Microvascular organ chip and microvascular network culture method

Abstract

The invention belongs to the technical field of organ chips, and particularly discloses a microvascular organ chip and a microvascular network culture method.The microvascular organ chip comprises a chip body, the chip body is internally provided with a microvascular culture channel and four culture medium channels, and the microvascular culture channel comprises a central culture cavity; the four culture medium channels are circumferentially arrayed around the central culture cavity and are respectively communicated with the central culture cavity, each of the four culture medium channels is provided with a first medium inlet and outlet hole, and the n culture medium channels are used as inflow channels of a culture medium; the 4-n culture medium channels are used as outflow channels of microvascular network outflow liquid in the microvascular culture channel, the inflow channels and the outflow channels form pressure differences to control interstitial flow of the microvascular network so as to promote complicated growth of the microvascular network, and n is larger than or equal to 1 and smaller than 4. Through the microvascular organ chip provided by the invention, a high-complexity and high-authenticity microvascular network can be cultivated.

Description

Technical Field

[0001] The present application belongs to the technical field of organ chips, and more specifically, relates to a microvascular organ chip and a microvascular network culture method. Background Art

[0002] Organ chip technology is a technology for three-dimensional cell culture in a chip. By constructing microchannels, microreactors and other functional components, the cells, fluids, gases and extracellular microenvironment in the chip are precisely controlled to generate microtissues and microorgans with biological functionality.

[0003] The construction of complex microvascular networks in vitro based on organ chips is of great significance in the fields of biomedicine and drug development. As a small blood vessel structure that is ubiquitous in the human body, the microvascular network undertakes key tasks such as transporting nutrients, removing waste, and exchanging oxygen and carbon dioxide. These microvessels are not only essential for maintaining the physiological functions of tissues, but also play a core role in the development of tumors, diabetes-related complications and other diseases.

[0004] For example, in specific physiological environments, such as pancreatic islets, the distribution and complexity of microvessels directly affect the structure and function of parenchymal tissues. In addition, microvascular structure has significant diagnostic value in cardiac hypertrophy and heart failure, pulmonary hypertension, diabetic retinopathy, and glaucoma. For example, fractal analysis of retinal vessels can predict the risk of death from future heart disease.

[0005] However, the morphological structure of the microvascular networks cultured by traditional organ chips is mostly relatively simple, and there are often large differences between them and real microvascular networks. It is difficult to simulate the real microvascular network and improvement is urgently needed. Summary of the invention

[0006] In response to the defects of the prior art or the need for improvement, the present application provides a microvascular organ chip and a microvascular network culture method, aiming to improve the problem that the microvascular network cultured in traditional organ chips has a simple morphological structure and is difficult to simulate the real microvascular network.

[0007] In a first aspect, the present application provides a microvascular organ chip, which specifically includes a chip body, wherein the chip body is provided with: a microvascular culture channel comprising a central culture chamber; Four culture medium channels, the four culture medium channels are arranged in a circular array around the central culture cavity and are respectively connected to the central culture cavity, and the four culture medium channels all have a first medium inlet and outlet hole connected to the outer surface of the chip body, and The n culture medium channels are used as inflow channels for the culture medium, and 4-n culture medium channels are used as outflow channels for the effluent of the microvascular network in the microvascular culture channel. The inflow channel and the outflow channel form a pressure difference to control the interstitial flow of the microvascular network to promote the complex growth of the microvascular network, wherein 1≤n<4.

[0008] Through the above technical scheme conceived by the present application, compared with the prior art, when the microvascular organ chip designed by the present scheme is used to culture the microvascular network, since the central culture cavity in the microvascular culture channel is connected to the four culture medium channels one by one, after inoculating cells in the microvascular culture channel, the growth culture of the microvascular network can be carried out by adjusting the medium pressure in the four culture medium channels to form a pressure difference. Based on the unique layout of the four culture medium channels, after setting the pressure between the four culture medium channels to form a pressure difference, the direction and size of the interstitial flow can be controlled, so that the interstitial flow direction with a large angle change can be formed in the present chip, thereby realizing the generation of complex interstitial flow. Based on the complex interstitial flow, the complexity and authenticity of the microvascular network can be significantly improved, and the construction of a complex microvascular network with high physiological relevance can be realized.

[0009] As further preferred, among the four culture medium channels, two culture medium channels that are opposite to each other simultaneously serve as inflow channels for the culture medium, and the other two culture medium channels that are opposite to each other simultaneously serve as outflow channels for the effluent of the microvascular network.

[0010] As further preferred, the culture medium channel is a U-shaped channel, the middle section of the U-shaped channel is connected to the edge of the central culture chamber, and both ends of the U-shaped channel are provided with the first medium inlet and outlet holes.

[0011] As further preferred, the microvessel culture channel further comprises a plurality of inoculation branches, wherein the inoculation branches are connected to the central culture chamber, and the inoculation branches have second medium inlet and outlet holes connected to the outer surface of the chip body.

[0012] As further preferred, the chip body comprises a chip body and a cover glass attached to the surface of the chip body, and a microvessel culture channel and a culture medium channel are formed between the chip body and the cover glass.

[0013] As further preferred, a phase-conducting structure is protruded from the chip body, and the phase-conducting structure is located at the connecting point between the central culture cavity and the culture medium channel.

[0014] In a second aspect of the present application, a microvascular network culture method is provided, which is based on any microvascular organ chip described in the first aspect, and specifically comprises the following steps: S1: Cell seeding in microvascular culture channels; S2: using n culture medium channels to perfuse the culture medium and 4-n culture medium channels to discharge the microvascular network effluent, so that a microvascular network is formed in the microvascular culture channel, wherein 1≤n<4.

[0015] As further preferred, the step S1 specifically includes: S11: mixing the cell suspension and the hydrogel raw material into a mixture in an environment of 0°C-4°C; S12: inoculating the mixture into the microvascular culture channel of the microvascular organ chip; S13: The inoculated microvascular organ chip is placed in a physiological temperature environment for hydrogel solidification.

[0016] As further preferred, the step S2 specifically includes: S21: using two of the culture medium channels that are disposed opposite to each other as inflow channels to perform perfusion of the culture medium, and using the remaining two culture medium channels that are disposed opposite to each other as outflow channels to allow outflow of the effluent of the microvascular network; S22: exchanging the inflow channel and the outflow channel in the previous step, perfusing the culture medium through the new inflow channel, and continuing to culture the microvascular network; S23: Repeat step S22 to culture and form a microvascular network.

[0017] As further preferred, in step S21, independent culture medium containers are connected at each first medium inlet and outlet hole of the culture medium channel, and the initial height of the liquid level in each culture medium container is adjusted to form a pressure difference, so that two of the culture medium channels serve as inflow channels and the other two culture medium channels serve as outflow channels.

[0018] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art: 1. This microvascular organ chip contains four culture medium channels with specific distribution characteristics. By setting the pressure between the four culture medium channels, the direction and size of the interstitial flow in the chip can be controlled, so that the interstitial flow direction with large angle changes can be formed in the chip, thereby realizing the generation of complex interstitial flow. Based on this complex interstitial flow, the complexity and authenticity of the microvascular network can be significantly improved.

[0019] 2. In this microvascular network culture method, by mixing the cell suspension and the hydrogel raw material in a low-temperature environment, then inoculating the mixture into the microvascular organ chip, and then quickly placing the chip in a physiological temperature environment for one-time rapid curing, a thicker microvascular network can be effectively produced.

[0020] 3. In the present microvascular network culture method, by constructing a hydrostatic pressure difference based on four culture medium channels, restoring the hydrostatic pressure difference and reversing the direction of the hydrostatic pressure difference when a preset time is reached, the complex formation of the microvascular network can be significantly promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the structure of a microvascular organ chip provided in an embodiment of the present application; Figure 2 This is a flow chart of a microvascular network culture method provided in an embodiment of the present application; Figure 3 It is a timeline diagram of a microvascular network culture method provided in an embodiment of the present application; Figure 4 is a schematic diagram of a hydrogel solidification scheme in a microvascular network culture method provided in an embodiment of the present application; Figure 5 A schematic diagram showing the change in interstitial flow direction under four different perfusion methods; Figure 6 The distribution diagrams of interstitial flow direction angles under four different perfusion modes are shown; Figure 7 It is a fluid characteristic data diagram under different experimental conditions and data indicators provided in the embodiments of the present application; Figure 8 This is a diagram of experimental simulation results for a square chip in one direction; Fig. 9 This is a diagram of experimental simulation results of a traditional parallel chip; Fig.10 It is a display of the observation results of three microvascular network culture schemes; Fig.11 This is a comparative analysis of the morphological characteristics of the microvascular network under three microvascular network culture schemes.

[0022] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1. Central culture chamber; 2. Culture medium channel; 3. First medium inlet and outlet holes; 4. Inoculation branch channel; 5. Second medium inlet and outlet holes; 6. Chip body; 7. Cover glass; 8. Phase-conducting structure. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] The following is combined with Figure 1-Figure 11 This application is described in further detail.

[0025] The present application discloses a microvascular organ chip. Figure 1 The microvascular organ chip includes a chip body, in which a microvascular culture channel and four culture medium channels 2 are arranged. The microvascular culture channel includes a central culture chamber 1; the four culture medium channels 2 are arranged in a circular array around the central culture chamber 1, the four culture medium channels 2 are respectively connected to the central culture chamber 1, and the four culture medium channels 2 have a first medium inlet and outlet hole 3 connected to the outer surface of the chip body; and n culture medium channels 2 are used as inflow channels of the culture medium, and 4-n culture medium channels 2 are used as outflow channels of the outflow liquid of the microvascular network in the microvascular culture channel, and the inflow channel and the outflow channel form a pressure difference to control the interstitial flow of the microvascular network to promote the complex growth of the microvascular network, wherein 1≤n<4.

[0026] When the microvascular organ chip designed in the present scheme is used to culture a microvascular network, since the central culture chamber 1 in the microvascular culture channel is connected to the four culture medium channels 2 one by one, after inoculating cells in the microvascular culture channel, the growth and culture of the microvascular network can be achieved by adjusting the medium pressure in the four culture medium channels 2 to form a pressure difference (generally speaking, the first medium inlet and outlet hole 3 of the culture medium channel 2 is externally connected to a culture medium container, and a pressure difference can be formed by differentially setting the liquid level heights of multiple culture medium containers).

[0027] Based on the unique layout of the four culture medium channels 2, the size and direction of the pressure difference between the four culture medium channels 2 are set, so that the direction and size of the interstitial flow in the chip can be controlled, so that the interstitial flow direction with a large angle change can be formed in the chip, thereby realizing the generation of complex interstitial flow. Based on the complex interstitial flow, the complexity and authenticity of the microvascular network can be significantly improved.

[0028] Preferably, in some embodiments, among the four culture medium channels 2, two culture medium channels 2 disposed opposite to each other simultaneously serve as inflow channels of the culture medium, and the other two culture medium channels 2 disposed opposite to each other simultaneously serve as outflow channels of the microvascular network outflow fluid. Generally speaking, under this design scheme, the interstitial flow direction angle changes the most, which can be conducive to the generation of complex interstitial flow.

[0029] Furthermore, in some embodiments, the microvessel culture channel also includes a plurality of inoculation branches 4, and the plurality of inoculation branches 4 are all connected to the central culture chamber 1, and the plurality of inoculation branches 4 have a second medium inlet and outlet hole 5 connected to the outer surface of the chip body.

[0030] Preferably, four inoculation branches 4 are provided, and the four inoculation branches 4 are distributed in a circular array around the central culture chamber 1 and are alternately arranged between the four culture medium channels 2, and the four inoculation branches 4 are respectively connected to the central culture chamber 1. In other embodiments, the number of inoculation branches 4 can also be set to any other number.

[0031] Under this design, users can inoculate cells into the central culture chamber 1 of the microvascular organ chip through the inoculation branch 4 for culture.

[0032] Generally speaking, hydrogel is used as a cell carrier material, and a mixture of cells and hydrogel raw materials is injected into the microvascular culture channel along the second medium inlet and outlet hole 5 of the inoculation branch 4 under a low temperature environment, so that the cells can be successfully inoculated into the central culture chamber 1; then, the chip is placed in a culture dish and transferred to an incubator for hydrogel solidification to form a three-dimensional cell-carrier complex in the microvascular culture channel of the chip. The three-dimensional cell-carrier complex is a porous structure, and cells can migrate and grow in the three-dimensional spatial structure of the carrier.

[0033] As a preferred example, Figure 1 As shown, in some embodiments, a phase-conducting structure 8 is protruded from the chip body, and the phase-conducting structure 8 is located at the connection point between the central culture cavity 1 and the culture medium channel 2 .

[0034] Under this design, the phase-conducting structure 8 can partially block the communicating opening between the central culture chamber 1 and the culture medium channel 2, but will not physically separate the central culture chamber 1 and the culture medium channel 2. When the micro-blood organ chip is actually used, due to the presence of the phase-conducting structure 8, the mixture of cells and carrier substances will be constrained in the central culture chamber 1 due to the surface tension effect and will not flow into the culture medium channel 2. After the mixture is gelled and solidified, the culture medium is introduced into the culture medium channel 2, and the culture medium can be in direct contact with the cell-carrier complex, ensuring the exchange of nutrients and waste between the culture medium and the microvascular network.

[0035] Furthermore, in some embodiments, the culture medium channel 2 is a U-shaped channel, the middle section of the U-shaped channel is connected to the edge of the central culture chamber 1, and the first medium inlet and outlet holes 3 are provided at both ends of the U-shaped channel.

[0036] Under this design, the culture medium channel 2 and the central culture chamber 1 have a relatively wide connecting area, which is beneficial to improving the material exchange efficiency between the culture medium in the culture medium channel 2 and the microvascular network in the central culture chamber 1, and is beneficial to the multi-directional and large-scale flow of interstitial flow.

[0037] Further, such as Figure 1As shown, in some embodiments, the chip body includes a chip body 6 and a cover glass 7 attached to the surface of the body, and the chip body 6 and the cover glass 7 form a microvessel culture channel and a culture medium channel 2.

[0038] Generally speaking, the bottom of the chip body 6 has an open cavity, and the top has a first medium inlet and outlet hole 3 and a second medium inlet and outlet hole 5 connected to the cavity. A cover glass 7 is set at the bottom of the chip body 6 to seal the bottom of the cavity to enclose a microvascular culture channel and four culture medium channels 2.

[0039] Furthermore, in some embodiments, the cross section of the central culture chamber 1 is approximately square, and in other embodiments, the cross section of the central culture chamber 1 may also be circular or other shapes.

[0040] Furthermore, in some embodiments, the channel height of the microvessel culture channel and the culture medium channel 2 is not less than 200 μm. Preferably, the channel height of the microvessel culture channel and the culture medium channel 2 is selected to be 200 μm.

[0041] The present application also discloses a method for cultivating a microvascular network based on any of the above-mentioned microvascular organ chips. Figure 2-Figure 4 As shown, the method comprises the following steps: S1: Cell seeding based on central culture chamber 1.

[0042] S2: Use n culture medium channels 2 to perfuse the culture medium, and use 4-n culture medium channels 2 to discharge the microvascular network effluent, so that a microvascular network is cultured and formed in the microvascular culture channel, wherein 1≤n<4.

[0043] Furthermore, in some embodiments, step S1 specifically includes: S11: In an environment of 0°C-4°C, the cell suspension and the hydrogel raw material are mixed into a mixture.

[0044] S12: The mixture is inoculated into the microvascular culture channel of the microvascular organ chip.

[0045] S13: placing the inoculated microvascular organ chip in a physiological temperature environment and performing hydrogel solidification treatment to form a cell-carrier complex in the microvascular culture channel.

[0046] Among them, physiological temperature refers to the normal core body temperature in humans or other organisms. Generally speaking, the physiological temperature is 36.1 ℃ to 37.2 ℃.

[0047] In some embodiments, step S2 specifically includes: S21: Use two of the culture medium channels 2 that are opposite to each other as inflow channels (i.e., high-pressure perfusion channels) to perfuse the culture medium, and use the remaining two culture medium channels 2 that are opposite to each other as outflow channels (i.e., low-pressure outflow channels) to allow the outflow of the effluent from the microvascular network.

[0048] S22: After the perfusion time reaches the preset time, the inflow channel and the outflow channel in the previous step are interchanged (i.e., the inflow channel in the previous step is changed to the outflow channel, and the outflow channel in the previous step is changed to the inflow channel), and the culture medium is perfused based on the new inflow channel to continue culturing the microvascular network.

[0049] S23: Repeat step S22 and regularly replace the culture medium to culture and form a microvascular network.

[0050] Preferably, in step S21, independent culture medium containers (generally a pipette tip is used as the culture medium container) are connected at each first medium inlet and outlet hole 3 of the culture medium channel 2, and a pressure difference is formed by adjusting the initial height of the liquid level in each culture medium container, so that two of the culture medium channels 2 serve as inflow channels and the other two culture medium channels 2 serve as outflow channels, thereby constructing a hydrostatic pressure difference and culturing the microvascular network.

[0051] Generally speaking, the culture medium containers corresponding to two of the oppositely disposed culture medium channels 2 contain culture medium with a high liquid level, and the culture medium containers corresponding to the other two oppositely disposed culture medium channels 2 contain culture medium with a low liquid level, or no culture medium, to form a pressure difference.

[0052] In step S21, the solidified hydrogel in the chip presents a porous structure, and the hydrostatic pressure applied at the inlet will gradually decrease over time and eventually tend to the equilibrium pressure between the four culture medium channels 2.

[0053] In step S22, after the preset time is reached, the amount of solution in the culture medium container is preferably redistributed, so that the culture medium container originally having a high liquid level culture medium is adjusted to have a low liquid level culture medium, and the culture medium container originally having a low liquid level culture medium is adjusted to have a high liquid level culture medium, thereby realizing the recovery of the hydrostatic pressure difference and the exchange of the direction of the hydrostatic pressure difference, thereby adjusting the direction of mass flow, promoting the generation of interstitial flow in complex directions, and thereby improving the complexity and authenticity of the microvascular network.

[0054] Of course, in some other embodiments, the use of pipeline pumping and other solutions is not excluded. By pumping and perfusing the culture medium into two relatively arranged culture medium channels 2, the effluent from the other two culture medium channels 2 is recovered, so that two of the culture medium channels serve as inflow channels (i.e., high-pressure perfusion channels) and the other two culture medium channels 2 serve as outflow channels (i.e., low-pressure outflow channels).

[0055] Example: A microvascular organ chip, wherein a microvascular culture channel and four culture medium channels 2 are formed in the chip body. The microvascular culture channel includes a square central culture cavity 1 with a side length of 3mm*3mm formed in the center of the chip body, and the four corners of the central culture cavity 1 are respectively connected to inoculation branches 4, the width of the inoculation branch 4 is 0.375mm, the inoculation branch 4 extends radially and away from the central culture cavity 1, and the end of the inoculation branch 4 away from the central culture cavity 1 has a second medium inlet and outlet hole 5 opened to the upper surface of the chip body, and the diameter of the second medium inlet and outlet hole 5 is 1.1mm.

[0056] The four culture medium channels 2 are dispersed around the perimeter of the central culture chamber 1, wherein the width of the culture medium channel 2 is 0.5 mm, and a first medium inlet and outlet hole 3 is provided at one end of the culture medium channel 2 away from the central culture chamber 1, and the diameter of the first medium inlet and outlet hole 3 is 2.5 mm. The channel height (i.e., the distance between the inner top wall and the inner bottom wall) of the microvascular culture channel and the four culture medium channels 2 is 200 μm. The phase-conducting structure 8 is located at the top wall of the channel, and its thickness is 50 μm and its width is 150 μm.

[0057] For ease of understanding, Figure 1 (a) shows the main structure of the microvascular organ chip. Figure 1 (b) shows the morphological layout of the microvessel culture channel and culture medium channel 2. Figure 1 (c) shows the cross-sectional structure of the interface between the hydrogel and the culture medium in the microvascular culture channel and the culture medium channel 2; for ease of understanding, Figure 1 In (c) of FIG. 1 , the position of the phase-conducting structure 8 is indicated by a frame line.

[0058] The method for making the microvascular organ chip is as follows: The PDMS matrix (polydimethylsiloxane) and the curing agent are mixed in a ratio of 10:1, poured on the square chip mold, and the bubbles are exhausted in a vacuum dryer, and then placed in an 80°C oven for curing for 2 hours. After complete curing, the chip body 6 is separated from the chip mold to obtain the chip body 6 (the relevant phase-conducting structure 8 is located in the chip body 6), and the first medium inlet and outlet holes 3 and the second medium inlet and outlet holes 5 are punched out using 2.5mm and 1.1mm biopsy punches. The chip body 6 is then ultrasonically cleaned, and then the chip body 6 and the cover glass 7 are placed in oxygen plasma gas for 90 seconds, and then the chip body 6 and the cover glass 7 are bonded and placed in an 80°C drying oven overnight to ensure that it is completely cured and tightly bonded to the cover glass 7.

[0059] Based on the microvascular organ chip, a method for culturing a microvascular network is: HUVEC-RFP (human umbilical vein endothelial cells immortalized-red fluorescent protein labeled) and HFL1 (human embryonic lung fibroblasts) were used to construct microvascular networks, and the standard culture and passage procedures were followed. HUVEC-RFP and HFL1 were cultured in T75 culture flasks at 37°C and 5% CO2. HUVEC-RFP used Endothelial Cell medium (an endothelial cell culture medium), and HFL1 used F12K medium (an animal cell culture medium). When the cells reached 80% confluence, they were digested and passaged. HUVEC-RFP and HFL1 were used between 4 and 8 generations. HUVEC-RFP was digested with 0.05% trypsin–EDTA (wt / vol) for 1 min and resuspended to a concentration of 1.8×107 cell / mL; HFL1 was digested with 0.25% trypsin–EDTA (wt / vol) for 1 min and resuspended to a concentration of 2×106 cell / mL. Add thrombin solution to the cell suspension and adjust the concentration to 4 U / mL. Take an equal volume of cell suspension and mix it with 8 mg / mL fibrinogen solution on ice and quickly inoculate it into the chip. Among them, Trypsin-EDTA (wt / vol) is a commonly used cell culture reagent, mainly used for cell digestion and passage.

[0060] like Figure 4 As shown, in order to reduce the sedimentation of cells during the hydrogel solidification process, after the cell inoculation is completed, a single chip is loaded into a culture dish and quickly placed in a 37°C incubator to wait for a preset time (usually 30 minutes) to complete the hydrogel solidification (during this process, components such as fibrinogen are cross-linked under the action of thrombin to form a stable hydrogel structure, which not only provides support and attachment points for cells, but also simulates the extracellular matrix environment in the body, which is conducive to cell growth and the formation of microvascular networks. ‌) Compared with the traditional Flip chip method with a fixed interval, this solidification method is more convenient and easier to operate, which can reduce the sedimentation of cells during the hydrogel solidification process and is conducive to increasing the thickness of the microvascular network.

[0061] After solidification, cut a 10 μL pipette tip and establish a hydrostatic pressure difference on the chip according to the screened culture scheme for culture (the culture medium is supplemented with 100 ng / mL VEGF-165 (vascular endothelial growth factor 165) and 6.5 μg / mL aprotinin, the hydrostatic pressure difference is restored and the direction of the hydrostatic pressure difference is adjusted every 12 hours, and a new culture medium is replaced every 24 hours.

[0062] For ease of understanding, Figure 5 A schematic diagram showing the changes in interstitial flow direction under four different perfusion methods. Figure 6The distribution diagram of interstitial flow direction angle under four different perfusion modes is shown, among which, Figure 6 (a) and Figure 5 corresponds to (a) in , and so on.

[0063] For ease of understanding, Figure 7 (a) in the figure shows the distribution of the average flow direction angle under four different injection methods. Figure 5 The perfusion scheme shown in (b) is the experimental operation scheme. Figure 7 (b) in the figure shows the relationship between the proportion of physiological interstitial flow rate and the inlet hydrostatic pressure in a certain experiment under this scheme. Figure 7 (c) shows the relationship between the proportion of physiological interstitial flow rate and the test time. Figure 2 (d) shows the relationship between the average interstitial flow velocity and the experimental time.

[0064] The proportion of physiological interstitial flow velocity refers to the ratio of the area in the physiological flow velocity range (i.e., 1.7 μm / s-11 μm / s) to all areas in the microvascular culture channel under the current pressure difference between the inlet and the outlet (referred to here as the inlet hydrostatic pressure).

[0065] It can be seen that in this experiment, when the inlet hydrostatic pressure is 60Pa, the physiological interstitial flow rate accounts for the highest proportion (about 91.8%). After about 12 hours, the proportion of the optimal physiological interstitial flow rate begins to be lower than 60%. Therefore, it can be set to restore the hydrostatic pressure at the inlet to 60Pa every 12 hours.

[0066] Considering that the nutrients and growth factors in the culture medium will be exhausted within a certain period of time, such as 24 hours, it is possible to replace the culture medium with a new one every 24 hours, and to change the direction of the hydrostatic pressure difference each time the culture medium is replaced.

[0067] In some embodiments, a specific culture protocol such as Figure 3 As shown, the columns A1, A2, A3 and A4 indicate the pressures at the first medium inlet and outlet 3 in the four culture medium channels, where A1 and A3 are relative, and A2 and A4 are relative. For example, when changing for the first time, the pressure at the positions indicated by A1 and A3 is set to 60Pa, and the pressure at the positions indicated by A2 and A4 is set to 0Pa, thereby generating a hydrostatic pressure difference of 60Pa; after 12 hours, A2 and A4 are adjusted to 60Pa, and A1 and A3 are 0Pa, and the cycle repeats.

[0068] Generally speaking, a hydrostatic pressure difference of 60Pa means a height difference of 6mm in the culture medium, and it is preferred to use a 10uL pipette tip inserted in the channel to establish the hydrostatic pressure difference. Generally speaking, the pipette tip needs to be trimmed or cut to adjust the length, shape or opening size of the tip to better fit the microvascular organ chip.

[0069] Furthermore, to facilitate understanding of the performance differences between the present microvascular organ chip and traditional chips, Figure 8 The fluid dynamics analysis diagram of a unidirectional square chip (i.e., a unidirectional flow square chip) is shown. Figure 8 (a) shows a schematic diagram of the change in interstitial flow direction when microvascular networks are cultured in a unidirectional square chip. Figure 8 (b) in the figure is the relationship between the corresponding interstitial flow direction angle and the number of interstitial flow streamlines.

[0070] Fig. 9 A conventional parallel chip fluid dynamics analysis diagram is shown. Fig. 9 (a) shows a schematic diagram of the change in interstitial flow direction when microvascular networks are cultured in parallel chips. Fig. 9 (b) in the figure is the relationship between the corresponding interstitial flow direction angle and the number of interstitial flow streamlines.

[0071] Figure 8 and Fig. 9 The results showed that the single flow direction square chip had no significant difference from the parallel chip in most vascular characteristic parameters, indicating that the area of ​​the microvascular culture channel (especially the central culture chamber) had no obvious effect on microvascular growth.

[0072] For ease of understanding, the microvascular organ chip is referred to as a square chip. Figure 10-11 As shown in the figure, the microvascular network culture results of the square chip of this design, the unidirectional square chip, and the traditional parallel chip were imaged and analyzed. It can be seen that the microvascular network on the square chip can grow normally, and the percentage of blood vessel area is better than that of the traditional parallel microvascular chip and the square chip with only a single flow direction. In addition, whether it is observed in vivo or after microvessel fixation, it can be seen that the microvascular network on the square chip can fully utilize the thickness of the chip hydrogel channel ( Fig.10 XZ and YZ parts), forming a thicker microvascular network ( Fig.10 The white arrows show the lumen structure of microvessels).

[0073] like Fig.11As shown in the figure, the morphological characteristics of microvessels on square chips, parallel chips and unidirectional square chips on the 5th day of culture were quantitatively analyzed. The microvessels on the square chip showed a decrease in vascular radius and length, and an increase in the number of branch points and endpoints, indicating that the microvessel branches of the square chip were richer per unit area. In addition, by comparing the standard deviation of vascular tortuosity on different chips (the larger the standard deviation, the more unstable the direction change of the vascular path and the higher the tortuosity). The direction of the vascular branches on the square chip changed the most, proving that the vascular structure it generated was the most complex.

[0074] Finally, by statistically comparing the differences in the ratio of vascular surface area to volume among the three chips (the larger the value, the higher the material exchange efficiency of the blood vessels), the results showed that the square chip had higher indicators than the other two chips. It can be seen that the multi-directional complex interstitial flow generated by the square chip promoted the formation of complex microvascular networks to a certain extent.

[0075] It should be understood that expressions such as "include" and "may include" used in this application indicate the existence of the disclosed functions, operations or constituent elements, and do not limit one or more additional functions, operations and constituent elements. In this application, terms such as "include" and / or "have" may be interpreted as indicating specific characteristics, numbers, operations, constituent elements, components or combinations thereof, but may not be interpreted as excluding the existence or possibility of adding one or more other characteristics, numbers, operations, constituent elements, components or combinations thereof.

[0076] It should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0077] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0078] In this application, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0079] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A microvascular organ chip, characterized in that: The chip body comprises: A microvessel culture channel comprising a central culture chamber (1); four culture medium channels (2), the four culture medium channels (2) are distributed in a circular array around the central culture chamber (1) and are respectively connected to the central culture chamber (1), and the four culture medium channels (2) all have a first medium inlet and outlet hole (3), and The n culture medium channels (2) are used as inflow channels of the culture medium, and 4-n culture medium channels (2) are used as outflow channels of the microvascular network effluent in the microvascular culture channel. The inflow channel and the outflow channel form a pressure difference to control the interstitial flow of the microvascular network to promote the complex growth of the microvascular network, wherein 1≤n<4.

2. The microvascular organ chip according to claim 1, characterized in that: Among the four culture medium channels (2), two culture medium channels (2) arranged opposite to each other simultaneously serve as inflow channels for the culture medium, and the other two culture medium channels (2) arranged opposite to each other simultaneously serve as outflow channels for the outflow fluid of the microvascular network.

3. The microvascular organ chip according to claim 1, characterized in that: The culture medium channel (2) is a U-shaped channel, the middle section of which is connected to the edge of the central culture chamber (1), and both ends of the U-shaped channel are provided with the first medium inlet and outlet holes (3).

4. The microvascular organ chip according to claim 1, characterized in that: The microvessel culture channel further comprises a plurality of inoculation branches (4), wherein the inoculation branches (4) are connected to the central culture chamber (1), and the inoculation branches (4) have second medium inlet and outlet holes (5) connected to the outer surface of the chip body.

5. The microvascular organ chip according to any one of claims 1 to 4, characterized in that: The chip body comprises a chip body (6) and a cover glass (7) attached to the surface of the chip body (6); a microvessel culture channel and a culture medium channel (2) are formed between the chip body (6) and the cover glass (7).

6. The microvascular organ chip according to any one of claims 1 to 4, characterized in that: A phase-conducting structure (8) is protruding from the chip body, and the phase-conducting structure (8) is located at the connection point between the central culture cavity (1) and the culture medium channel (2).

7. A method for culturing a microvascular network, based on a microvascular organ chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: Cell seeding in microvascular culture channels; S2: using n culture medium channels (2) to perfuse the culture medium and 4-n culture medium channels (2) to discharge the microvascular network effluent, so that a microvascular network is formed in the microvascular culture channel, wherein 1≤n<4.

8. The microvascular network culture method according to claim 7, characterized in that: The step S1 comprises: S11: mixing the cell suspension and the hydrogel raw material into a mixture in an environment of 0°C-4°C; S12: inoculating the mixture into the microvascular culture channel of the microvascular organ chip; S13: The inoculated microvascular organ chip is placed in a physiological temperature environment for hydrogel solidification.

9. The microvascular network culture method according to claim 7 or 8, characterized in that: The step S2 comprises: S21: using two of the culture medium channels (2) disposed opposite to each other as inflow channels for perfusing the culture medium, and using the remaining two culture medium channels (2) disposed opposite to each other as outflow channels for allowing the outflow of the effluent from the microvascular network; S22: exchanging the inflow channel and the outflow channel in the previous step, perfusing the culture medium through the new inflow channel, and continuing to culture the microvascular network; S23: Repeat step S22 to culture and form a microvascular network.

10. The microvascular network culture method according to claim 9, characterized in that: In step S21, independent culture medium containers are connected at each first medium inlet and outlet hole (3) of the culture medium channel (2), and the initial height of the liquid level in each culture medium container is adjusted to form a pressure difference, so that two oppositely arranged culture medium channels (2) serve as inflow channels and the other two oppositely arranged culture medium channels (2) serve as outflow channels.

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