least comprises the following steps: placing the 3D printed osteosarcoma organoid of any of claims 1-2 in an organoid culture zone; and injecting fluid into the micro-flow channel and the organoid culture area through the liquid inlet to obtain the micro-fluidic chip.
8. A osteosarcoma model organoid dynamic culture model comprising the microfluidic chip of claim 6 and a microfluidic pump in communication with a liquid inlet and a liquid outlet in the microfluidic chip for powering fluid flow within the microfluidic chip.
9. Use of a 3D printed osteosarcoma organoid model according to any of claims 1-2 or a microfluidic chip according to claim 6 or a osteosarcoma model organoid dynamic culture model according to claim 8 for screening anti-osteosarcoma drugs.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210839348.4A CN115651909B (en) | 2022-07-14 | 2022-07-14 | Osteosarcoma organoid model, construction method and application |
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| WO2019122351A1 (en) * | 2017-12-22 | 2019-06-27 | Cellink Ab | Tissue-specific human bioinks for the physiological 3d-bioprinting of human tissues for in vitro culture and transplantation |
| CN110575567A (en) * | 2019-08-20 | 2019-12-17 | 苏州诺普再生医学有限公司 | scaffold composite material and preparation method and application thereof |
| CN111808817A (en) * | 2020-07-24 | 2020-10-23 | 上海昊佰生物科技有限公司 | Culture method of osteosarcoma organs and bone tumor culture medium thereof |
| WO2022035275A1 (en) * | 2020-08-14 | 2022-02-17 | 주식회사 넥스트앤바이오 | Method for manufacturing organoid using microfluidic chip |
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| WO2019079292A1 (en) * | 2017-10-16 | 2019-04-25 | President And Fellows Of Harvard College | Methods of forming three-dimensional tissues scaffolds using biological fiber inks and methods of use thereof |
| KR102213931B1 (en) * | 2019-09-16 | 2021-02-05 | 포항공과대학교 산학협력단 | Methods to develop the reconstituted miniature tissues with multiple cell types from patient-derived stem or tumor cells |
| CN115651909B (en) * | 2022-07-14 | 2023-09-19 | 上海交通大学医学院附属第九人民医院 | Osteosarcoma organoid model, construction method and application |
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| WO2019122351A1 (en) * | 2017-12-22 | 2019-06-27 | Cellink Ab | Tissue-specific human bioinks for the physiological 3d-bioprinting of human tissues for in vitro culture and transplantation |
| CN110575567A (en) * | 2019-08-20 | 2019-12-17 | 苏州诺普再生医学有限公司 | scaffold composite material and preparation method and application thereof |
| CN111808817A (en) * | 2020-07-24 | 2020-10-23 | 上海昊佰生物科技有限公司 | Culture method of osteosarcoma organs and bone tumor culture medium thereof |
| WO2022035275A1 (en) * | 2020-08-14 | 2022-02-17 | 주식회사 넥스트앤바이오 | Method for manufacturing organoid using microfluidic chip |
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