CN116814547A - In-vitro tumor vascular model and method for detecting compound by using same - Google Patents
In-vitro tumor vascular model and method for detecting compound by using same Download PDFInfo
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Abstract
The invention provides an in-vitro tumor vascular model and a method for detecting compounds by the in-vitro tumor vascular model, and belongs to the technical field of biological tissue engineering. The in vitro tumor vascular model of the invention comprises: the artificial blood vessel group comprises an artificial blood vessel, an artificial blood vessel inner runner for independently culturing and/or perfusion of the inside of the artificial blood vessel, and an artificial blood vessel outer runner for culturing and/or perfusion of the outside of the artificial blood vessel; the tumor microsphere group comprises single or a plurality of tumor microspheres and a tumor microsphere runner for culturing and/or perfusing the single or a plurality of tumor microspheres, and the tumor microsphere runner is connected with the artificial blood vessel outflow runner. The invention establishes a tumor blood vessel model in vitro, can realize the microenvironment construction of drug and nutrient substance through blood vessel absorption and tumor metastasis invasion, and has the functions of blood circulation, blood vessel filtration and the like.
Description
Technical Field
The invention belongs to the technical field of biological tissue engineering, and particularly relates to an in-vitro tumor vascular model and a method for detecting a compound by using the in-vitro tumor vascular model.
Background
In the research of pharmacokinetics, pharmacodynamics and drug toxicity of anti-tumor drugs, the traditional method mainly comprises animal experiments and two-dimensional cell culture, and although a plurality of achievements are achieved, the traditional method is limited by factors such as cycle, cost, precision, ethics and the like, and the actual effect of the drugs is difficult to accurately and effectively evaluate.
Therefore, there is an urgent need to establish an effective tumor drug screening and drug evaluation model.
The organ chip is combined with various subjects such as cell biology, engineering, biological materials and the like to construct a three-dimensional tumor microenvironment in vitro, and is used as a screening model of tumor drugs. However, constructing a single organ chip as a drug screening model has certain limitations in function and application because the complexity, the function change and the integrity of the organ function of the organism cannot be reflected.
Disclosure of Invention
The invention aims at solving at least one of the technical problems existing in the prior art and provides an in-vitro tumor blood vessel model and a method for detecting a compound by using the in-vitro tumor blood vessel model.
In one aspect of the present invention, there is provided an in vitro tumor vascular model comprising:
artificial blood vessel group, and
a group of tumor microspheres, which are prepared from the following materials,
the artificial blood vessel group comprises an artificial blood vessel, an artificial blood vessel inner runner for independently culturing and/or perfusion of the inside of the artificial blood vessel, and an artificial blood vessel outer runner for culturing and/or perfusion of the outside of the artificial blood vessel;
the tumor microsphere set comprises single or a plurality of tumor microspheres and a tumor microsphere runner for culturing and/or perfusing the single or the plurality of tumor microspheres, and the tumor microsphere runner is connected with the artificial blood vessel outflow runner.
Preferably, the vascular prosthesis comprises an endothelial layer and a smooth muscle layer; and/or the number of the groups of groups,
the width of the artificial blood vessel ranges from 2mm to 40mm.
Preferably, the length range of the in-vitro tumor blood vessel model is 10 mm-50 mm, and the width range is 5 mm-40 mm.
Preferably, the tumor microsphere diameter in the single or multiple tumor microspheres is in the range of 900 μm to 1300 μm.
Preferably, the in vitro tumor vascular model further comprises a set of individual tumor microspheres,
the independent tumor microsphere group comprises single or a plurality of independent tumor microspheres and independent tumor microsphere flow channels for independently culturing and/or perfusing the single or the plurality of independent tumor microspheres.
Preferably, the independent tumor microspheres are symmetrically arranged with the tumor microspheres.
Preferably, the artificial blood vessel group further comprises an artificial blood vessel introduction port communicated with the artificial blood vessel inflow channel;
the tumor microsphere group also comprises a tumor microsphere introduction port communicated with the tumor microsphere flow channel;
the independent tumor microsphere group also comprises an independent tumor microsphere introduction port communicated with the independent tumor microsphere flow passage.
In another aspect of the present invention, there is provided a method for detecting a compound using the in vitro tumor vascular model described above, comprising the specific steps of:
introducing each culture medium into each flow channel to culture or perfuse tumor microspheres and/or artificial blood vessels;
introducing a test compound into the vascular prosthesis;
and obtaining the regulation result of the compound to be tested on the tumor microspheres and/or the artificial blood vessels.
Preferably, the obtaining the result of the adjustment of the tumor microsphere and/or the artificial blood vessel by the compound to be tested includes:
obtaining the cell activity and/or cell migration result of the tumor microsphere cultured by the artificial blood vessel; and/or the number of the groups of groups,
obtaining the shape of an artificial blood vessel;
screening out a compound to be tested matched with the tumor based on the cell activity and/or cell migration result of the tumor microsphere; and/or the number of the groups of groups,
and based on the morphology of the artificial blood vessel, obtaining the regulation result of the compound to be tested on the artificial blood vessel.
Preferably, the obtaining the result of the adjustment of the tumor microsphere and/or the artificial blood vessel by the compound to be tested further comprises:
obtaining the cell activity and/or cell migration result of the independent tumor microspheres;
based on the cell activity and/or cell migration results of the independent tumor microspheres and the tumor microspheres, the regulation result of the test compound on the tumor microspheres after artificial blood vessel absorption is obtained.
The invention establishes a tumor blood vessel model in vitro, realizes the microenvironment construction of drug and nutrient substance through blood vessel absorption and tumor metastasis invasion, and has the functions of blood circulation, blood vessel filtration and the like. By utilizing the in-vitro tumor vascular biological model, on one hand, drugs and nutrient substances are absorbed by blood vessels to act on tumors and/or directly act on the tumors, so that more accurate, efficient and convenient test of anti-tumor drugs is realized; on the other hand, the method can realize accurate, efficient and convenient test of the influence of the anti-tumor drug on the artificial blood vessel (for example, the anti-tumor drug can influence the blood vessel to cause vascular related diseases such as vasculitis, hypertension, thrombosis and the like).
Drawings
FIG. 1 is a schematic diagram showing the overall structure of an in vitro tumor vascular model according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a culture layer according to an embodiment of the invention;
FIG. 3 is a schematic diagram of a sealing layer according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a connection layer according to an embodiment of the present invention;
FIG. 5 is a schematic diagram showing a culture system according to an embodiment of the present invention;
FIG. 6 is an image of NCI-H23 tumor microspheres according to an embodiment of the present invention;
FIG. 7 is a graph showing the cell activity of NCI-H23 tumor microspheres as a function of days of drug action according to an embodiment of the present invention;
FIG. 8 is a graph showing the variation of NCI-H23 tumor microspheres with days of drug action according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail below with reference to the drawings and detailed description for the purpose of better understanding of the technical solution of the present invention to those skilled in the art. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention belong to the protection scope of the present invention.
As shown in fig. 1 to 5, in one aspect of the present invention, there is provided an in vitro tumor vascular model comprising: an artificial blood vessel group and a tumor microsphere group, wherein the artificial blood vessel group comprises an artificial blood vessel, an artificial blood vessel inner runner for independently culturing and/or perfusion of the inside of the artificial blood vessel, and an artificial blood vessel outer runner for culturing and/or perfusion of the outside of the artificial blood vessel; the tumor microsphere group comprises single or a plurality of tumor microspheres and a tumor microsphere runner for culturing and/or perfusing the single or a plurality of tumor microspheres, wherein the tumor microsphere runner is connected with an artificial blood vessel outflow runner, i.e. the tumor microsphere with blood vessel function can be formed based on the model.
The tumor vascular model of the embodiment can realize vascular absorption of medicines and nutrients, has blood circulation and vascular filtration functions, constructs a tumor metastasis invasion microenvironment, can truly reflect the complexity, functionality and integrity of organism organ functions, more truly simulate in-vivo microenvironment and improve the experimental data accuracy of the model in application.
In some preferred embodiments, the vascular prosthesis comprises an endothelial layer and a smooth muscle layer, i.e. the inner wall of the vascular prosthesis conduit is an endothelial layer consisting of a layer of endothelial cells and the outer side of the inner wall is a smooth muscle layer consisting of at least one layer of smooth muscle cells.
The vascular endothelial cells of the vascular prosthesis of this example were closely aligned (no gaps between vascular endothelial cells), and the alignment had directionality and intact cell morphology.
The endothelial layer of the inner wall of the blood vessel of the embodiment has the capability of sensing the flow velocity and direction of the fluid in the blood vessel; smooth muscle cells outside the inner wall of the blood vessel can feel stimulation (such as epinephrine and the like produce contraction, etc.), realize the functions of vasoconstriction and dilation, and evaluate the toxic influence of drugs on the blood vessel, for example, when the drugs promote the blood vessel to excessively contract, hypertension can be caused; when the medicine promotes the blood vessel to be excessively dilated, the blood pressure is reduced and the like; when the drug promotes smooth muscle cell hyperproliferation, vasculitis, plaque formation, and the like may result.
In other preferred embodiments, the width of the vascular prosthesis ranges from 2mm to 40mm.
As a further preferable scheme, the width of the artificial blood vessel is preferably in the range of 2 mm-7 mm so as to simulate the vein of the human body.
The embodiment realizes the high simulation of the human blood vessel by adjusting the width of the artificial blood vessel to simulate the thickness of the human real blood vessel.
Further, in some preferred embodiments, the in vitro tumor vascular model has a length in the range of 10mm to 50mm and a width in the range of 5mm to 40mm.
The model of this embodiment is whole less, can improve the convenient degree of experimental operation.
Still further, in some preferred embodiments, the single or multiple tumor microspheres may be spherical in structure, with the tumor microspheres ranging in diameter from 900 μm to 1300 μm.
The tumor microsphere of the embodiment has the functional characteristics of deep middle color, light edge and clear outline.
In this embodiment, the kind of the single or multiple tumor microspheres is not particularly limited, and for example, one or more of lung cancer tumor microspheres, liver cancer tumor microspheres, intestinal cancer tumor microspheres, skin cancer tumor microspheres, and throat cancer tumor microspheres may be selected.
Specifically, as shown in fig. 1 to 3, the tumor microsphere flow path includes a tumor microsphere culture inlet B2, a tumor microsphere inlet flow path B2-1, a blood vessel culture chamber B13, a blood vessel culture chamber outlet flow path B2-2, a micro-mixing flow path B10, a first group of culture chambers B12, a tumor microsphere outlet flow path B1-1 and a tumor microsphere culture outlet B1, which are sequentially arranged on the culture layer B in the fluid flow direction.
Further, as shown in fig. 1 to 3, the artificial blood vessel inner flow path includes a blood vessel inflow port C3, a blood vessel inlet flow path C3-1, a blood vessel inlet connection hole C3-2, a blood vessel outlet connection hole C4-2, a blood vessel outlet flow path C4-1, and a blood vessel outflow port C4, which are provided in this order on the sealing layer C in the fluid flow direction. Wherein the sealing layer C is laminated below the culture layer B, the artificial blood vessel E is arranged in a blood vessel culture chamber B13 on the culture layer B, two ends of the artificial blood vessel E are respectively communicated with a blood vessel outlet connecting hole C4-2 through a blood vessel inlet connecting hole C3-2 to form an artificial blood vessel inner runner for culturing and/or perfusion of the inside of the artificial blood vessel, and the rest space except the artificial blood vessel in the blood vessel culture chamber forms an artificial blood vessel outer runner.
It should be noted that, in this embodiment, how the artificial blood vessel is disposed in the blood vessel culturing chamber is not particularly limited, as long as the fluid communication between the artificial blood vessel inner flow passage and the artificial blood vessel outer flow passage can be realized.
For example, in order to facilitate the installation of the artificial blood vessel, as shown in fig. 1 and 3, the two ends of the artificial blood vessel E are respectively penetrated with a connecting pipe D2, the other ends of the connecting pipes D2 are respectively penetrated on the fixed blocks D1, the two fixed blocks D1 are fixedly arranged on the culture layer B, and the two fixed blocks D1 are respectively positioned at the two ends of the blood vessel culture chamber B13. And, a runner communicated with the connecting pipe D2, the vessel inlet connecting hole C3-2 or the vessel outlet connecting hole C4-2 is arranged in each fixing block D1 so as to form an artificial blood vessel inner runner.
Based on the specific structures of the tumor microsphere group and the artificial blood vessel group, the culture or perfusion principle of the artificial blood vessel and the tumor microsphere is as follows: and in combination with the illustration of fig. 5, a first culture medium and a second culture medium are respectively introduced into the tumor microsphere flow channel and the artificial blood vessel flow channel, so that when the tumor microsphere and the artificial blood vessel are cultured or perfused, the second culture medium based on the artificial blood vessel flow channel contains a compound to be tested, thus, the culture mediums in the two flow channels are subjected to substance exchange in the blood vessel culture chamber due to concentration difference, pressure difference and other reasons, namely, the compound to be tested in the artificial blood vessel flow channel seeps out after being absorbed by an endothelial layer, smooth muscle layer, filtration and other effects of the artificial blood vessel, and enters the blood vessel culture chamber of the artificial blood vessel outer flow channel, and the compound to be tested enters the micro-mixing flow channel along with the first culture medium and flows into the first group of culture chambers, so that the tumor microsphere with blood vessel function is obtained, and meanwhile, the effect of the drug on the blood vessel can be obtained.
In other preferred embodiments, the in vitro tumor vascular model further comprises an independent set of tumor microspheres comprising single or multiple independent tumor microspheres, and independent tumor microsphere channels for independent culture and/or perfusion of single or multiple independent tumor microspheres, for comparison with tumor microspheres having vascular function.
Specifically, as shown in fig. 2, the independent tumor microsphere flow channel comprises an independent culture inlet B5, an independent inlet flow channel B5-1, a second group of culture chambers B7, an independent outlet flow channel B6-1 and an independent culture outlet B6 which are sequentially arranged on the culture layer B according to the fluid flowing direction.
Further, the second group of culture chambers are correspondingly arranged with the first group of culture chambers, and comprise corresponding positions and numbers, the two groups of culture chambers are symmetrically arranged on two sides of the culture layer along the width direction of the culture layer, and the two groups of culture chambers comprise five sub-culture chambers so as to realize co-culture of a plurality of tumor microspheres.
Illustratively, as shown in FIG. 2, the first group of culture chambers B12 includes five sub-culture chambers arranged at equal intervals, each of which is in spaced communication with the sub-flow path B12-2 through the sub-flow path B12-1. The second group of culture chambers B7 also comprises five sub-culture chambers which are arranged at equal intervals, and each sub-culture chamber is communicated with the sub-flow channel B7-2 at intervals through the sub-flow channel B7-1.
It should be noted that, when the plurality of tumor microspheres are organ tumor microspheres, the parallel repetition principle of the experiment can be ensured, and of course, the plurality of tumor microspheres can also be a plurality of organ tumor microspheres so as to realize the co-culture of multiple organs.
Furthermore, when the tumor microsphere flow channel and each flow channel in the independent tumor microsphere flow channel are arranged on different planes of the culture layer, the culture layer is also provided with a fluid switching hole so as to switch the direction of the fluid and enable each flow channel on different surfaces to be communicated.
For example, referring to fig. 2, the outlet flow channel B2-2 of the vascular culture chamber is disposed on the upper surface of the culture layer B, and the micro-mixing flow channel B10 is disposed on the lower surface of the culture layer B, with the first fluid switching hole B9 formed between the two flow channels. When the inlet flow channels B12-2 of the first group of culture chambers B12 are provided on the upper surface of the culture layer B, a second fluid switching hole B11 is also formed between the flow channels and the micro-mixing flow channel B10. When the outlet flow passages B7-2 of the second group of culture chambers B7 are provided on the upper surface of the culture layer B and the independent outlet flow passages B6-1 are provided on the lower surface of the culture layer, a third fluid switching hole B8 is formed between the two flow passages.
In other preferred embodiments, to enhance the ease of addition of the test compound, the artificial blood vessel set further comprises an artificial blood vessel inlet in communication with the artificial blood vessel flow path; the tumor microsphere group also comprises a tumor microsphere introduction port communicated with the tumor microsphere flow channel; the independent tumor microsphere group also comprises independent tumor microsphere inlet communicated with the independent tumor microsphere flow channels so as to introduce the corresponding culture medium through each inlet, and an outlet is also arranged to lead out the culture medium of each flow channel.
Illustratively, as shown in fig. 1 to 4, an artificial blood vessel inlet A3, an artificial blood vessel outlet A4, a tumor microsphere inlet A2, a tumor microsphere outlet A1, an independent tumor microsphere inlet A5, and an independent tumor microsphere outlet A6 are provided on the connection layer a laminated over the culture layer B. Wherein, the artificial blood vessel leading-in port A3 is communicated with the blood vessel inflow port C3 through the artificial blood vessel culturing inlet B3 on the culturing layer B, and the artificial blood vessel leading-out port A4 is communicated with the blood vessel outflow port C4 through the artificial blood vessel culturing inlet B4 on the culturing layer B. And secondly, a tumor microsphere inlet A2 is communicated with a tumor microsphere culture inlet B2, and a tumor microsphere outlet A1 is communicated with a tumor microsphere culture outlet B1. Furthermore, the independent tumor microsphere inlet A5 is communicated with the independent culture inlet B5, and the independent tumor microsphere outlet A6 is communicated with the independent culture outlet B6.
According to the embodiment, the independent inlet and outlet are arranged on each runner, so that the culture medium and the compound to be tested can be conveniently introduced into the corresponding runner, and convenience in obtaining the influence results of different drugs on tumor microspheres and artificial blood vessels is improved.
In another aspect of the present invention, there is provided a method for detecting a compound using the in vitro tumor vascular model described above, comprising the specific steps of:
firstly, introducing culture mediums into each runner to culture or perfuse tumor microspheres and/or artificial blood vessels;
secondly, introducing a compound to be tested into the artificial blood vessel group;
thirdly, obtaining the regulation result of the compound to be tested on tumor microspheres and/or artificial blood vessels.
It should be understood that when the above-described model is used for culturing or perfusion of tumor microspheres, the model is further required to be connected to a culture pipeline, as shown in fig. 5, the culture pipeline comprises a first culture flask 1, a second culture flask 2, a third culture flask 3, and a first culture medium, a second culture medium and a third culture medium which are contained in the respective culture flasks, each culture flask is correspondingly provided with a pump M1, a pump M2 and a pump M3, the first culture flask 1 is communicated with a tumor microsphere runner, the second culture flask 2 is communicated with an artificial blood vessel runner, the third culture flask 3 is communicated with an independent tumor microsphere runner, and each culture medium is led into each runner from each culture flask through the pump on each culture pipeline.
In some preferred embodiments, the detection method specifically includes:
firstly, introducing a first culture medium into a tumor microsphere group so as to culture or perfuse tumor microspheres;
secondly, introducing a second culture medium into the artificial blood vessel group to culture or perfuse the artificial blood vessel, and simultaneously introducing a compound to be tested into the artificial blood vessel group;
thirdly, analyzing the tumor microspheres of the tumor microsphere group to obtain the cell activity and the cell migration result of the tumor microspheres cultured by the artificial blood vessel; and/or analyzing the morphology of the artificial blood vessel to obtain morphology changes of the artificial blood vessel;
based on the cell activity and/or cell migration result of the tumor microsphere, screening the compound to be tested matched with the tumor, and/or based on the morphological change of the artificial blood vessel, obtaining the regulation result of the compound to be tested on the artificial blood vessel.
It should be noted that, in order to ensure that the tumor microsphere has the preset functional characteristics, the first activity analysis can be performed on the artificial blood vessel and the tumor microsphere cultured in the second culture medium without the compound to be tested, so as to ensure that the vascular endothelial cells are closely arranged and have directionality, and the tumor microsphere has good functional characteristics. And then, performing secondary activity analysis on the tumor microspheres and the artificial blood vessels of the second culture medium added with the compound to be tested to obtain the regulation results of the compound to be tested on tumors and blood vessels.
It will be appreciated that, as a result of the addition of the test compound to the second medium of this example, drug exchange occurs in the vascular culture chamber, drug acts on the tumor microspheres with the first medium, tumor cell activity decreases after administration, and tumor cells solidify or scatter after administration, as well as vascular morphology changes after administration.
In this embodiment, appropriate drugs for tumor cells of different organs can be selected based on the cell activity and cell migration results of the tumor microsphere set, and the effect of the anti-tumor drugs on the artificial blood vessel can be obtained, for example, the effect of the anti-tumor drugs on the blood vessel has vasculitis, hypertension, thrombosis, etc.
In other preferred embodiments, the detection method specifically further comprises:
firstly, introducing a third culture medium into the independent tumor microsphere group to culture or perfuse the independent tumor microspheres;
secondly, analyzing the tumor microspheres in the independent tumor microsphere group to obtain the cell activity and the cell migration result of the independent tumor microspheres;
based on the cell activity and/or cell migration results of the tumor microspheres and the independent tumor microspheres, the regulation result of the test compound on the tumor microspheres after artificial blood vessel absorption is obtained.
It should be understood that, since no drug exchange occurs in this example, no drug acts on the individual tumor microspheres, their cell activity increases, and the individual tumor microspheres proliferate normally, the tumor cell diameter increases or the tumor cells migrate, forming a control group with the tumor microsphere group described above, to obtain the effect of the test compound on the tumor.
The invention simulates interaction of human blood vessels and single organ tumor microspheres or multi-organ tumor microspheres in vitro, establishes a tumor blood vessel model, can more truly reflect the action effect of a compound to be tested in human body, realizes vascular absorption of medicines and nutrients and microenvironment construction of tumor metastasis invasion, and can be used for screening tumor medicines and obtaining the effect result of the tumor medicines on vascular toxicity through analyzing the tumor microspheres.
The in vitro tumor vascular model and its specific applications will be further described in conjunction with the specific examples below:
example 1
The present example illustrates a method for detecting a compound using an in vitro tumor vascular model, comprising the steps of:
s1, obtaining a model with an artificial blood vessel group, a tumor microsphere group and an independent tumor microsphere group.
S2, culturing the artificial blood vessel by using Huvec endothelial cells until the artificial blood vessel has functional characteristics, such as compact arrangement and directionality of the endothelial cells.
S3, preparing 3D tumor microspheres by using human lung cancer cells NCI-H23, and culturing until the NCI-H23 tumor microspheres show functional characteristics.
S4, under the aseptic culture environment, taking the sterilized model and the cultured artificial blood vessel, placing the cultured artificial blood vessel into a blood vessel culture chamber, and inserting the artificial blood vessel into a fixing block and a connecting pipe to fix the artificial blood vessel.
S5, in a sterile culture environment, the cultured NCI-H23 tumor microspheres are sequentially placed in a first group of culture chambers and a second group of culture chambers.
In this example, lung cancer cell NCI-H23 is taken as an example, and it is needless to say that tumor cells of other organs may be selected and cultured in different subculture chambers.
S6, sealing and connecting the connecting layer and the culture layer by using a double-sided adhesive tape without biological toxicity to form a closed artificial blood vessel inner runner, a closed tumor microsphere runner and a closed independent tumor microsphere runner.
And in a sterile environment, connecting the assembled model into a culture pipeline, and sterilizing the model and the culture pipeline by adopting ethylene oxide to form a culture system.
As shown in fig. 5, the first medium in the first culture flask 1 is connected to the tumor microsphere flow path via a pump M1, the second medium in the second culture flask 2 is connected to the artificial blood vessel flow path via a pump M2, and the third medium in the third culture flask 3 is connected to the independent tumor microsphere flow path via a pump M3.
S7, respectively placing 15mL of corresponding culture mediums into each culture bottle (1/2/3), starting a culture system, and placing the model and the culture pipelines into a sterile incubator at 37 ℃ after each culture medium is filled with the corresponding culture chamber and forms a flowing perfusion loop; and (3) continuously pouring and culturing for 24 hours, taking out the model together with a culture pipeline, and performing activity analysis on the artificial blood vessels and tumor microspheres in the model by using a high content image analysis system to ensure that the artificial blood vessels and the tumor microspheres have good functional characteristics, the vascular endothelium is closely arranged, the tumor microspheres have certain directivity after continuous pouring and culturing, the middle color of the tumor microspheres is deep, the edges are shallow and the contours are clear, and a tumor microsphere image acquired by the high content image analysis system is shown in figure 6.
Further, in a sterile environment, replacing a second culture medium corresponding to the artificial blood vessel with a culture medium of doxorubicin (Dox), placing the model together with a culture system into a sterile incubator at 37 ℃, and continuously culturing and perfusing for 10 days to obtain the tumor microsphere with the blood vessel function.
Further, the obtained tumor microspheres were further analyzed by using a high content system, as shown in fig. 7 and 8, and after the above-mentioned continuous culture, perfusion culture was performed on days 1, 3, 5, 7 and 10, respectively, the activity of the 5 NCI-H23 tumor microspheres in the 5 subculture chambers of the first group of culture chambers B12 in the drug test zone (corresponding to Dox-1 in fig. 7) and the 5 subculture chambers of the second group of culture chambers B7 in the independent control zone (corresponding to Dox-2 in fig. 7) was reduced to 20% of the activity of the 5 NCI-H23 tumor microspheres in the second group of culture chambers B7 after the drug action was found, which is consistent with the effect of doxorubicin on human lung cancer cells NCI-H23, which means that the doxorubicin can affect the tumor microspheres after absorption, filtration, and the like of the artificial blood vessel, and the toxicity of the human lung cancer cells NCI-H23 were generated.
In the same manner as described above, docetaxel at a concentration of 40umol/L was also used for the test, and the experimental data obtained in the same manner are shown in FIG. 8, which illustrates that docetaxel (corresponding to DMSO in FIG. 8) can exert a toxic effect on NCI-H23 lung cancer cells in the model of the present invention.
The invention provides an in-vitro tumor blood vessel model and a method for detecting a compound by using the in-vitro tumor blood vessel model, which have the following beneficial effects:
firstly, the invention establishes a tumor blood vessel model in vitro, realizes the absorption of medicines and nutrient substances through blood vessels and the construction of microenvironment for tumor metastasis invasion, and has the functions of blood circulation, blood vessel filtration and the like;
secondly, the model of the invention can realize multi-organ co-culture and reflect the complexity, functional change and integrity of the actual organism organ functions;
thirdly, the in-vitro tumor vascular model of the invention has the effects on tumors and/or directly on tumors after the drugs and nutrient substances are absorbed by blood vessels, so that more accurate, efficient and convenient test of anti-tumor drugs is realized; on the other hand, the method can realize accurate, efficient and convenient test of the influence of the anti-tumor drug on the blood vessel (for example, the anti-tumor drug can influence the blood vessel to cause vascular related diseases such as vasculitis, hypertension, thrombosis and the like).
It is to be understood that the above embodiments are merely illustrative of the application of the principles of the present invention, but not in limitation thereof. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the invention, and are also considered to be within the scope of the invention.
Claims (10)
1. An in vitro tumor vascular model comprising:
artificial blood vessel group, and
a group of tumor microspheres, which are prepared from the following materials,
the artificial blood vessel group comprises an artificial blood vessel, an artificial blood vessel inner runner for independently culturing and/or perfusion of the inside of the artificial blood vessel, and an artificial blood vessel outer runner for culturing and/or perfusion of the outside of the artificial blood vessel;
the tumor microsphere set comprises single or a plurality of tumor microspheres and a tumor microsphere runner for culturing and/or perfusing the single or the plurality of tumor microspheres, and the tumor microsphere runner is connected with the artificial blood vessel outflow runner.
2. The model of claim 1, wherein the vascular prosthesis comprises an endothelial layer and a smooth muscle layer; and/or the number of the groups of groups,
the width of the artificial blood vessel ranges from 2mm to 40mm.
3. The model of claim 1, wherein the in vitro tumor vascular model has a length in the range of 10mm to 50mm and a width in the range of 5mm to 40mm.
4. The model of claim 1, wherein the tumor microsphere diameter of the single or plurality of tumor microspheres is in the range of 900 μιη to 1300 μιη.
5. The model of claim 1, wherein the in vitro tumor vascular model further comprises a set of individual tumor microspheres,
the independent tumor microsphere group comprises single or a plurality of independent tumor microspheres and independent tumor microsphere flow channels for independently culturing and/or perfusing the single or the plurality of independent tumor microspheres.
6. The model of claim 5, wherein the individual tumor microspheres are symmetrically disposed with respect to the tumor microspheres.
7. The model of claim 1, wherein the artificial blood vessel set further comprises an artificial blood vessel inlet in communication with the artificial blood vessel flow path;
the tumor microsphere group also comprises a tumor microsphere introduction port communicated with the tumor microsphere flow channel;
the independent tumor microsphere group also comprises an independent tumor microsphere introduction port communicated with the independent tumor microsphere flow passage.
8. A method for detecting a compound using the in vitro tumor vascular model according to any one of claims 1 to 7, comprising the specific steps of:
introducing each culture medium into each flow channel to culture or perfuse tumor microspheres and/or artificial blood vessels;
introducing a test compound into the vascular prosthesis;
and obtaining the regulation result of the compound to be tested on the tumor microspheres and/or the artificial blood vessels.
9. The method of claim 8, wherein the obtaining of the modulation of tumor microspheres and/or vascular prostheses by the test compound comprises:
obtaining the cell activity and/or cell migration result of the tumor microsphere cultured by the artificial blood vessel; and/or the number of the groups of groups,
obtaining the shape of an artificial blood vessel;
screening out a compound to be tested matched with the tumor based on the cell activity and/or cell migration result of the tumor microsphere; and/or the number of the groups of groups,
and based on the morphology of the artificial blood vessel, obtaining the regulation result of the compound to be tested on the artificial blood vessel.
10. The method of claim 9, wherein the obtaining of the modulation of tumor microspheres by the test compound further comprises:
obtaining the cell activity and/or cell migration result of the independent tumor microspheres;
based on the cell activity and/or cell migration results of the independent tumor microspheres and the tumor microspheres, the regulation result of the test compound on the tumor microspheres after artificial blood vessel absorption is obtained.
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