Artery-like vascular organ chip for in-situ measurement of mechanical properties of endothelial cells

Abstract

The invention discloses an artery-like vascular organ chip for in-situ measurement of mechanical properties of endothelial cells. The artery-like vascular organ chip comprises a vascular endothelial cell culture layer, a thin film layer, a pulsating flow layer, a micro-contraction channel layer and a substrate which are sequentially arranged from top to bottom, the vascular endothelial cell culture layer is internally provided with a first inlet, a cell adherence chamber, a first outlet, a second inlet, a mixing channel unit and a second outlet; the film layer is provided with a first through hole; a second through hole as well as a liquid inlet, a liquid storage chamber and a liquid outlet which are communicated in sequence are formed in the pulsating flow layer, and the liquid storage chamber is opposite to the cell attachment chamber; a cell inlet, a micro-contraction channel unit and a cell outlet which are communicated in sequence are arranged in the micro-contraction channel layer; the second outlet, the first through hole, the second through hole and the cell inlet are correspondingly arranged in sequence. The chip disclosed by the invention is simple in structure, shear stress and cyclic tensile stress stimulation can be applied to endothelial cells in vitro, and meanwhile, in-situ measurement of cell mechanical properties can be realized.

Description

Technical Field

[0001] This invention relates to the field of organoid chip technology, and more particularly to an arterial vascular organoid chip for in-situ measurement of the mechanical properties of endothelial cells. Background Technology

[0002] Vascular endothelial cells are an important component of human blood vessels and the inner lining cells that make up arteries, veins, and capillaries. They are in direct contact with blood flow within the blood vessels. Endothelial cells form a protective layer between the blood flow and various tissues, playing a vital role in protecting human tissues. They are the first line of defense against cardiovascular disease and also control the degree of vasodilation and vasoconstriction, as well as the extravasation of solutes, fluids, macromolecules, hormones, platelets, and blood cells.

[0003] Organ-on-a-chip technology is a highly promising three-dimensional cell culture method. It allows for accurate and efficient cell culture in vitro and, through the connection of external mechanical devices, can simulate the internal environment of an animal. The vascular system is the most abundant organ system in the human body; therefore, it plays a crucial role in maintaining bodily stability and ensuring optimal organ function. Thus, using organ-on-a-chip technology to study the human vascular system is very helpful in understanding the microenvironment and physiological function of blood vessels within the body. The occurrence of cardiovascular diseases is often related to changes in the endothelial cell cytoskeleton. Existing research shows a high correlation between changes in the cytoskeleton and changes in cell mechanical properties. Therefore, studying the mechanical properties of vascular endothelial cells is of great significance for understanding, preventing, and treating cardiovascular diseases.

[0004] Current vascular organ-on-a-chip devices can only apply one type of mechanical stimulation in vitro, resulting in poor physiological relevance. Furthermore, there is still a lack of devices capable of on-chip in-situ measurement of cellular mechanical properties, making it impossible to link endothelial cell function with its mechanical characteristics. Therefore, it is necessary to develop a vascular organ-on-a-chip that can achieve both in vitro dynamic culture and on-chip in-situ measurement of mechanical properties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an arterial vascular organ-on-a-chip for in-situ measurement of the mechanical properties of endothelial cells.

[0006] To achieve the above objectives, an embodiment of the present invention provides the following technical solution:

[0007] An arterial vascular organ-on-a-chip for in-situ measurement of the mechanical properties of endothelial cells includes, from top to bottom, a vascular endothelial cell culture layer, a thin film layer, a pulsatile flow layer, a microcontraction channel layer, and a substrate;

[0008] The vascular endothelial cell culture layer is provided with a first inlet, a cell adhesion chamber, a first outlet, a second inlet, a mixing channel unit, and a second outlet. The first inlet, the cell adhesion chamber, and the first outlet are connected in sequence. The first outlet and the second inlet are both connected to one end of the mixing channel unit, and the other end of the mixing channel unit is connected to the second outlet.

[0009] The thin film layer is provided with a first through hole;

[0010] The pulsating flow layer is provided with a second through hole and a liquid inlet, a liquid storage chamber, and a liquid outlet connected in sequence, with the liquid storage chamber facing the cell adhesion chamber;

[0011] The micro-contraction channel layer is provided with a cell inlet, a micro-contraction channel unit, and a cell outlet that are connected in sequence.

[0012] The second outlet, the first through hole, the second through hole, and the cell inlet are arranged in sequence.

[0013] As a further improvement of the present invention, the mixing channel unit includes a plurality of mixing channels arranged side by side, and adjacent mixing channels are connected to each other. Each mixing channel includes a first channel sidewall and a second channel sidewall opposite to each other. The first channel sidewall forms at least one first arcuate portion protruding toward the second channel sidewall, and the second channel sidewall forms at least one second arcuate portion protruding toward the first channel sidewall. The first arcuate portion and the second arcuate portion are arranged alternately.

[0014] As a further improvement of the present invention, the distance between the top of the first arc-shaped portion and the side wall of the second channel is 20 μm, and the distance between the top of the second arc-shaped portion and the side wall of the first channel is 20 μm.

[0015] As a further improvement of the present invention, the mixing channel is U-shaped.

[0016] As a further improvement of the present invention, the plurality of mixing channels include a first group of mixing channels and a second group of mixing channels. The first group of mixing channels includes a plurality of first mixing channels with gradually decreasing widths. The second group of mixing channels includes a plurality of second mixing channels with the same width.

[0017] As a further improvement of the present invention, the cell-adhering chamber has a length of 6 mm and a width of 1 mm.

[0018] As a further improvement of the present invention, the thickness of the thin film layer is 98-102 μm.

[0019] As a further improvement of the present invention, the first inlet is connected to a microfluidic pump, the flow rate of which is controlled at 0-20 μl / min, and the liquid inlet is connected to a pressure pump, the pressure of which is controlled at 0-150 mbar.

[0020] As a further improvement of the present invention, the micro-contraction channel unit includes a plurality of micro-contraction channel components arranged side by side. Each micro-contraction channel component includes a U-shaped channel and a micro-contraction channel that are connected to each other. One end of the U-shaped channel is connected to the cell inlet, and the other end is connected to one end of the micro-contraction channel. The other end of the micro-contraction channel is connected to the cell outlet.

[0021] As a further improvement of the present invention, the length of the micro-shrinkage channel is 100 μm and the width is 6 μm.

[0022] The beneficial effects of this invention are:

[0023] (1) The arterial vascular organ chip provided by the present invention is an integrated microfluidic chip. The manufacturing process is simple and the materials are readily available. At the same time, it reduces the experimental cycle, improves experimental efficiency, and avoids the problem of inaccurate experimental data caused by multiple intermediate steps.

[0024] (2) It can apply shear stress and cyclic tensile stress to endothelial cells in vitro to simulate different physiological and pathological internal environments of the human body.

[0025] (3) The setting of the first and second arc-shaped parts of the mixing channel makes the flow rate of the two solutions, trypsin and culture medium, faster and changes the original laminar flow state when they flow through the mixing channel, thus accelerating the mixing effect, effectively improving the solution mixing efficiency, quickly terminating the excessive digestion of cells by trypsin, and avoiding damage to cells.

[0026] (4) The micro-contraction channel structure functional area meets the requirement that a single cell is completely compressed. At the same time, the arrayed micro-contraction channel structure effectively avoids chip blockage. Combined with image recognition algorithm, the chip can complete the function of integrating cell dynamic culture and cell mechanical property measurement, complete the acquisition of cell elastic modulus data, realize in-situ measurement of cell mechanical properties, and link the changes in endothelial cell function with the changes in its mechanical properties. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is an exploded structural diagram of a preferred embodiment of the present invention;

[0029] Figure 2 A schematic diagram of the structure of the vascular endothelial cell culture layer with a mixing channel unit according to a preferred embodiment of the present invention;

[0030] Figure 3 A schematic diagram of the structure of the pulsating flow layer of the present invention, showing the inlet, storage chamber and outlet;

[0031] Figure 4 This is a schematic diagram of the structure of the micro-shrinkage channel layer with micro-shrinkage channel units according to a preferred embodiment of the present invention;

[0032] Figure 5 An experimental effect diagram of an arterial vascular organ-on-a-chip, representing a preferred embodiment of the present invention, displayed under a microscope.

[0033] In the figure: 1. Vascular endothelial cell culture layer, 11. First inlet, 12. Cell adhesion chamber, 13. First outlet, 14. Second inlet, 15. Mixing channel unit, 151. First channel sidewall, 1511. First arc-shaped portion, 152. Second channel sidewall, 1521. Second arc-shaped portion, 153. First mixing channel, 154. Second mixing channel, 16. Second outlet, 2. Thin film layer, 21. First through-hole, 3. Pulsating flow layer, 31. Second through-hole, 32. Liquid inlet, 33. Liquid storage chamber, 34. Liquid outlet, 4. Microcontraction channel layer, 41. Cell inlet, 42. Microcontraction channel unit, 421. Microcontraction channel assembly, 4211. U-shaped channel, 4212. Microcontraction channel, 43. Cell outlet, 5. Substrate. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0035] Please see Figures 1-4This application discloses an arterial vascular organ-on-a-chip for in-situ measurement of the mechanical properties of endothelial cells, comprising, from top to bottom, a vascular endothelial cell culture layer 1, a thin film layer 2, a pulsating flow layer 3, a microcontraction channel layer 4, and a substrate 5. The vascular endothelial cell culture layer 1 has a first inlet 11, a cell adhesion chamber 12, a first outlet 13, a second inlet 14, a mixing channel unit 15, and a second outlet 16. The first inlet 11, the cell adhesion chamber 12, and the first outlet 13 are sequentially connected. The first outlet 13 and the second inlet 14 are both connected to one end of the mixing channel unit 15, and the other end of the mixing channel unit 15 is connected to the second outlet 16. The thin film layer 2 has a first through-hole 21. The pulsating flow layer 3 has a second through-hole 31 and sequentially connected liquid inlet 32, liquid storage chamber 33, and liquid outlet 34. The liquid storage chamber 33 faces the cell adhesion chamber 12. The micro-contraction channel layer 4 is provided with a cell inlet 41, a micro-contraction channel unit 42, and a cell outlet 43 that are connected in sequence. The second outlet 16, the first through hole 21, the second through hole 31, and the cell inlet 41 are arranged in a corresponding manner. In this way, the cells after cell wall detachment enter the micro-contraction channel unit 42 through the second outlet 16, the first through hole 21, the second through hole 31, and the cell inlet 41 in sequence.

[0036] Please see Figure 2 The first inlet 11 serves as the seeding inlet for endothelial cells and the inlet for perfusion culture medium and trypsin. Preferably, the first inlet 11 is connected to a microfluidic pump (not shown in the figure), with the flow rate controlled at 0-20 μl / min. The cell adhesion chamber 12 serves as the cell adhesion culture area, allowing cells to transition from a suspension state to an adherent state, enabling cells to attach to the bottom of the cell adhesion chamber 12. Preferably, the cell adhesion chamber 12 is 6 mm long and 1 mm wide to better facilitate cell adhesion culture and the application of biochemical and physical stimuli within it. The first outlet 13 serves as the outlet for culture medium. After a certain period of cell culture, the first outlet 13 is sealed to prevent cells from flowing out of the first outlet 13, ensuring that all cells flow towards the mixing channel unit 15. The second inlet 14 is used to introduce culture medium to terminate the excessive digestion of cells by trypsin and avoid cell damage.

[0037] Please see Figure 1 Preferably, the thickness of the thin film layer 2 is 98-102 μm, which facilitates deformation and better enables the adherent cells in the cell adhesion chamber 12 to be subjected to cyclic tensile stress. More preferably, the thickness of the thin film layer 2 is 100 μm.

[0038] Please see Figure 1 , Figure 3A pressure pump (not shown in the figure) is connected to the inlet 32 ​​of the pulsating flow layer 3 for injecting PBS solution. The PBS solution enters the reservoir 33. The pressure of the pressure pump is controlled at 0-150 mbar, causing deformation of the membrane layer 2 and thus subjecting the endothelial cells above it to cyclic tensile stress. The pressure pump controller can communicate with a computer via serial port. The pressure pump controller outputs pressure of different amplitudes, which acts on the reservoir containing the PBS solution. The PBS solution flows under pressure from the inlet 32 ​​into the reservoir 33. The outlet 34 is connected to a waste liquid tank for easy collection of discharged PBS solution.

[0039] Please see Figure 2 The mixing channel unit 15 includes multiple mixing channels arranged side by side, with adjacent mixing channels interconnected. Each mixing channel includes a first channel sidewall 151 and a second channel sidewall 152 facing each other. The first channel sidewall 151 forms at least one first arcuate portion 1511 protruding towards the second channel sidewall 152, and the second channel sidewall 152 forms at least one second arcuate portion 1521 protruding towards the first channel sidewall 151. The first arcuate portions 1511 and second arcuate portions 1521 are staggered. The arrangement of the first arcuate portions 1511 and second arcuate portions 1521 increases the flow rate of the trypsin and culture medium solutions when they flow through the mixing channel, changes the original laminar flow state, accelerates the mixing effect, quickly terminates the over-digestion of trypsin, and avoids damage to cells.

[0040] Preferably, the distance between the top tip of the first arc-shaped portion 1511 and the side wall 152 of the second channel is 20 μm, and the distance between the top tip of the second arc-shaped portion 1521 and the side wall 151 of the first channel is also 20 μm. This arrangement spatially disperses cell clumps without compressing the cells, thus avoiding data interference and improving the accuracy of cell measurements.

[0041] Further optimization is to use a U-shaped mixing channel.

[0042] Preferably, the multiple mixing channels include a first group of mixing channels and a second group of mixing channels. The first group of mixing channels includes multiple first mixing channels 153, the width of which gradually decreases. The second group of mixing channels includes multiple second mixing channels 154, the width of which is the same. The gradually decreasing width of the multiple first mixing channels 153 is used to compress the cell space, break up cell clumps as much as possible, and avoid data loss.

[0043] Please see Figure 4The micro-contraction channel unit 42 includes a plurality of micro-contraction channel components 421 arranged side by side. Each micro-contraction channel component 421 includes a U-shaped channel 4211 and a micro-contraction channel 4212 connected to each other. One end of the U-shaped channel 4211 is connected to the cell inlet 41, and the other end is connected to one end of the micro-contraction channel 4212. The other end of the micro-contraction channel 4212 is connected to the cell outlet 43.

[0044] The preferred length of the microcontraction channel 4212 is 100 μm and the width is 6 μm. This configuration ensures that there is a sufficiently long channel to allow the cells to be completely compressed within the microcontraction channel 4212.

[0045] The preferred vascular endothelial cell culture layer 1, thin film layer 2, pulsating flow layer 3, and microcontraction channel layer 4 are all made of PDMS material, and the substrate 5 is made of glass material.

[0046] vascular endothelial cell culture layer 1, pulsating flow layer 3, and microcontraction channel layer 4 were fabricated using soft photolithography. These layers, along with the substrate 5, were then bonded together using a bonding process to obtain an arterial-like vascular organ chip. The chip experimental steps are as follows:

[0047] (1) Chip pretreatment. Before the experiment, soak the chip and consumables such as capillary tubes in alcohol and air dry overnight. Sterilize with ultraviolet light for half an hour before the experiment.

[0048] (2) Microarray treatment before seeding endothelial cells. Human umbilical vein endothelial cells (HUVECS) were used for the experiment. HUVECS were cultured for 4 to 9 generations to achieve better experimental results. Fibronectin was introduced to cover the cell adhesion chamber 12, and the microarray was placed in an incubator overnight. Fibronectin can more effectively promote cell adhesion.

[0049] (3) Cell seeding. Centrifuged human umbilical vein endothelial cells were divided into 1×10⁶ cells. 7A cell suspension of cells / mL is introduced into the vascular endothelial cell culture layer 1 through an external microfluidic pump via the first inlet 11. After observing that the cell adhesion chamber 12 is filled with cells, the chip is placed in an incubator for static culture for 4-6 hours to allow the cells to adhere to the bottom of the cell adhesion chamber 12. After cell adhesion, culture medium is injected through the first inlet 11 via the microfluidic pump to provide nutrients. The perfusion flow rate is controlled at 0, 5 μl / min, 10 μl / min, and 20 μl / min to subject the cells to shear stress. The first outlet 13 serves as the outlet for the culture medium. An external pressure pump is connected to the inlet 32 ​​of the pulsating flow layer 3, and the pressure is controlled at 0, 50 mbar, 100 mbar, and 150 mbar to control the amplitude of the pulsating flow in the pulsating flow layer 3. This causes deformation of the PDMS film layer 2, thereby subjecting the endothelial cells on it to cyclic tensile stress.

[0050] (4) Cell trypsin treatment. Before measuring mechanical properties, the microcontraction channel layer 4 was treated with 0.01% BSA to reduce cell adhesion to the channel. Trypsin was introduced into the first inlet 11 of the vascular endothelial cell culture layer 1 to allow the endothelial cells to detach and flow downstream. The first outlet 13 was sealed to prevent cell loss. At the same time, complete culture medium containing fetal bovine serum was introduced into the second inlet 14 through the mixing channel unit 15 to allow the culture medium and trypsin solution to react fully. After cell detachment, the excessive digestion of trypsin was terminated. Finally, the cell suspension flowed into the microcontraction channel unit 42 of the microcontraction channel layer 4 through the second outlet 16, the first through-hole 21, the second through-hole 31, and the cell inlet 41.

[0051] (5) Measurement of mechanical properties by micro-shrinkage. The cell suspension mixed through the mixing channel unit 15 flows into the micro-shrinkage channel unit 42. The endothelial cells are squeezed and deformed by the squeezing structure of the micro-shrinkage channel 4212. The cell deformation parameters are obtained by image recognition algorithm, and the elastic modulus of the experimental group cells is calculated to characterize the mechanical properties of the group of cells.

[0052] The arterial vascular organ-on-a-chip provided in this embodiment of the invention was placed under a microscope to demonstrate the experimental results, as shown in the figure below. Figure 5 As shown. Figure 5 As shown in (i), the seeded cell suspension fills the culture chamber of the chip. After 4 hours of adhesion, the cells change from a suspension state to a dense cell monolayer, as shown in (i). Figure 5 -(ii) is shown. The mixing channel unit 15, with an arc-shaped structure having a first arc-shaped portion 1511 and a second arc-shaped portion 1521, facilitates the mixing of trypsin and culture medium. Figure 5As shown in (iii), it is evident that the two solutions initially maintain a laminar flow state. The arc-shaped structure effectively disrupts the fluid flow, thereby promoting the mixing of the trypsin and culture medium solutions. The mixing efficiency was verified by examining changes in image grayscale values. Three different regions of the mixing channel unit 15 were selected, as shown... Figure 5 Regions I, II, and III of (iii) were obtained by measuring grayscale values ​​using ImageJ software. Figure 5 As shown in (iv), the grayscale value of the red curve corresponding to region I shows a sudden change, indicating laminar flow; the curves corresponding to regions II and III are flat, indicating that mixing has been completed at these regions. Figure 5 As shown in (v), the micro-contraction channel unit 42 squeezes the cell to deform it and combines the image recognition algorithm to identify the size change of the cell before and after being squeezed.

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0054] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An arterioform vessel organ chip for in-situ measurement of mechanical properties of endothelial cells, characterized by, The blood vessel endothelial cell culture layer, the membrane layer, the pulsatile flow layer, the micro-constriction channel layer and the base are sequentially arranged from top to bottom. The first inlet, the cell adhesion chamber, the first outlet are sequentially connected, the first outlet and the second inlet are both communicated to one end of the mixing channel unit, and the other end of the mixing channel unit is communicated to the second outlet. The membrane layer is provided with a first through hole. The pulsatile flow layer is provided with a second through hole and a liquid inlet, a liquid storage chamber and a liquid outlet which are sequentially connected, and the liquid storage chamber faces the cell adhesion chamber. The micro-constriction channel layer is provided with a cell inlet, a micro-constriction channel unit and a cell outlet which are sequentially connected. The second outlet, the first through hole, the second through hole and the cell inlet are sequentially arranged.

2. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 1, characterized in that, The mixing channel unit includes a plurality of mixing channels arranged side by side, and adjacent mixing channels are communicated, each mixing channel includes opposite first and second channel side walls, the first channel side wall forms at least one first arc portion protruding towards the second channel side wall, and the second channel side wall forms at least one second arc portion protruding towards the first channel side wall, and the first arc portion and the second arc portion are staggered.

3. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 2, characterized in that, The distance between the top end of the first arc portion and the second channel side wall is 20μm, and the distance between the top end of the second arc portion and the first channel side wall is 20μm.

4. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 2, characterized in that, The mixing channel is U-shaped.

5. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 2, characterized in that, The plurality of mixing channels includes a first group of mixing channels and a second group of mixing channels, the first group of mixing channels includes a plurality of first mixing channels, the width of the plurality of first mixing channels gradually decreases, and the second group of mixing channels includes a plurality of second mixing channels, the width of the plurality of second mixing channels is the same.

6. The arterially-like vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 1, characterized in that, The length of the cell adhesion chamber is 6mm, and the width is 1mm.

7. The arterially-like vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 1, characterized in that, The thickness of the membrane layer is 98-102μm.

8. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 1, characterized in that, The micro flow pump connected to the first inlet has a flow rate control of 0-20μl / min, and the pressure pump connected to the liquid inlet has a pressure control of 0-150mbar.

9. The arterially-like vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 1, wherein, The micro-constriction channel unit includes a plurality of micro-constriction channel assemblies arranged side by side, each micro-constriction channel assembly includes a U-shaped channel and a micro-constriction channel which are communicated, one end of the U-shaped channel is communicated to the cell inlet, the other end of the U-shaped channel is communicated to one end of the micro-constriction channel, and the other end of the micro-constriction channel is communicated to the cell outlet.

10. The arterioid vessel organ-chip for in-situ measurement of mechanical properties of endothelial cells according to claim 9, characterized in that, The length of the micro-constriction channel is 100μm, and the width is 6μm.

Images