CN114591836A

CN114591836A - In-vitro vascular bed micro-fluidic chip and application thereof

Info

Publication number: CN114591836A
Application number: CN202210152808.6A
Authority: CN
Inventors: 马少华; 胡志伟; 曹远雄; 埃德加·艾力克斯; 赵浩然
Original assignee: Shenzhen International Graduate School of Tsinghua University
Current assignee: Shenzhen International Graduate School of Tsinghua University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-06-07

Abstract

The invention discloses an in vitro vascular bed micro-fluidic chip and application thereof, wherein the micro-fluidic chip comprises: the flow regulating device comprises a first flow passage, a second flow passage, a third flow passage and a fourth flow passage, wherein first flow limiting parts are arranged between the second flow passage and the first flow passage and between the third flow passage and the first flow passage; the first flow restriction portion is configured to allow a cell culture medium injected into the second flow channel and the third flow channel to flow through the vascular bed culture chamber while restricting the endothelial cell gel in the vascular bed culture chamber; a second flow restriction is disposed between the fourth flow channel and the second flow channel, the second flow restriction being configured to restrict the fibroblasts in the fourth flow channel while allowing secretions of the fibroblasts to diffuse into the second flow channel. The density of blood vessels in the formed vascular bed is high, the functional expression factor of the blood vessels is up-regulated, and the thickness of the periphery of the large blood vessel with the middle thickness of the vascular bed is thinner. Compared with the traditional single blood vessel, the chip can better simulate the structure of the blood vessel network when being used for carrying out blood vessel culture, and has higher quality.

Description

In-vitro vascular bed micro-fluidic chip and application thereof

Technical Field

The application relates to the technical field of tissue engineering, in particular to an in-vitro vascular bed microfluidic chip and application thereof.

Background

At present, the technology of the vascularization micro-fluidic chip is developed rapidly, and various means are developed, including vascularization organoid chips under the stimulation of fluid, 3D printing vascularization micro-fluidic chips, single-layer 3D blood vessel network micro-fluidic chips, micro-tissue and blood vessel co-culture micro-fluidic chips and the like. The technology of vascularizing organoid chips under fluid stimulation is to provide dynamic culture environment for organoids such as those derived from human pluripotent stem cells by using microfluidic chips, so as to generate a vascular network. However, the vascular network formed in this way is unevenly distributed and highly random, and it is difficult to simulate the transport of substances through blood vessels into organoids. The 3D printing technology is a common strategy for manufacturing the vascularized microfluidic chip at present, and the method can be used for preparing the vascularized in-vitro model of thick tissues and presenting certain vascular structure and functions. However, the structure and function of the biological ink used in the method cannot meet the requirements of cell growth and differentiation, so that the printed blood vessel network only has partial functional structure and cannot effectively play the functions of blood vessel endogenesis and tissue growth and development promotion. Meanwhile, incubation times of more than 40 days are generally required due to the printing material. In contrast, although the single-layer 3D vascular network microfluidic chip can culture a single-layer vascular network with a good structural function within one week, due to the size limitation, such vascular chip models can only culture tissues with a size of about 100 μm, and cannot be used for long-term culture of larger tissues. The micro-fluidic chip for co-culturing the micro-tissue and the blood vessel utilizes a micro-fluidic chip platform to provide dynamic fluid shear force in a mode of mixed culture of tissue cells and endothelial cells, thereby constructing a vascularized organoid culture model. The method successfully constructs a vascularized in-vitro tissue model, and can effectively realize the function of vascular substance transportation. However, the size of the micro-tissue in the scheme is only about 200 μm, and the growth process of the micro-tissue is not uniform, so that the larger-scale and more uniform vascularization culture cannot be met. Meanwhile, the culture operation is complex, the cost is high, and the universality is not realized. In addition, the practices of the four prior arts find that the vascular network structure constructed by the chips is intermittent and poor in quality, and the simulation degree needs to be improved.

Disclosure of Invention

The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides an in vitro vascular bed microfluidic chip capable of constructing a more complex vascular model and application thereof. The microfluidic chip can be used for culturing a vascular network with a larger size and constructing a more complex vascular model so as to effectively simulate the nutrition supply of the vascular network in vivo to tissues.

In a first aspect of the present application, there is provided a microfluidic chip comprising:

a first flow channel having a vascular bed culture chamber for containing endothelial cell gel;

the first flow channel and the second flow channel are respectively arranged on two sides of the first flow channel, and first flow limiting parts are arranged between the first flow channel and the second flow channel and between the first flow channel and the third flow channel; the first flow restriction portion is configured to allow a cell culture medium injected into the second flow channel and the third flow channel to flow through the vascular bed culture chamber while restricting the endothelial cell gel in the vascular bed culture chamber;

the fourth flow channel is positioned on one side, far away from the first flow channel, of the second flow channel, a second flow limiting part is arranged between the fourth flow channel and the second flow channel, and the fourth flow channel is used for culturing fibroblasts; the second flow restriction is configured to restrict the fibroblasts in the fourth flow channel while allowing secretions of the fibroblasts to diffuse into the second flow channel.

The microfluidic chip according to the embodiment of the application has at least the following beneficial effects:

(1) when the micro-fluidic system is adopted for culture, at the beginning stage of culture, a vascular network in a vascular bed culture chamber is not formed, a cell culture medium in the second flow channel flows to the third flow channel through the tiny gaps of endothelial cell gel to form gap flow, and the mechanical force generated in the process can further stimulate the vascularization process of endothelial cells. After the vascular network is formed, the cell culture medium flows from one side to the other side through the 3D vascular cavity, and the shearing force formed in the vascular cavity continuously stimulates the division and proliferation of endothelial cells to form a richer and more bionic vascular network.

(2) Through setting up solitary vascular bed culture chamber and fourth runner in this scheme, separate culture fibroblast and endothelial cell, make the multiple bioactive factor that fibroblast paracrine effect produced diffuse to vascular bed culture chamber through the cell culture medium in, when accelerating vascular network maturity, when having avoided both mixed culture, the blood vessel can lead to the problem that vascular structure is intermittent and continuous because of fibroblast's excessive growth to form complicated intensive vascular network.

(3) Through the combination of the two modes, the finally formed blood vessels in the vascular bed have high density, the functional expression factors of the blood vessels are up-regulated, the middle thickness of the vascular bed can reach 500-1000 microns, and the peripheral thickness of the blood vessels is thinner only about 100 microns. And the thickness of the blood vessel can be dynamically adjusted and changed in a gradient manner according to the adjustment of a culture medium and the like in the culture process, compared with the condition that the thickness of the traditional single blood vessel is low and incomplete, the blood vessel network structure can be better simulated by utilizing the chip to culture the blood vessel, and the quality is higher.

In some embodiments of the present application, the vascular bed culture chamber is provided with an upwardly open culture well. Open culture wells are provided in the vascular bed culture chamber so that additional microtissue can be introduced for co-culture after the formation of a separate dynamic vascular bed, thereby building a more complex dynamic model system.

In some embodiments of the present application, the depth of the culture well is 1 to 3mm, and further 2 to 3 mm. The middle thickness of the formed vascular bed can be further adjusted by controlling the depth of the culture hole, so that a more effective vascular bed-blood vessel peripheral structure with thick middle and thin edge is achieved.

In some embodiments of the present application, the first flow restriction and the second flow restriction comprise a plurality of flow restricting pillars arranged at intervals. The endothelial cell gel is ensured to be limited in the vascular bed culture chamber through the spacing current limiting columns, and the cell culture medium can be circulated in the vascular bed culture chamber to form gap flow.

In some embodiments of the present application, the device further includes a fifth flow channel, the fifth flow channel is located on a side of the third flow channel away from the first flow channel, a third flow restriction portion is disposed between the fifth flow channel and the third flow channel, and the fifth flow channel is used for culturing fibroblasts; the third flow restriction is configured to be able to restrict the fibroblasts in the fifth flow channel while allowing secretions of the fibroblasts to diffuse into the third flow channel. By arranging the fourth flow channel and the fifth flow channel which are opposite to each other on two sides, the paracrine of the fibroblast is introduced into the second flow channel and the third flow channel, so that the stimulation effect on the vascular bed network is improved, and the maturation of the vascular bed network is further accelerated.

In some embodiments of the present application, the third flow restriction comprises a plurality of flow restricting pillars arranged at intervals.

In a second aspect of the present application, there is provided a method for performing in vitro vascular bed culture by using the aforementioned microfluidic chip, comprising the following steps:

injecting the endothelial cell suspension into a vascular bed culture chamber, and solidifying to form endothelial cell gel;

injecting fibroblast suspension into the fourth flow channel, and fixing to form fibroblast gel;

respectively injecting different amounts of cell culture medium into the second flow channel and the third flow channel, and enabling the liquid level of the second flow channel to be higher than that of the third flow channel, so that the cell culture medium flows from the second flow channel to the third flow channel through the vascular bed culture chamber under the action of gravity, a gap flow for stimulating endothelial cell gel is formed, and the vascular bed is obtained through culture.

In some embodiments of the present application, the incubation time to form the vascular bed is 3-8 days.

In some embodiments of the present application, the manner of immobilization of the endothelial cell gel and the fibroblast gel is at least one of enzymatic immobilization or thermal immobilization.

It will be appreciated that the gel stock components used in the endothelial and fibroblast gels may be any one or more of the hydrogel components well known in the art, and may be specifically selected according to the particular vascular bed culture requirements.

In some embodiments of the present application, the endothelial cell suspension and the fibroblast cell suspension each independently comprise at least one of fibrin, collagen, matrigel. After the cell suspension containing the components is adopted to form the gel, the vascular bed can be ensured to be capable of efficiently supplying nutrient substances after the gel is formed, and the vascular bed with high density and high thickness can be formed.

In a third aspect of the present application, a vascular bed model cultured according to the aforementioned method is provided. The model includes a vascular bed and peripheral structures of the blood vessel.

In some specific embodiments, the vascular bed further comprises a micro-tissue located on the vascular bed.

In some specific embodiments, the microtissue comprises at least one of organoids, primary tissue, cell pellets, cell suspensions.

In a fourth aspect of the present application, there is provided the use of the aforementioned vascular bed model in tissue culture, drug screening, drug discovery.

In conclusion, the microfluidic chip provided by the embodiment of the application can construct a vascular bed model in a short time, and further can be rapidly allowed to be co-cultured with a micro-tissue. After the vascular bed is formed, the open vascular model is allowed to be co-cultured with various different micro-tissues, so that various different vascularized in-vitro micro-tissue models are formed, and the opening degree is higher. In addition, the model is applicable to various functional materials such as fibrin, collagen, matrigel and the like. Based on an in-vitro vascularized micro-tissue model, the invention can be applied to the fields of tissue culture, drug screening, drug discovery, immunotherapy research and other multiple production and study. The vascular structure at the periphery of the blood vessel can effectively simulate the vascular network in the normal tissue structure, and the more complex and dense network in the vascular bed can effectively simulate the vascular network rich in the biological tissue in the body. Meanwhile, the vascular structure in the periphery of the blood vessel is tightly connected with the vascular bed to form a dynamic substance exchange transportation network, thereby effectively simulating the substance transportation condition of the vascular network in vivo. In addition, different gels can be selected according to actual requirements in the using process, and the vascular network model with specific structural functions can be obtained in a customized mode.

Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.

Drawings

Fig. 1 is a schematic diagram of the structure of a microfluidic chip and the culture process and culture results in an embodiment of the present application.

Fig. 2 is a partial schematic view of a microfluidic chip according to an embodiment of the present disclosure, with a top view and a bottom view in front view.

Fig. 3 is an experimental result of the vascular bed cultured in example 1 of the present application.

Fig. 4 is a schematic structural diagram of the vascularized tumor microspheres prepared in the examples of the present application and a 3D Z axial scan image of the corresponding vascular bed and the periphery of the blood vessels.

FIG. 5 shows the results of an experiment for measuring the transport ability of a substance in example 2 of the present application.

Fig. 6 is an experimental result of a drug screening experiment in example 3 of the present application.

Reference numerals: the culture device comprises a first flow channel 100, a blood vessel bed culture chamber 110, a culture hole 111, a first flow channel first liquid storage tank 120, a first flow channel second liquid storage tank 130, a second flow channel 200, a second flow channel first liquid storage tank 210, a second flow channel second liquid storage tank 220, a third flow channel 300, a third flow channel first liquid storage tank 310, a third flow channel second liquid storage tank 320, a fourth flow channel 400, a fourth flow channel first liquid storage tank 410, a fourth flow channel second liquid storage tank 420, a fifth flow channel 500, a fifth flow channel first liquid storage tank 510, a fifth flow channel second liquid storage tank 520, a first flow limiting part 600, a second flow limiting part 710 and a third flow limiting part 720.

Detailed Description

The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.

The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.

In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Referring to fig. 1, a microfluidic chip for in vitro vascular bed culture provided in an embodiment of the present application is shown, including a first flow channel 100, a second flow channel 200, a third flow channel 300, and a fourth flow channel 400. The first channel 100 has a vascular bed culture chamber 110 for containing endothelial cell gel therein, the second channel 200 and the third channel 300 are respectively located at both sides of the first channel 100, and first flow restrictions 600 are provided between the first channel 100 and the second channel 200 and between the first channel 100 and the third channel 300. The fourth flow channel 400 is located on a side of the second flow channel 200 away from the first flow channel 100, and a second flow restriction 710 is disposed between the fourth flow channel 400 and the second flow channel 200.

Wherein the vascular bed culture chamber 100 of the first flow channel 100 is used for culturing endothelial cells, the second flow channel 200 and the third flow channel 300 are used for containing cell culture medium, and the first flow restriction part 600 between the two and the first flow channel 100 can circulate the cell culture medium injected into the second flow channel 200 and the third flow channel 300 through the vascular bed culture chamber 110, and can restrict the endothelial cell gel in the vascular bed culture chamber 100 from flowing into the second flow channel 200 or the third flow channel 300. The fourth flow channel 400 is used for culturing fibroblasts, and the second flow restriction part 710 disposed therebetween can restrict the fibroblasts in the fourth flow channel 400, and simultaneously allow secretions of the fibroblasts to diffuse into the second flow channel 200 and further enter the vascular bed culture chamber 110 during the circulation of the cell culture medium in the second flow channel 200 so as to promote the maturation of the vascular bed.

At the beginning of the culture, the vascular network in the vascular bed culture chamber 110 is not formed, and the cell culture medium in the second flow channel 200 flows to the third flow channel 300 through the tiny slits of the endothelial cell gel, forming a gap flow, and the mechanical force generated in the process further stimulates the vascularization process of the endothelial cells. After the vascular network is formed, the cell culture medium flows from one side to the other side through the 3D vascular cavity, and the shearing force formed in the vascular cavity continuously stimulates the division and proliferation of endothelial cells to form a richer and more bionic vascular network. Through the arrangement of the independent vascular bed culture chamber 110 and the fourth flow channel 400, fibroblasts and endothelial cells are separately cultured at different positions, so that a plurality of bioactive factors generated by the paracrine action of the fibroblasts are diffused into the vascular bed culture chamber 110 through a cell culture medium, the maturation of the vascular network is accelerated, and simultaneously, the problem that the vascular structure is discontinuous due to the overgrowth of the fibroblasts in the mixed culture of the two is avoided, and a complex and dense vascular network is formed. The finally formed blood vessels in the vascular bed have high density, the functional expression factors of the blood vessels are up-regulated, the middle thickness of the vascular bed is large, and the peripheral thickness of the blood vessels is thinner. And the thickness of the blood vessel can be dynamically adjusted and changed in a gradient manner according to the adjustment of a culture medium and the like in the culture process, compared with the condition that the thickness of the traditional single blood vessel is low and incomplete, the blood vessel network structure can be better simulated by utilizing the chip to culture the blood vessel, and the quality is higher.

Referring to fig. 1, in some embodiments, vascular bed culture chamber 110 is provided with an upwardly open culture well 111. By providing the culture well 111 with an opening in the vascular bed culture chamber 110, it is possible to construct a more complex dynamic model system by introducing additional micro-tissues from the culture well 111 for co-culture after culturing to form a single dynamic vascular bed. Specific types of micro-tissues include, but are not limited to, organoids, primary tissues, cell clusters, cell suspensions, etc., for different co-culture requirements. During the culture of the vascular bed in the vascular bed culture chamber 110 with the culture holes 111, the middle thickness of the vascular bed in the vascular bed culture chamber can be further increased, and endothelial cells can grow into the micro-tissue through co-culture to form a vascularized dynamic micro-tissue system.

Referring to fig. 2, a schematic diagram of a portion of a microfluidic chip according to some embodiments of the present application is shown, in which the top view is a top view and the bottom view is a front sectional view. In some specific embodiments, in conjunction with fig. 2 and 1, the first flow restriction 600 and the second flow restriction 710 include a plurality of spaced flow restriction columns. The endothelial cell gel is ensured to be limited in the vascular bed culture chamber through the spacing current limiting columns, and the cell culture medium can be circulated in the vascular bed culture chamber to form gap flow.

In some specific embodiments, the device further comprises a fifth flow channel 500, the fifth flow channel 500 is located on a side of the third flow channel 300 away from the first flow channel 100, a third flow restriction portion 720 is provided between the fifth flow channel 500 and the third flow channel 300, and the fifth flow channel 500 is also used for culturing fibroblasts; the third flow restriction 720 is configured to be able to restrict the fibroblasts in the fifth flow channel 500 while allowing the secretions of the fibroblasts to diffuse into the third flow channel 300. By arranging the fourth flow channel 400 and the fifth flow channel 500 which are opposite to each other on two sides, the second flow channel 200 and the third flow channel 300 are introduced with the paracrine of the fibroblast, thereby improving the stimulation effect on the vascular bed network and further accelerating the maturation of the vascular bed network. It will be appreciated that the third flow restriction may also comprise a plurality of spaced flow restricting posts.

In some specific embodiments, a first channel first reservoir 120, a first channel second reservoir 130, a second channel first reservoir 210, a second channel second reservoir 220, a third channel first reservoir 310, a third channel second reservoir 320, a fourth channel first reservoir 410, a fourth channel second reservoir 420, a fifth channel first reservoir 510, and a fifth channel second reservoir 520 are respectively disposed at two ends of the first channel 100, the second channel 200, the third channel 300, the fourth channel 400, and the fifth channel 500, respectively, and are used for introducing or removing a culture medium, a suspension, and the like into or from the corresponding channels.

It can be understood that the lengths, widths, and thicknesses of the first flow channel 100, the second flow channel 200, the third flow channel 300, the fourth flow channel 400, and the fifth flow channel 500 in the microfluidic chip, and the widths, spacings, and the like of the flow-limiting pillars in the first flow-limiting part 600, the second flow-limiting part 710, and the third flow-limiting part 720 can be adaptively adjusted according to actual culture needs.

In some specific embodiments, the width of the vascular bed culture chamber in the first flow channel 100 is 2-5 mm, the width of the second flow channel 200 and the third flow channel 300 is 0.5-2 mm, and the width of the fourth flow channel 400 and the fifth flow channel 500 is 0.5-1 mm.

In some embodiments, the heights of the first flow channel 100, the second flow channel 200, the third flow channel 300, the fourth flow channel 400, and the fifth flow channel 500 are 50 to 200 μm.

In some specific embodiments, the culture wells are circular, have a diameter of 1-3 mm, and have a depth of 2-3 mm.

In some embodiments, the width of the current-limiting pillars is 100-500 μm, and the pitch of the current-limiting pillars is 50-200 μm.

In a second aspect of the present application, there is provided a method for performing in vitro vascular bed culture by using the microfluidic chip, the method comprising the following steps:

In some specific embodiments, the vascular bed is formed in a culture period of 3 to 8 days. In some specific embodiments, the endothelial cell gel and the fibroblast gel are immobilized by at least one of enzymatic or thermal immobilization. In some specific embodiments, the endothelial cell suspension and the fibroblast cell suspension each independently comprise at least one of fibrin, collagen, and matrigel. The cell suspension containing the components can ensure that the vascular bed can efficiently supply nutrient substances after gel is formed, and is favorable for forming the vascular bed with high density and high thickness.

In some specific embodiments, the endothelial cells include, but are not limited to, human umbilical vein endothelial cells, vascular endothelial cells, lymphatic endothelial cells, and the like.

The embodiment of the application also provides a vascular bed model obtained by culturing according to the method and application of the vascular bed model in tissue culture, drug screening and drug discovery.

The embodiments of the present application will be described below with reference to specific examples.

Example 1

The embodiment provides a microfluidic chip, which is shown in fig. 1 and includes a first flow channel 100, a second flow channel 200, a third flow channel 300, a fourth flow channel 400 and a fifth flow channel 500, wherein a vascular bed culture chamber 110 is disposed in the first flow channel and is communicated with the second flow channel 200 and the third flow channel 300 through a plurality of flow limiting columns on two sides, and the second flow channel 200 and the third flow channel 300 are respectively communicated with the fourth flow channel 400 and the fifth flow channel 500 through a plurality of flow limiting columns. The flow passages are provided at both ends thereof with a pair of liquid reservoirs, respectively. A culture hole 111 is also provided in the middle of the vascular bed culture chamber 110.

In this example, the width of the vascular bed culture chamber 110 was 2.5mm, the widths of the second flow channel 200 and the third flow channel 300 were 1mm, the widths of the fourth flow channel 400 and the fifth flow channel 500 were 800 μm, and the heights of the first flow channel, the second flow channel 200, and the third flow channel 300 were 100 μm. The current-limiting pillars of the first, second, and third current-limiting

parts

600, 710, and 730 are triangular prisms having a width of 300 μm, a length of 200 μm, and a pitch of 100 μm. The culture hole 111 is circular, and has a diameter of 2mm and a depth of 2-3 mm.

The present embodiment also provides a method for culturing a vascular bed, which includes the following steps:

1) cell gel suspension injection: uniformly mixing endothelial cell (HUVECs) suspension with hydrogel, and injecting the mixture into the microfluidic chip through liquid storage tanks at two ends of the first flow channel; and in the same way, the fibroblast suspension and the hydrogel are uniformly mixed and injected into the microfluidic chip through the liquid storage tanks at the two ends of the fourth flow channel and the fifth flow channel, the two cell gel suspensions can be effectively limited in the respective flow channel areas by the limiting columns designed in the flow channels, and the cell gel is solidified and formed under the action of enzyme reaction to obtain the endothelial cell gel positioned in the vascular bed culture chamber and the fibroblast gel positioned in the fourth flow channel and the fifth flow channel.

2) Introducing a culture medium: and introducing the cell culture medium into the second flow channel and the third flow channel through liquid storage tanks at two ends of the second flow channel and the third flow channel so that the cell culture medium can be fully contacted with the cell gel solidified at two sides.

3) Construction of dynamic culture environment: and controlling the liquid level height of the cell culture medium in the second flow channel to be higher than that of the cell culture medium in the third flow channel to form a liquid level difference, and enabling the culture medium to flow to the third flow channel through the blood vessel bed culture chamber through the second flow channel by utilizing the gravity principle to construct a dynamic culture environment. At the beginning, a vascular network is not formed, and the cell culture medium flows from the side with the high liquid level to the side with the low liquid level through the tiny slits of the hydrogel to form interstitial flow. The mechanical forces generated at this point further stimulate the vascularization process of the endothelial cells. After the vascular network is formed, the culture medium flows from one side to the other side through the 3D vascular cavity, and the shearing force formed by the fluid in the vascular cavity continuously stimulates the division and proliferation of endothelial cells to form a richer and more bionic vascular network.

4) And (3) carrying out micro-tissue vascularization co-culture: the micro-fluidic chip is cultured for about 5 days to form a dynamic blood vessel network. Subsequently, the microtissue (Eca-109 cell microspheres) was added to the vascular bed via culture wells. Vascular endothelial cells in the vascular bed can grow into the microtissue through coculture to form a vascularized dynamic microtissue system.

Referring to fig. 1, there is also shown a block diagram of a vascular bed model formed according to the method, including a centrally located vascular bed and peripherally located vascular structures.

FIG. 3 shows the specific results of the vascular bed prepared in this example, wherein A represents the culture conditions of the Vascular Periphery (VP) and the Vascular Bed (VB) on

days

1, 3 and 5. B is the quantification of the vessel diameter in the VP and VB regions at different culture times. C is the quantification of the fluorescence area of VP and VB regions. D is a 3D structural map of the vascular bed and the vessel peripheral junction, with the arrows marking the 3D vascular structures on the vascular bed where the vessels grow upward. E is the result of immunostaining of the VP and VB junction with CD31 fluorescent dye and was amplified in 1(VP), 2(VP and VB junction) and 3(VB) of F. It can be seen that the protein expression level of VB is obviously higher than 1, which indicates that the vascular structure and permeability in the vascular bed are superior to the periphery of the blood vessel. Also, 2 showed high CD31 expression at the VB and VP junctions, indicating frequent mass transport around the blood vessels and in the vascular bed. G is the quantification of the area of the CD31 fluorescent protein in VB and VP, and it can be seen that there is a significant difference in the expression of the two (p < 0.05). H is a schematic diagram of the tumor microspheres planted on the vascular bed after 10 days of culture and the fluorescence of different cells after slicing, and it can be found from the diagram that the red fluorescence of endothelial cells appears in the green fluorescence of the tumor microspheres, which indicates that blood vessels have endogenously entered the tumor microspheres.

Fig. 4 is a schematic structural view of the vascularized tumor micro-tissue prepared in the examples of the present application and a 3D Z axial scan image of the corresponding vascular bed and the periphery of the blood vessels. Referring to fig. 4 in conjunction with fig. 1, the vascularized tumor microtissue includes upper tumor microspheres (microtissue) and lower vascular network structure including a vascular bed in the middle of the vascular bed culture chamber and peripheral blood vessel periphery. From the scanning results in the figures, whether the vascular bed or the periphery of the blood vessel, the blood vessel network part constructed by the embodiment of the application can meet the requirement of complex, dense and continuous integrity, and can not cause the interruption and incompleteness of the vascular structure caused by the overgrowth of the fibroblasts due to the mixed culture of the endothelial cells and the fibroblasts in the prior art.

In the vascular bed model constructed in the embodiment, the single-layer 3D vascular network at the periphery of the blood vessel can effectively simulate the structure of normal vascular tissue, and the culture holes above the vascular bed can allow the planting and co-culture of micro-tissues with the thickness of more than 500 micrometers, so that the vascularization of the micro-tissues is realized. The vascular bed tissue and the monolayer 3D vascular network at the periphery of the blood vessel have close material transportation and exchange, and the nutrition supply of the vascular network in vivo to the tissue is effectively simulated. In addition, the vascularization chip has simple manufacturing process, can form a blood vessel network with a good functional structure within 5 days, and allows the co-culture with various micro tissues. Therefore, the vascularization chip can be widely used for constructing an in vitro vascularization dynamic culture model, thereby providing a more bionic in vitro model.

Example 2: substance transport capacity detection

Constructing a tumor microtissue-vascular bed model according to the method provided in example 1, and setting an experimental group (+ DMOG) and a control group (-DMOG), wherein the experimental group is added with Dimethyloxalglycine (DMOG) in a cell culture medium, the control group is not added with DMOG, the two groups are repeated three times, and blue fluorescent beads with a diameter of 1 μm are added in the cell culture medium after 10 days of culture, and the result is shown in fig. 5, wherein a is an image of tumor microspheres of the experimental group and the control group after 30 minutes of fluorescent bead perfusion, and the scale bars above and below the same group are 100 μm (20 times) and 50 μm (60 times), respectively; b and C are the quantification of fluorescent beads deposited in tumor microspheres and blood vessels, respectively. Combining the results of a-C of fig. 5, the blue fluorescence in the tumor microspheres in the experimental group was much higher than the blue fluorescence in the tumor microspheres in the control group, indicating that DMOG can effectively increase the number of fluorescent microbeads transported to the tumor spheres via the blood vessels.

Example 3: drug screening test

The vascularized tumor microspheres on the microfluidic chip treated with DMOG of example 2 were used to test the sensitivity of tumor cells to paclitaxel and cisplatin under different conditions, with three replicates in each group. The results are shown in FIG. 6, where A is the fluorescence staining results after treatment of the vascularized tumor microspheres with paclitaxel (8.5 μ M) or cisplatin (7.7 μ M), respectively, the arrows indicate the apoptotic endothelial cells or tumor cells, and B and C are the apoptosis rates of the endothelial cells and tumor cells, respectively, with a scale of 100 μ M. From the results in the figure, it can be seen that the apoptosis rate of HUVEC endothelial cells in the vascularized tumor microspheres is increased after the DMOG treatment, and the apoptosis rate of Eca-109 tumor cells is increased after the DMOG treatment, both in the case of paclitaxel and cisplatin treatment, indicating that the model can be used for related drug screening.

The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

Claims

1. A microfluidic chip, comprising:

a first flow channel having a vascular bed culture chamber for containing an endothelial cell gel;

the second flow channel and the third flow channel are respectively arranged on two sides of the first flow channel, and first flow limiting parts are arranged between the second flow channel and the first flow channel and between the third flow channel and the first flow channel; the first flow restriction portion is configured to allow a cell culture medium injected into the second flow channel and the third flow channel to flow through the vascular bed culture chamber while restricting the endothelial cell gel to the vascular bed culture chamber;

the fourth flow channel is positioned on one side, far away from the first flow channel, of the second flow channel, a second flow limiting part is arranged between the fourth flow channel and the second flow channel, and the fourth flow channel is used for culturing fibroblasts; the second flow restriction is configured to be able to confine the fibroblasts in the fourth flow channel while allowing secretions of the fibroblasts to diffuse into the second flow channel.

2. The microfluidic chip according to claim 1, wherein the vascular bed culture chamber is provided with culture wells that are open upward.

3. The microfluidic chip according to claim 1, wherein the first current-limiting portion and the second current-limiting portion comprise a plurality of current-limiting pillars spaced apart from each other.

4. The microfluidic chip according to any one of claims 1 to 3, further comprising a fifth flow channel, wherein the fifth flow channel is located on a side of the third flow channel away from the first flow channel, a third flow restriction portion is disposed between the fifth flow channel and the third flow channel, and the fifth flow channel is used for culturing the fibroblasts; the third flow restriction is configured to enable fixation of the fibroblast cells in the fifth flow channel while allowing secretion of the fibroblast cells to diffuse to the third flow channel.

5. The microfluidic chip according to claim 4, wherein the third current-limiting portion comprises a plurality of current-limiting pillars spaced apart from each other.

6. The method for culturing the blood vessel bed in vitro by using the microfluidic chip of any one of claims 1 to 5, is characterized by comprising the following steps:

respectively injecting different amounts of cell culture medium into the second flow channel and the third flow channel, and enabling the liquid level of the second flow channel to be higher than that of the third flow channel, so that the cell culture medium flows from the second flow channel to the third flow channel through the vascular bed culture chamber under the action of gravity, a gap flow for stimulating endothelial cell gel is formed, and the vascular bed is cultured to obtain the vascular bed.

7. The method of claim 6, further comprising the steps of: after the vascular bed is formed in the vascular bed culture chamber, microtissue is injected onto the vascular bed.

8. The method according to any one of claims 6 to 7, wherein the endothelial cell suspension and the fibroblast suspension each independently comprise at least one of fibrin, collagen, matrigel.

9. A vascular bed model grown by the method of any one of claims 6 to 8.

10. Use of the vascular bed model of claim 9 in tissue culture, drug screening, drug discovery.