CN116836911A - Construction method and application of vascularized organoid - Google Patents
Construction method and application of vascularized organoid Download PDFInfo
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- C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
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Abstract
The invention discloses a microporous array type polymer membrane based on 3D biological printing for constructing vascular organoids. The vascular organoid is successfully constructed by using a micropore array type polymer membrane as a main supporting material and using a composite of endothelial cells, pericytes and matrix gel as elastic biological ink. The blood vessel organoid has normal blood vessel structure and blood vessel function. Clinically, the vascular targeting drug has the function of inhibiting the vascular function of the vascular organoid. The vascular organoid has good bionic degree and high clinical compliance, and can be used as a screening model of clinical vascular targeting drugs.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a construction method and application of vascularized organoids.
Background
Vascular diseases are various, and the number of vascular related diseases patients in China is large and rises year by year. For the treatment of vascular diseases, the use of vascular targeting drugs is an extremely effective means. However, in the development process of vascular targeting drugs, the lack of an effective drug screening model has become one of the difficulties in development.
The drug screening models commonly used in the current drug development process mainly comprise a traditional 2D cell model, a traditional animal model, a non-human primate model, a PDX model and the like. The traditional 2D cell model has unstable genome, can not simulate the functions of real organs and can not accurately reflect the physiological environment of a human body; traditional animal models such as mouse models belong to complete individual models, but have the defects of species difference, poor prediction capability on actual human body reaction, ethical problems, limitation of imaging observation and the like; although the non-human primate model is closest to human on a phylogenetic tree, has highly similar brain structure and cognitive function of an immune system, but is expensive, and has the defects of ethical limitation, imaging observation limitation, uncontrollable complex variables, difficulty in high-throughput screening of medicines and the like; although the PDX model has been proved to better simulate the growth and metastasis of tumors, the human tumor microenvironment cannot be rebuilt, and the PDX also has the problems of low success rate of transplantation, high construction cost, long period, limitation of high-throughput screening of medicines and the like. Therefore, the development of the drug screening model with good clinical compliance and high bionic degree has important significance.
Organoids are a new development reagent replacement in the last decade, which is a three-dimensional aggregate with specific functions composed of different tissue cells (such as vascular cells, parenchymal cells, etc.) according to a certain structure, and has the basic functions of real organs. Compared with the traditional drug screening model, the organoid model has obvious advantages of humanization and near physiology, and is also a research hotspot of the current drug screening model.
The current commercial blood vessel organoid drug screening model is a blood vessel organoid chip produced by the netherlands mimeta company, but the blood vessel organoid drug screening model cannot reach the index consistent with the blood vessel of a human body in the aspect of blood vessel integrity and physiological function: only has endothelial cells, no expansion and contraction ability, no elasticity and low water permeability. Therefore, developing a blood vessel organoid with physiological functions is a crucial technical point for constructing a blood vessel organoid drug screening model.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a construction method of vascularized organoids with good bionic degree and high clinical compliance.
The invention also aims to provide the application of the vascular organoid in screening the vascular targeting drugs, and provides a screening model for developing the vascular targeting drugs for clinic.
The aim of the invention is achieved by the following technical scheme:
the invention provides a vascularized organoid based on 3D printing, which is obtained by taking a microporous array type polymer membrane printed by 3D as a biological bracket;
furthermore, the vascularized organoid based on 3D printing is obtained by composite culture of endothelial cells-pericyte-matrix gel.
The invention provides a vascular organoid obtained based on a 3D printing technology by taking tumor vascular endothelial cells and pericytes derived from clinical samples as materials. The clinical-source tumor vascular endothelial cells and pericytes are obtained through separation and extraction of clinical tumor tissues and through flow cell sorting identification.
The retractility, elasticity and permeability are inherent functions of blood vessels. In order to realize similar regulation and control effects in 3D printed vascular organoids, the applicant adopts a micropore array type polymer membrane as a biological bracket, and sprays matrix gel containing vascular endothelial cells and pericytes, so that the biological functions of blood vessels can be obviously reserved;
further, the vascularized organoids based on 3D printing include primary endothelial cells and pericytes extracted and separated from various tissues, preferably tumor-derived endothelial cells and pericytes; wherein the amount of type I collagen added is 9mg/ml, and the concentration of methacrylic anhydride gelatin is 40mg/ml.
The vascular elasticity of 3D printed vascular organoids is formed by matrix gel in early culture and mainly by collagen secreted by endothelial cytoskeleton and pericytes in later culture. By analyzing the blood vessel formed by the 3D printing blood vessel organoid, the organoid blood vessel is found to be 77% of normal blood vessel in elasticity, and the formed blood vessel has a lumen.
The technical means for constructing blood vessels by the 3D biological printer mainly comprises bionics and self-assembly. Although the appearance of the vascular structure obtained by adopting the 3D biological printer according to the bionics principle is very similar to that of normal tissue blood vessels, the adherence and vascularization degree of the vascular endothelial cells are difficult to control because the individual vascular endothelial cells are not controlled, and the growth direction of the endothelial cells cannot be ensured to completely grow according to the direction of the stent structure. The self-assembly technology takes cells as main initial factors of tissue generation, drives biological printing tissues to develop according to embryo mechanisms by adjusting the microenvironment of the cells, realizes the regulation and control of angiogenesis by a self-assembly principle by changing the microenvironment of endothelial cells, and the formed blood vessel is a blood vessel structure spontaneously formed by the endothelial cells, accords with the development process of life, has more reliable structure and function of a vascular network and is more closely related to solid cells.
Furthermore, the vascularized organoid constructed based on 3D biological printing takes a micropore array type polymer film as a supporting runner in the spraying and loading process, and the polymer film takes polycaprolactone PCL and gelatin as frames and is loaded into a micropore array type through 3D printing.
The second invention provides application of the 3D printed vascular organoid in vascular targeting drugs, and provides a screening model for developing vascular targeting drugs for clinic.
Further, the screening model of the vascular targeting drug is used for evaluating the change of the structure, the relaxation and the elastic capacity of the vascular organoid after the drug treatment.
Drawings
In order to clearly show the technical scheme of the implementation of the invention, the construction, the function evaluation and the application of the vascular organoid in the embodiment of the invention are briefly described below.
FIG. 1 is a flow chart depicting the results of the extraction of isolated primary endothelial cells and pericytes according to an embodiment of the present invention.
Fig. 2 is a flow chart of the construction of 3D-printed vascular organoids using endothelial cell-pericyte-type i collagen-matrix gel as a biomaterial according to an embodiment of the present invention.
FIG. 3 is a diagram illustrating the structure and elastic capabilities of a blood vessel formed in a vascular organoid according to an embodiment of the present invention.
FIG. 4 is a comparison of vasodilation and elastic capacity of a vascular organoid of an embodiment of the present invention with a normal tumor.
FIG. 5 shows the application of the vascularized organoids of the present invention in the screening of vascular targeting drugs, exemplified by bevacizumab.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The material sources involved in the embodiments of the present invention are:
matrigel high concentration Matrigel available from Corning corporation of usa.
ECM medium, available from Gibco, usa.
MDEM medium, available from Gibco, U.S.A.
PM medium, available from Gibco, inc., USA.
Fetal bovine serum, available from us Thermo Fisher Scientific company.
pancreatin/EDTA digest was purchased from us Thermo Fisher Scientific company.
Methacrylic acid anhydrified gelatin, available from the smart device company, yongqing, suzhou.
Hyaluronic acid, available from us Thermo Fisher Scientific company.
Glycerol, available from Sigma Aldrich, usa.
R-GEN 200D bioprinter available from regenHU, switzerland.
Collagen I, available from the company wuhansai wilt.
Polycaprolactone PCL, available from Sigma Aldrich, usa.
EXAMPLE 1 extraction and isolation of primary endothelial cells and pericytes from tumor tissue
The experimental method comprises the following steps: taking fresh human colorectal cancer sample, cutting into 1-2mm in ultra clean bench 3 After the pellet was washed 3 times with 1% PS in PBS, the tissue pellet was transferred to geneMACS containing 5mL DMEM basal medium TM And C, adding a tissue lyase according to the instruction of the tumor tissue dissociation kit, and putting the kit on a fully-automatic tissue processor for dissociation. Filtering cells after tissue dissociation is completed with 100 μm filter screen, centrifuging at 1500rpm for 3min at room temperature, discarding supernatant, adding appropriate amount of erythrocyte lysate for resuspension, centrifuging at 1500rpm for 3min, discarding supernatant, adding fresh DMEM culture medium for resuspension cell precipitation, performing cell count with common optical microscope, collecting 1×10 7 After the supernatant was discarded, the cells were resuspended using 80. Mu.L of flow-through cell stain, incubated for 1h with CD31 and PDGFR. Beta. Flow-through antibody in the absence of light, washed once with PBS, centrifuged at 300 Xg for 5min at room temperature, the supernatant was discarded, and then 400. Mu.L of flow-through stain was added and detected on the machine.
From the experimental results shown in the table, it can be seen that: the flow cytometry results show that the isolated human colorectal cancer vascular endothelial cells and human colorectal cancer perivascular cells respectively express an endothelial cell marker CD31 and a perivascular cell marker PDGFR beta (see figure 1).
Example 2 construction of endothelial cell-pericyte-matrix gel composite bioenhas
The experimental method comprises the following steps: human Endothelial Cells (ECs) were cultured in ECM medium (Gibco), and human Pericytes (PCs) were isolated in pericyte medium (Gibco) supplemented with 20% fetal bovine serum and 1% fbs. Respectively taking 3×10 5 Endothelial cells and pericytes were seeded in 6cm diameter plates37℃、5％CO 2 Culturing in a saturated humidity incubator with culture medium for 24 hr. ECM high sugar medium containing 10% glycerol and 3mg/ml hyaluronic acid was transferred to a centrifuge tube, and then type I collagen and methacrylic acid anhydrified gelatin were added, wherein the amount of type I collagen added was 9mg/ml, and the concentration of methacrylic acid anhydrified gelatin was 40mg/ml. Perivascular cells were isolated from vascular endothelial cells 1:5, adding the mixture into a matrix gel solution containing the type I collagen and gelatin, and placing the mixture in a 37 ℃ incubator to be solidified for 30 minutes to construct the composite biological material with endothelial cells and pericytes embedded in the matrix gel.
From the table it can be seen that: composite bio-ink was prepared from endothelial cell-pericyte-type i collagen-matrix gel as biomaterial (see fig. 2).
Example 33D printing of microporous array Polymer Membrane biological scaffolds to construct vascular organoids
The experimental method comprises the following steps: the polymer membrane takes polycaprolactone PCL and gelatin as frames, and is printed into a micropore array type by using a 3D biological printer, so that the micropore array type polymer membrane biological stent is successfully constructed. And simultaneously extruding cell ink (inner nozzle) carrying endothelial cells-matrix gel and cell gel ink (outer nozzle) carrying pericyte-matrix gel by using a coaxial nozzle, wherein a micropore array type polymer film is used as a supporting runner during printing, a biological bracket is melted to form a smooth runner network during culturing, and endothelial cells are released from the biological bracket and adhered to the inner wall of the runner to be vascularized, so that the transition from the runner network to the vascularized network is realized.
From the experimental results shown in the figures, it can be seen that: based on this method, printing of vascularized organoids (. Gtoreq.1 cm) and in vitro long-time culture (. Gtoreq.20 days) are realized, and the structure of blood vessels formed in the vascularized organoids (see FIG. 3). And the blood vessel has good dilating and elastic functions (see fig. 3 and 4).
EXAMPLE 4 screening action of vascularized organoids on vascular targeting drugs
The experimental method comprises the following steps: the prepared vascularized organoids were cultured in DMEM containing 1% PS and 10% fbs, and 0, 1, 5, 10 μm regorafenib was added to the culture for 24 hours, respectively, and the vascular elasticity of each group of vascularized organoids after drug treatment was evaluated.
From the experimental results shown in the figures, it can be seen that: evaluation of the results of the vasoconstrictor-diastolic function experiments showed that regorafenib dose-dependent inhibited the physiological elastic capacity of vascularized organoids (see fig. 5).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. A method for constructing vascularized organoids, comprising the steps of mixing perivascular cells with vascular endothelial cells 1:5, adding the mixture into a matrix gel solution containing type I collagen and gelatin to construct composite biological ink, printing a micropore array type polymer membrane biological bracket by using a polymer membrane and taking polycaprolactone PCL and gelatin as a frame, and spraying the biological ink in the biological bracket by using a coaxial spray head of a 3D biological printer to construct the vascularized organoid.
2. The clinical sample-derived tumor vascular endothelial cells and pericytes according to claim 1, wherein the steps of extracting and isolating the primary endothelial cells and pericytes are as follows: cutting tumor tissue, cleaning with PBS, performing tissue dissociation, filtering to obtain cell suspension, centrifuging to remove supernatant, performing erythrocyte lysis, centrifuging, cleaning with PBS, adding corresponding antibody, incubating, and separating CD31 by flow cytometry + Endothelial cells and pdgfrβ + Pericytes.
3. The method of claim 1, wherein the bioprinting ink is prepared from endothelial cell-pericyte-matrix gel in a ratio.
4. The method for constructing vascularized organoids according to claim 1, wherein the microporous array type polymer membrane biological scaffold is printed into a microporous array type by using a 3D biological printer by using a polymer membrane with polycaprolactone PCL and gelatin as frames.
5. A vascular targeting drug screening method based on vascularized organoids is characterized by comprising the following specific operation steps: the vascularized organoids are cultured by DMEM containing 1% PS and 10% FBS, and the vascularized organoids are cultured for 24-48 hours by adding medicines with certain gradient concentration, and the vascular elastic capacity of the vascularized organoids after the medicines are treated is evaluated, so that the change of vascular physiological functions is observed.
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