Double-channel vascularized organ chip and method
By using a dual-channel structure and PDMS material to create a vascularized organ-on-a-chip, the problems of uneven channel design and insufficient tissue interaction have been solved, enabling complex interactions and vascularized simulations between multiple tissues, which is suitable for drug screening and disease model research.
Patent Information
- Application Number
- CN202510973066.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-12-02
AI Technical Summary
Existing multi-channel organ-on-a-chip systems lack fluid dynamics considerations in their channel design, resulting in uneven fluid distribution, insufficient tissue interaction, and inadequate vascularization simulation, making it difficult to realistically simulate the complex interactions between multiple tissues in vivo.
The vascularized organ-on-a-chip employs a dual-channel structure, comprising a stacked channel layer and a bottom layer. It uses polydimethylsiloxane (PDMS) material and designs serpentine channels and micropillar structures. Pressure is regulated by capillary burst valves to achieve co-culture of different cell types and construction of vascular networks.
It enables material exchange and signal transmission between cell culture chambers, simulates complex interactions between multiple tissues in vivo, optimizes fluid distribution, supports the construction and functional simulation of vascular networks, and is suitable for drug screening and disease model research.
Smart Images
Figure CN121046201A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biomedical engineering and microfluidics, specifically relating to a dual-channel vascularized organ-on-a-chip and its method. Background Technology
[0002] Organ-on-a-chip is a microfluidic cell culture system manufactured using microfabrication technology. It can reproduce the physiological and pathological characteristics of organs in vivo by constructing in vitro models. Using organ-on-a-chip can help researchers better understand the physiological and pathological processes of tissues and organs, and provide powerful tools for drug screening, in vitro disease models, and immune co-culture.
[0003] Early research on organ-on-a-chip mainly focused on simulating single-function tissues, such as liver-on-a-chip, lung-on-a-chip, and kidney-on-a-chip. These organ-on-a-chips achieved preliminary simulations of single organ functions by culturing specific types of cells in microfluidic channels. However, the functions of human organs often depend on complex interactions between multiple tissues, such as metabolic interactions between the liver and intestines, and gas exchange between the lungs and blood. Therefore, organ-on-a-chips with single-channel and single-cell-type structures have significant limitations in simulating complex physiological processes.
[0004] To better simulate multi-tissue interactions and inter-organ synergy, multi-channel and multi-cell organ-on-a-chip technologies have attracted increasing attention. For example, dual-channel or multi-channel organ-on-a-chip designs enable the co-culture of different cell types within the same chip and simulate material exchange between different tissues through independent channel systems. However, existing multi-channel organ-on-a-chips still have some design and functional limitations, mainly manifested in the following ways: 1. Inadequate channel design: Existing channel designs often lack in-depth consideration of fluid dynamics, leading to uneven fluid distribution within the chip and affecting cell growth and function; 2. Insufficient tissue interaction: The mechanisms for material exchange and signal transduction between different channels in multi-channel organ-on-a-chips are still imperfect, making it difficult to realistically simulate the complex interactions between multiple tissues in vivo; 3. Insufficient vascularization simulation: The vascular system is an important component of human organs, responsible for transporting oxygen, nutrients, and metabolic products. However, existing organ-on-a-chip technology still faces significant challenges in simulating vascularized structures, making it difficult to achieve effective vascular network construction and functional simulation. Summary of the Invention
[0005] To address the shortcomings of current organ-on-a-chip technology, such as insufficient inter-tissue interaction, inadequate vascularization simulation, and insufficient optimization of flow channel design, this invention provides a dual-flow channel vascularized organ-on-a-chip and its method.
[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A dual-channel vascularized organ-on-a-chip includes a stacked channel layer and a bottom layer, with the lower surface of the channel layer and the bottom layer bonded and sealed. The lower surface of the channel layer has culture medium channels, with symmetrically arranged serpentine tubes along these channels. A first injection channel and a second injection channel are respectively located at both ends of the culture medium channels. The lower surface of the channel layer also has a first cell culture chamber and a second cell culture chamber connected to the culture medium channels. The vascularized organ-on-a-chip is injection molded from a high-molecular-weight polymer material. The vascularized organ-on-a-chip uses polydimethylsiloxane (PDMS), which is softer than glass and allows for better cell fusion. The vascularized organ-on-a-chip is transparent.
[0007] Preferably, the culture medium channel includes a first culture medium channel and a second culture medium channel, with the two ends of the first culture medium channel connected to the two ends of the second culture medium channel via a third culture channel and a fourth culture channel, respectively; serpentine tubes are respectively arranged on the first culture medium channel and the second culture medium channel; a first injection channel is arranged at the end of the first culture medium channel away from the second culture medium channel; a second injection channel is arranged at the end of the second culture medium channel away from the first culture medium channel; both the first culture medium channel and the second culture medium channel are C-shaped channels.
[0008] Preferably, the first cell culture chamber is connected to the third culture channel via a first microcolumn with multiple gaps, the first cell culture chamber is connected to the second cell culture chamber via a second microcolumn with multiple gaps, and the second cell culture chamber is connected to the fourth culture channel via a third microcolumn; the distance between the first microcolumns is 80 μm; the distance between the third microcolumns is 80 μm; the first and second cell culture chambers between the microcolumns constitute the required cell culture space, and the first and second cell culture chambers are rhomboid chambers; the first and third microcolumns can block the passage of hydrogel containing cells but allow small molecules in the culture medium to pass through and exchange material information, and the second microcolumn can block the passage of hydrogel containing cells but allow material information exchange between hydrogels containing cells.
[0009] Preferably, the first cell culture chamber is provided with a first gel dispensing channel and a first gel injection channel at both ends; the second cell culture chamber is provided with a second gel dispensing channel and a second gel injection channel at both ends.
[0010] Preferably, a capillary burst valve is provided on the lower surface of the flow channel layer, and the two ends of the capillary burst valve are respectively connected to the first gel injection channel and the second gel injection channel.
[0011] Preferably, the center of the capillary rupture valve is connected to the rupture valve orifice disposed on the flow channel layer.
[0012] Preferably, the flow channel layer is provided with a first injection hole communicating with the first injection channel, a second injection hole communicating with the second injection channel, a first gel injection hole communicating with the first gel injection channel, a first gel injection hole communicating with the first gel injection channel, a second gel injection hole communicating with the second gel injection channel, and a second gel injection hole communicating with the second gel injection channel.
[0013] Preferably, the upper surface of the flow channel layer is provided with glass tubes that are connected to the first injection hole and the second injection hole; the first injection channel and the second injection channel are rhomboid injection cavities.
[0014] Preferably, the widths of the first gel injection channel and the first gel dispensing channel gradually decrease along the first gel injection hole and the first gel dispensing hole toward the first cell culture chamber, respectively; the widths of the second gel injection channel and the second gel dispensing channel gradually decrease along the second gel injection hole and the second gel dispensing hole toward the second cell culture chamber, respectively.
[0015] Preferably, the thickness of the flow channel layer is 1-2 mm; the thickness of the culture medium channel is 100 μm; the diameter of the first injection hole is 1-1.5 mm; the diameter of the second injection hole is 1-1.5 mm; the diameter of the first gel outlet hole is 1-1.5 mm; the diameter of the second gel outlet hole is 1-1.5 mm; the diameter of the first gel injection hole is 1-1.5 mm; the diameter of the second gel injection hole is 1-1.5 mm; the diameter of the burst valve hole is 1-1.5 mm; the length of the serpentine tube is 4-5 mm; the length of the culture medium channel is 10-12 mm and the width is 0.5 mm; the diameter of the glass tube is 8 mm and the height is 11 mm; and the thickness of the bottom layer is 1-2 mm.
[0016] A method for a dual-channel vascularized organ-on-a-chip, comprising the steps of: S1. The structure of each channel on the channel layer is drawn using drawing software, a mask is made, a channel layer silicon wafer is made by photolithography, and a monolithic structure is formed by casting with PDMS (polydimethylsiloxane). Then, by drilling holes, bonding the channel layer to the bottom layer, and attaching glass tubes to the surface of the channel layer, a vascularized organ-on-a-chip is obtained.
[0017] S2. Sterilize the vascularized organ-on-a-chip for at least 30 minutes before use.
[0018] S3. Inject collagen modification through the first or second injection hole. The collagen travels along the first or second injection hole through the culture medium channel and serpentine pipe to the first and second culture chambers and emerges from the second or first injection hole. Then, place it in an incubator for 10 minutes.
[0019] S4. Remove the vascularized organ-on-a-chip from the incubator, perfuse the first gel injection well with hydrogel containing endothelial cells, fibroblasts and thrombin, and perfuse the second gel injection well with hydrogel containing liver cancer cells and thrombin. Place the vascularized organ-on-a-chip in a cell culture incubator for a period of time to allow the cells to adhere and grow, in order to prevent the cells from being washed away when the culture medium is injected.
[0020] S5. After the cells adhere to the wall, remove the vascularized organochip and add EGM-2 (endothelial cell culture medium) to the two glass tubes respectively. Place the vascularized organochip back into the incubator for further culture and obtain the final experimental results.
[0021] Preferably, in S3, the amount of collagen is 10 μl; the CO2 content in the incubator is 5%, and the temperature is 37°C.
[0022] Preferably, in S4, the concentration of endothelial cells in the cell suspension is 0.7 × 10⁻⁶. 6 / ml, the concentration of fibroblasts was 0.6×10 6 / ml, the concentration of liver cancer cells was 0.2×10 5 / ml; incubation time is 6-12 hours.
[0023] Preferably, in S5, the culture medium in one glass tube is 1.2 ml and the culture medium in the other glass tube is 0.2 ml.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention employs a double-layer structure of a flow channel layer and a bottom layer, featuring simple structure, convenient operation, and easy chip fabrication. The first and second cell culture chambers are separated by a second micropillar. The gap between the second micropillars enables interaction between different types of cell tissues in the first and second cell culture chambers, thus realistically simulating the complex interactions between multiple tissues in vivo. Simultaneously, the culture medium can flow into the first and second cell culture chambers through the gap between the first and third micropillars, facilitating vascular network construction and functional simulation while culturing cells, meeting the needs of cell culture. The culture medium channel and serpentine pipe design of this invention take into account fluid dynamics, facilitating uniform distribution of the culture medium in the chip, significantly reducing the impact on cell growth and function, and exhibiting good biocompatibility.
[0025] 2. This invention perfuses a hydrogel containing endothelial cells, fibroblasts, and thrombin into the first gel injection well, and a hydrogel containing liver cancer cells and thrombin into the second gel injection well. By perfusing the hydrogel, liver cancer cells can be co-cultured with endothelial cells and fibroblasts. Liver cancer cells will migrate to the buds of new blood vessels, and a continuous vascular network will form around the liver cancer cells. They will absorb nutrients from the vascular network, ultimately realizing the interaction between the vascularized network and the liver cancer cells.
[0026] 3. The organ-on-a-chip of the present invention comprises, from top to bottom, a channel layer and a bottom layer. The first and second injection holes in the channel layer are connected to a trapezoidal first and second cell culture chamber via serpentine conduits and culture medium channels for co-culturing two different target cell types. The method of the present invention includes assembly; injecting cell suspensions into each suspension hole; perfusing a hydrogel containing endothelial cells, fibroblasts, and thrombin into the first gel injection hole; perfusing a hydrogel containing liver cancer cells and thrombin into the second gel injection hole; and perfusing culture medium, which flows sequentially into the first and second cell culture chambers. The present invention can achieve vascularization and co-culturing with other cells, and can be used for drug screening, in vitro disease models, immune co-culture, etc. Attached Figure Description
[0027] Figure 1 This is an exploded structural diagram of the present invention in Embodiment 1; Figure 2 This is a schematic diagram of the lower surface structure of the flow channel layer in Example 1; Figure 3 This is a perspective structural diagram of the present invention in Embodiment 1; Figure 4 This is a schematic diagram of the overall structure of the present invention in Embodiment 1; Figure 5 This is a schematic diagram of the experimental results in Example 3; Figure 6 This is a schematic diagram of the culture medium channel in Example 4; Figure 7 This is a schematic diagram of the experiment involving blood vessels and cancer cells in Example 5.
[0028] In the figure, the flow channel layer is 1; the bottom layer is 2; the glass tube is 3; the serpentine tube is 12; the first cell culture chamber is 13; the second cell culture chamber is 14; the first microcolumn is 15; the second microcolumn is 16; the capillary burst valve is 17; the third microcolumn is 18; the first injection channel is 111; the second injection channel is 112; the first injection hole is 113; the second injection hole is 114; the first culture medium channel is 115; the third culture channel is 116; the fourth culture channel is 117; the second culture medium channel is 118; the first gel dispensing channel is 131; the first gel injection channel is 132; the first gel dispensing hole is 133; the first gel injection hole is 134; the second gel dispensing channel is 141; the second gel injection channel is 142; the second gel dispensing hole is 143; the second gel injection hole is 144; and the burst valve hole is 171. Detailed Implementation
[0029] The present invention will be further described in detail below through specific embodiments, but this does not limit the scope of the present invention.
[0030] Example 1 A dual-channel vascularized organ-on-a-chip, its structure is as follows: Figures 1-4 As shown, it includes a layered flow channel layer 1 and a bottom layer 2, with the lower surface of the flow channel layer 1 and the bottom layer 2 sealed by bonding; the lower surface of the flow channel layer 1 is provided with a culture medium channel, and serpentine pipes 12 are symmetrically arranged on the culture medium channel, with a first injection channel 111 and a second injection channel 112 respectively at both ends of the culture medium channel; the lower surface of the flow channel layer 1 is provided with a first cell culture chamber 13 and a second cell culture chamber 14 that are connected to the culture medium channel.
[0031] The culture medium channels include a first culture medium channel 115 and a second culture medium channel 118. The two ends of the first culture medium channel 115 are connected to the two ends of the second culture medium channel 118 through a third culture channel 116 and a fourth culture channel 117, respectively. A serpentine tube 12 is respectively disposed on the first culture medium channel 115 and the second culture medium channel 118. A first injection channel 111 is disposed at the end of the first culture medium channel 115 away from the second culture medium channel 118. A second injection channel 112 is disposed at the end of the second culture medium channel 118 away from the first culture medium channel 115.
[0032] The first cell culture chamber 13 is connected to the third culture channel 116 via the first microcolumn 15, the first cell culture chamber 13 is connected to the second cell culture chamber 14 via the second microcolumn 16, and the second cell culture chamber 14 is connected to the fourth culture channel 117 via the third microcolumn 18.
[0033] The first cell culture chamber 13 is provided with a first gel dispensing channel 131 and a first gel injection channel 132 at both ends; the second cell culture chamber 14 is provided with a second gel dispensing channel 141 and a second gel injection channel 142 at both ends.
[0034] A capillary burst valve 17 is provided on the lower surface of the flow channel layer 1. The two ends of the capillary burst valve 17 are connected to the first gel injection channel 132 and the second gel injection channel 142, respectively. The center of the capillary burst valve 17 is connected to the burst valve hole 171 provided on the flow channel layer 1.
[0035] The flow channel layer 1 is provided with a first injection hole 113 connected to the first injection channel 111, a second injection hole 114 connected to the second injection channel 112, a first gel injection hole 133 connected to the first gel injection channel 131, a first gel injection hole 134 connected to the first gel injection channel 132, a second gel injection hole 143 connected to the second gel injection channel 141, and a second gel injection hole 144 connected to the second gel injection channel 142.
[0036] The upper surface of the flow channel layer 1 is provided with glass tubes 3 that are connected to the first injection hole 113 and the second injection hole 114 respectively; the first injection channel 111 and the second injection channel 112 are rhomboid injection cavities.
[0037] The widths of the first gel injection channel 132 and the first gel dispensing channel 131 gradually decrease along the first gel injection hole 134 and the first gel dispensing hole 133 toward the first cell culture chamber 13, respectively; the widths of the second gel injection channel 142 and the second gel dispensing channel 141 gradually decrease along the second gel injection hole 144 and the second gel dispensing hole 143 toward the second cell culture chamber 14, respectively.
[0038] A method for a dual-channel vascularized organ-on-a-chip, comprising the steps of: S1. Using AutoCAD drawing software, the structure of each channel on the channel layer 1 is drawn, a mask is made, and a channel layer silicon wafer is made by photolithography. Then, a monolithic structure is formed by casting with PDMS (polydimethylsiloxane). Then, by drilling holes at the positions of the first injection hole 113, the second injection hole 114, the first gel outlet hole 133, the first gel injection hole 134, the second gel outlet hole 143, the second gel injection hole 144, and the burst valve hole 171, bonding the channel layer 1 to the bottom layer 2, and attaching the glass tube 3 to the surface of the channel layer 1, a vascularized organ chip is obtained.
[0039] S2. Sterilize the vascularized organ-on-a-chip for at least 30 minutes before use.
[0040] S3. Inject collagen modification through the first injection hole 113 or the second injection hole 114. The collagen flows along the first injection channel 111 or the second injection channel 112 through the first culture medium channel 115 or the second culture medium channel 118, the serpentine pipe 12, the third culture channel 116, and the fourth culture channel 117 to reach the first culture chamber and the second culture chamber, and emerges from the second injection hole 114 or the first injection hole 113. Then, place it in an incubator for 10 minutes. The amount of collagen is 10 μl. The CO2 content in the incubator is 5%, and the temperature is 37°C.
[0041] S4. Remove the vascularized organ-on-a-chip from the incubator. Infuse hydrogel containing endothelial cells, fibroblasts, and thrombin into the first gel injection well 134. The hydrogel in the first gel injection well 134 flows along the first gel injection channel 132 into the first cell culture chamber 13. Air bubbles and other impurities are expelled through the first gel outflow channel 131 and the first gel outflow well 133, ensuring the hydrogel infused into the first gel injection well 134 fills the first cell culture chamber 13. Infuse hydrogel containing liver cancer cells and thrombin into the second gel injection well 144. The hydrogel in the second gel injection well 144 flows along the second gel injection channel 142. The second cell culture chamber 14 is filled with hydrogel. Air bubbles and other impurities are expelled through the second gel injection channel 141 and the second gel injection orifice 143, allowing the hydrogel injected through the second gel injection orifice 144 to fill the second cell culture chamber 14. This enables the co-culture of liver cancer cells with endothelial cells and fibroblasts. A capillary burst valve 17 is connected to the external atmospheric pressure through a burst valve orifice 171, used to regulate and release the pressure during the injection of cell-containing hydrogel. The vascularized organ-on-a-chip is placed in a cell culture incubator for a period of time to allow the cells to adhere and grow, preventing the cells from being washed away during the injection of culture medium. The concentration of endothelial cells in the cell suspension is 0.7 × 10⁻⁶. 6 / ml, the concentration of fibroblasts was 0.6×10 6 / ml, the concentration of liver cancer cells was 0.2×10 5 / ml; incubation time is 6-12 hours.
[0042] S5. After the cells adhere to the wall, remove the vascularized organochip and inject an appropriate amount of culture medium into the second injection well 114. The culture medium flows along the second injection channel 112, the second culture channel, the serpentine tube 12, the third culture channel 116, the fourth culture channel 117, and the first culture medium channel 115 to the first injection well 113. Observe whether the culture medium emerges from the first injection well 113. After observing that the culture medium emerges, inject 1.2 ml and 0.2 ml of EGM-2 culture medium into the two glass tubes 3 respectively. Under the action of gravity, the culture medium is constantly perfused into the culture medium channel. Place the vascularized organochip back into the incubator for culture. The culture medium in the third culture channel 116 and the fourth culture channel 117 provides nutrients to the first cell culture chamber 13 and the second cell culture chamber 14 through the gap between the first microcolumn 15 and the third microcolumn 18, thereby obtaining experimental results.
[0043] Example 2 A method for a dual-channel vascularized organ-on-a-chip, comprising the steps of: The structural drawings of the flow channel layer 1 and the bottom layer 2 of this dual-channel vascularized organ-on-a-chip were drawn using AutoCAD software. After being processed into a mask, a silicon wafer with a microchannel structure (the selected culture medium channel height is 200μm) was fabricated using soft photolithography. This wafer was then attached to a culture dish with double-sided adhesive to create a mold. The chip configuration in the mask is as follows: Figure 2 As shown; a PDMS film with microchannel structure is obtained by uniformly mixing polydimethylsiloxane (PDMS) prepolymer and curing agent in a 10:1 ratio and pouring the mixture into a mold. Vacuuming, bubble blowing, drying, and peeling are then performed. The first injection hole 113, the second injection hole 114, the first gel outlet hole 133, the first gel injection hole 134, the second gel outlet hole 143, the second gel injection hole 144, and the burst valve hole 171 are then perforated. For the preparation and selection of the bottom layer 2, a PDMS film with a thickness of 2 mm is used. Specifically, the PDMS prepolymer and curing agent are uniformly mixed in a 10:1 ratio, vacuumed, poured onto the lid of a petri dish, and then spin-coated and dried.
[0044] Plasma bonding was performed on the flow channel surface of the lower surface of the flow channel layer 1 and the bottom layer 2. After bonding, a vascularized organ chip was obtained. It was placed on a hot plate and baked for 5 minutes to strengthen the bonding between the flow channel layer 1 and the bottom layer 2. Finally, a small amount of PDMS was applied to the first injection hole 113 and the second injection hole 114 using a glass tube 3. The vascularized organ chip was then placed in an oven for curing for 2 hours at a curing temperature of 75°C. Finally, a dual-channel vascularized organ chip with a liquid storage function glass tube 3 was obtained. This vascularized organ chip needs to be sterilized by high temperature and ultraviolet light before the experiment and stored in a clean bench.
[0045] Example 3 A method for a dual-channel vascularized organ-on-a-chip, comprising the steps of: When using this vascularized organ-on-a-chip for experiments, the confluence of cells in the culture state should reach approximately 70%-80%. First, trypsin is used to convert the cells from an adherent state to a floating state, and this process is terminated with a solution containing 10% FBS. After centrifugation, the supernatant is collected and added to the cell culture medium to prepare a cell density of 1×10⁻⁶ cells / mL. 6 Cell suspension of cells / ml; human umbilical vein endothelial cell suspension and human lung fibroblast suspension were mixed in a centrifuge tube at a ratio of 7:6, centrifuged, and the supernatant was discarded. 100 μl of filtered hydrogel was added; liver cancer cells were mixed with the filtered hydrogel to obtain a hydrogel-cell mixture.
[0046] Thrombin and the obtained hydrogel cell mixture were mixed evenly at a ratio of 1:10 (within 20 seconds). An organ-on-a-chip without an injection system was selected, and hydrogel containing endothelial cells, fibroblasts and thrombin was perfused into the first gel injection well 134. Liver cancer cells were perfused into the second gel injection well 144. Liver cancer cells were co-cultured with endothelial cells and fibroblasts by perfusing hydrogel. After the cell-containing gel was successfully injected into the vascularized organ-on-a-chip, 10 μl of laminin was injected into the first injection well 113, and the vascularized organ-on-a-chip was placed in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 10 minutes to observe whether the culture medium flow channels were smooth.
[0047] Then inject an appropriate amount of culture medium into the second injection hole 114 and observe whether the culture medium emerges from the first injection hole 113. After observing that the culture medium emerges, inject 1200 μl and 200 μl of EGM-2 culture medium into the two glass tubes 3 respectively. Since the volume of the culture medium in the two glass tubes 3 is different, the culture medium can be continuously perfused into the culture medium channel under the action of gravity. The culture medium provides nutrition to the first cell culture chamber 13 and the second cell culture chamber 14 through the first microcolumn 15 and the third microcolumn 18 respectively. The second microcolumn 16 facilitates the interaction of cells in the first cell culture chamber 13 and the second cell culture chamber 14. Place the vascularized organ chip in a cell culture incubator with a temperature of 37°C and a carbon dioxide concentration of 5% for 4 hours and observe whether there are air bubbles in the culture medium channel.
[0048] If air bubbles are present, remove them using a 10 μl pipette tip, then place the tube in a cell culture incubator at 37°C and 5% CO2 concentration for further culture. The medium in glass tube 3 of the organ-on-a-chip needs to be changed daily. Specifically, the culture medium in the two glass tubes 3 should be alternately added. For example, if 200 μl of culture medium is added to the left glass tube 3 and 1200 μl to the right glass tube 3 on the first day, then 200 μl of culture medium should be added to the right glass tube 3 and 1200 μl to the left glass tube 3 on the second day. Maintaining a hydrostatic pressure difference in the culture medium within the two glass tubes 3 continuously stimulates the cells. Experimental results are as follows: Figure 5 As shown, on the fifth and seventh days, it can be clearly observed that blood vessels migrate into the liver cancer cell cavities, and the liver cancer cells begin to grow along the shape of the blood vessels, turning into long filaments.
[0049] Example 4 A method for a dual-channel vascularized organ-on-a-chip differs from Example 1 in that its structure is as follows: Figure 6 As shown, the first cell culture chamber 13 is a straight channel, and the second cell culture chamber 14 is a circular channel with continuous boundaries; the second cell culture chamber 14 can also adopt a straight channel, a diamond-shaped channel, or other types of channels.
[0050] Example 5 A method for dual-channel vascularized organ-on-a-chip, differing from Example 3, involves a first cell culture chamber 13 being a straight channel and a second cell culture chamber 14 being a circular channel with continuous boundaries. Blood vessels are cultured in the first cell culture chamber 13, and cancer cells are cultured in the second cell culture chamber 14 to study the interaction between blood vessels and cancer cells. Figure 7 As shown, on the sixth day, it was clearly observed that the blood vessels in the first cell culture chamber 13 formed a network and migrated into the cancer cell channels in the second cell culture chamber 14. After FITC perfusion was performed on this chip, it can be seen that the formed blood vessels have a tubular structure.
[0051] The above description is only a preferred embodiment of the present invention, but is not limited to the above examples. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A dual-channel vascularized organ-on-a-chip, characterized in that, It includes a layered flow channel and a bottom layer, with the lower surface of the flow channel and the bottom layer sealed by bonding; the lower surface of the flow channel is provided with a culture medium channel, and serpentine pipes are symmetrically arranged on the culture medium channel, with a first injection channel and a second injection channel at each end of the culture medium channel; the lower surface of the flow channel is provided with a first cell culture chamber and a second cell culture chamber that are connected to the culture medium channel.
2. The dual-channel vascularized organ-on-a-chip according to claim 1, characterized in that, The culture medium channel includes a first culture medium channel and a second culture medium channel. The two ends of the first culture medium channel are connected to the two ends of the second culture medium channel through a third culture channel and a fourth culture channel, respectively. The serpentine tubes are respectively arranged on the first culture medium channel and the second culture medium channel. The first injection channel is arranged at the end of the first culture medium channel away from the second culture medium channel. The second injection channel is arranged at the end of the second culture medium channel away from the first culture medium channel.
3. The dual-channel vascularized organ-on-a-chip according to claim 2, characterized in that, The first cell culture chamber is connected to the third culture channel via the first microcolumn, the first cell culture chamber is connected to the second cell culture chamber via the second microcolumn, and the second cell culture chamber is connected to the fourth culture channel via the third microcolumn.
4. The dual-channel vascularized organ-on-a-chip according to claim 3, characterized in that, The first cell culture chamber is provided with a first gel dispensing channel and a first gel injection channel at both ends; the second cell culture chamber is provided with a second gel dispensing channel and a second gel injection channel at both ends.
5. The dual-channel vascularized organ-on-a-chip according to claim 4, characterized in that, The lower surface of the flow channel layer is provided with a capillary burst valve, and the two ends of the capillary burst valve are respectively connected to the first gel injection channel and the second gel injection channel.
6. The dual-channel vascularized organ-on-a-chip according to claim 5, characterized in that, The center of the capillary burst valve is connected to the burst valve orifice disposed on the flow channel layer.
7. The dual-channel vascularized organ-on-a-chip according to claim 6, characterized in that, The flow channel layer is provided with a first injection hole connected to the first injection channel, a second injection hole connected to the second injection channel, a first gel injection hole connected to the first gel injection channel, a first gel injection hole connected to the first gel injection channel, a second gel injection hole connected to the second gel injection channel, and a second gel injection hole connected to the second gel injection channel.
8. The dual-channel vascularized organ-on-a-chip according to claim 7, characterized in that, The upper surface of the flow channel layer is provided with glass tubes that are connected to the first injection hole and the second injection hole; the first injection channel and the second injection channel are rhomboid injection cavities.
9. The dual-channel vascularized organ-on-a-chip according to claim 8, characterized in that, The widths of the first gel injection channel and the first gel dispensing channel gradually decrease along the first gel injection hole and the first gel dispensing hole toward the first cell culture chamber, respectively; the widths of the second gel injection channel and the second gel dispensing channel gradually decrease along the second gel injection hole and the second gel dispensing hole toward the second cell culture chamber, respectively.
10. A method for dual-channel vascularized organ-on-a-chip according to any one of claims 1-9, characterized in that the step include: S1. Draw the structure of each channel on the channel layer, make a mask, make a channel layer silicon wafer by photolithography, and cast it to form a monolithic structure. Then, by drilling holes, bonding the channel layer to the bottom layer, and attaching glass tubes to the surface of the channel layer, the vascularized organ chip is obtained. S2. Sterilize the vascularized organ-on-a-chip for 30 minutes and then set it aside. S3. Inject collagen modification through the first injection hole or the second injection hole. The collagen reaches the first culture chamber and the second culture chamber through the culture medium channel and the serpentine tube along the first injection hole or the second injection hole and emerges from the second injection hole or the first injection hole. Then place it in the incubator for 10 minutes. S4. Remove the vascularized organ-on-a-chip from the incubator, perfuse the first gel injection well with hydrogel containing endothelial cells, fibroblasts and thrombin, and perfuse the second gel injection well with hydrogel containing liver cancer cells and thrombin. Place the vascularized organ-on-a-chip in a cell culture incubator and culture it for a period of time to allow the cells to adhere and grow, in order to prevent the cells from being washed away when the culture medium is injected. S5. After the cells adhere to the wall, remove the vascularized organochip, add EGM-2 culture medium to the two glass tubes respectively, and put the vascularized organochip back into the incubator for culture to obtain the experimental results.