Annular organ-on-a-chip and methods

Abstract

This invention belongs to the field of microfluidic chip technology, specifically disclosing a mechanically stretchable ring-shaped organ-on-a-chip and its method. It includes a thin film layer, a channel layer and a bottom layer disposed on both sides of the thin film layer, and a culture chamber disposed on the channel layer. The culture chamber has a groove structure with its ends connected, and a reservoir is connected to the culture chamber through a cell channel. The channel layer and the thin film layer have communicating pores, and the bottom layer has an air cavity communicating with the pores. The air cavity has a groove structure, and the projection of the edge of the air cavity onto the channel layer is located between the inner and outer edges of the culture cavity. This invention provides a mechanically stretchable ring-shaped organ-on-a-chip and its method for forming experimental and control groups in the same chip. In the control and experimental chip, it avoids the influence of uneven mixing of cell suspension on experimental results; it also enables uniform stretching of cells, facilitating observation of cell growth trends.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a ring-shaped organ-on-a-chip and its method. Background Technology

[0002] Microfluidic chips use chips as an operating platform, with life sciences being their primary application. Organ-on-a-chip is one of the main applications of microfluidics, using microfabrication processes to create microfluidic cell culture systems, aiming to reproduce the physiological and pathological characteristics of organs in vivo by constructing in vitro models.

[0003] Compared to other culture methods, although organ-on-a-chip is more difficult to operate at some levels, its advantages are significant. 2D models cannot accurately describe and simulate the rich environment and complex processes observed in vivo. Animal models and humans are prone to significant deviations due to species differences, which can lead to the failure of test drugs. Organ-on-a-chip can achieve tissue and organ function levels that conventional 2D or conventional 3D culture systems cannot reach, thus obtaining more accurate test data.

[0004] However, a key challenge currently facing organ-on-a-chip technology is how to construct an in vitro microenvironment that closely resembles the in vivo environment, suitable for cell growth. Under physiological conditions, cell behavior is influenced by numerous physical stimuli, such as electrical stimulation, fluid shear forces, and mechanical stretching. These physical signals are crucial factors in maintaining physiological function or inducing disease; simply allowing cells to grow within an organ-on-a-chip is far from sufficient. Some cells, such as heart cells and muscle cells, naturally thrive in a dynamic growth environment. Whether these cells depend on such an environment for growth, whether movement stimulation promotes cell proliferation, and what effects it has on our bodies remain unknown.

[0005] Currently, the main methods for stimulating cells within organ-on-a-chip include gas pressure stimulation and electrical stimulation. Adri'an López-Canosa et al. combined electrospun nanofibers with electrical stimulation in a microfabrication system to control the degree of anisotropy in cardiac tissue within a microdevice; Anna Marsano et al. designed a heart-on-a-chip and constructed a uniaxial cyclic strain pneumatic actuation system to predict signs of cardiac phenotypic hypertrophy by pressurizing the bottom compartment to deform PDMS.

[0006] However, existing chips have some drawbacks. For example, during the cell suspension injection process, the control group chip and the experimental group chip may not mix evenly, resulting in a significant deviation in the number of cells. The straight air chamber does not provide uniform mechanical stretching of the cells attached to the membrane in the straight culture chamber, and the different stretching directions of each cell make it difficult to observe the growth trend. The external injection system is difficult to fix on the pores of the organ chip, which can cause the organ chip to flip over or the needle to slip out. Summary of the Invention

[0007] The purpose of this invention is to provide a circular organ-on-a-chip and method for forming experimental and control groups in the same chip, which avoids the impact of uneven mixing of cell suspension on experimental results in the control chip and experimental chip; it also enables uniform stretching of cells, making it convenient to observe cell growth trends.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A ring-shaped organ-on-a-chip includes a thin film layer, a channel layer and a bottom layer disposed on both sides of the thin film layer, a culture chamber disposed on the channel layer, the culture chamber having a groove structure with the ends connected, and a liquid storage chamber connected to the culture chamber through a cell channel; pores communicating with each other are opened on the channel layer and the thin film layer, and an air cavity communicating with the pores is opened on the bottom layer; the air cavity has a groove structure, and the projection of the edge of the air cavity on the channel layer is located between the inner edge and the outer edge of the culture cavity.

[0009] Furthermore, the flow channel layer, thin film layer, and bottom layer are relatively fixed, and the flow channel layer and thin film layer are sealed by bonding / adhesion, and the thin film layer and bottom layer are sealed by bonding / adhesion.

[0010] Furthermore, the pores are connected to the center of the air cavity.

[0011] Furthermore, the culture chamber has an annular groove structure, the air chamber has a circular groove structure, and the pores have a circular hole structure. The diameter of the air chamber is larger than the inner diameter of the culture chamber but smaller than the outer diameter of the culture chamber.

[0012] Furthermore, the film layer is a biocompatible transparent opaque film.

[0013] Furthermore, the thickness of the flow channel layer is 4~5mm, the thickness of the bottom layer is 2~3mm, and the thickness of the thin film layer is 100μm~300μm.

[0014] Furthermore, the diameter of the through hole and the air hole is 1~1.5mm, and the diameter of the air cavity is 1.5mm~2.5mm.

[0015] Furthermore, the cell channel includes a pair of culture channels communicating with the culture chamber, and also includes two through holes opened on the flow channel layer communicating with the flow channel. Each culture channel and through hole is connected to a serpentine tube with a groove structure. Each through hole is connected to a liquid storage tank at the end away from the serpentine tube.

[0016] Furthermore, the serpentine tube is 4-5 mm long, the culture channel is 10-12 mm long and 0.5 mm wide.

[0017] A cell stretching method using the above-described ring-shaped organ-on-a-chip includes the following steps: Step 1: Inject collagen into one well to modify the cell channel until the collagen overflows from another well after passing through the serpentine tube, flow channel and culture chamber. Then place the organ-on-a-chip in an incubator.

[0018] Step 2: Inject the prepared cell suspension through the through-hole, then place the organ-on-a-chip in an incubator for 6-12 hours. After the cells grow tightly against the film layer, remove the organ-on-a-chip to prevent the injected culture medium from washing away the cells. Add culture medium through the through-hole and place the organ-on-a-chip in the incubator.

[0019] Step 3: Connect the injection pump to the vent, start the injection pump to continuously pump and push, periodically stretch and stimulate the cells on the thin film layer, and observe the results.

[0020] Furthermore, in step 3, when observing the results, the cells corresponding to the area inside the air cavity and the cells corresponding to the area outside the air cavity are divided into experimental group and control group.

[0021] Furthermore, in step 2, a cell suspension is prepared using human umbilical vein endothelial cells, with an endothelial cell concentration of 0.8 × 10⁻⁶. 6 ~1.5×10 6 / ml.

[0022] Furthermore, in step 2, the volume ratio of culture medium added to the two storage tanks is 6:1.

[0023] Furthermore, in step 3, the injection pump is connected to the air port via a metal tube needle.

[0024] Compared with the prior art, this application has the following beneficial effects: 1. The size of the air cavity is larger than the inner edge of the culture chamber but smaller than the outer edge, meaning the air cavity divides the culture chamber into an inner and outer ring. Cells in the inner ring adhere to the membrane and are located above the air cavity; under the influence of the air cavity, the cells in this region can be stretched. Cells in the outer ring adhere to the membrane and are bonded to the underlying layer outside the air cavity, unaffected by the air cavity. This directly forms a control group in the chip, allowing the effect of mechanical stimulation on cell growth to be demonstrated in the same chip.

[0025] 2. Some annular gas chambers have perfusion ports located at the edges, which cannot achieve uniform stimulation of the cells above the gas chamber. This application injects gas from the center of the gas chamber, providing mechanical stimulation to the cells within the culture chamber, resulting in a more uniform stimulation.

[0026] 3. Cells affected by mechanical stimulation grow in the same direction as the stretching direction. The direction of cell growth is affected by stimulation. When mechanical stimulation is applied to the cells in the culture chamber through the air cavity, the growth direction of the cells will be in a certain circular radius direction. Setting the air cavity as a circle makes it easier to observe later.

[0027] 4. Gas is infused and drawn through a metal syringe using a syringe pump. Furthermore, the culture chamber provides a more uniform force distribution from the center compared to other rectangular flow channel structures, enabling uniform pressure stimulation of cells in the upper flow channel layer and achieving effective and continuous cell movement. Because the metal syringe is located in the center of the chip, and the relatively thick flow channel layer provides support, the metal syringe is less likely to detach from the chip.

[0028] This application provides a ring-shaped organ-on-a-chip for stimulating cells without direct contact. It has a simple structure, is easy to operate, is easy to manufacture, and has good biocompatibility. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the internal structure of Embodiment 1 of the present invention; Figure 3 This is an exploded view of Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the lower surface of the flow channel layer in Embodiment 1 of the present invention; Figure 5 This is a schematic diagram of the culture medium perfusion in Example 2 of the present invention; Figure 6 This is a schematic diagram of the needle in Embodiment 2 of the present invention; Figure 7 This is a schematic diagram of the connection between the syringe and the injection pump in Embodiment 2 of the present invention; Figure 8 This is a schematic diagram of cells before air pressure stimulation in Embodiment 3 of the present invention; Figure 9 This is a schematic diagram of cells after air pressure stimulation in Embodiment 3 of the present invention; Figure 10 This is a schematic diagram of Embodiment 5 of the present invention; Figure 11 This is an exploded view of Embodiment 5 of the present invention; Figure 12 This is a schematic diagram of Embodiment 6 of the present invention; Figure 13 This is an exploded view of Embodiment 6 of the present invention; Figure 14 This is a schematic diagram of Embodiment 7 of the present invention; Figure 15 This is an exploded view of Embodiment 7 of the present invention.

[0030] In the figure: Flow channel layer 1, film layer 2, bottom layer 3, liquid storage tank 11, culture chamber 12, serpentine tube 13, culture channel 14, air chamber 31, first through hole 111, second through hole 112, air hole 113, third through hole 121, fourth through hole 122. Detailed Implementation

[0031] Example 1 A type of ring-shaped organ-on-a-chip, such as Figure 1-4 As shown, it includes a thin film layer 2 in the middle, and a flow channel layer 1 and a bottom layer 3 disposed on the upper and lower sides of the thin film layer 2. The flow channel layer 1, the thin film layer 2 and the bottom layer 3 are all horizontally disposed and relatively fixed. The flow channel layer 1 and the thin film layer 2, and the thin film layer 2 and the bottom layer 3 are connected by bonding or PDMS adhesive method. The material of the thin film layer 2 is PDMS film.

[0032] A culture chamber 12 is provided on the lower surface of the flow channel layer 1. The culture chamber 12 has a circular groove structure and is connected to a liquid storage tank 11 through a cell channel. The flow channel layer 1 and the film layer 2 have interconnected pores 113, which have a circular pore structure. The upper surface of the bottom layer 3 has an air cavity 31 that communicates with the pores 113. The air cavity 31 has a circular groove structure. The culture chamber 12, the pores 113 and the air cavity 31 are arranged coaxially. The diameter of the air cavity 31 is larger than the inner diameter of the culture chamber 12 and smaller than the outer diameter of the culture chamber 12.

[0033] like Figure 4 As shown, the cell channel includes a pair of culture channels communicating with the culture chamber 12, and two through holes on the flow channel layer 1 communicating with the culture channels 14. The through holes have a stepped hole structure with a lower diameter larger than the upper diameter. The upper parts of the two through holes are the first through hole 111 and the second through hole 112, respectively, and the lower parts of the two through holes are the third through hole 121 and the fourth through hole 122, respectively. The through holes and the air vents 113 are both vertically arranged. A serpentine tube 13 is connected between each culture channel and the through hole. The serpentine tube 13 has a serpentine groove structure, and both the serpentine tube 13 and the culture channel are located on the lower surface of the flow channel layer 1. A liquid storage chamber 11 is connected to the end of each through hole away from the serpentine tube 13. The liquid storage chamber 11 is located on the upper surface of the flow channel layer 1 and is a glass tube coaxial with the through hole. The depth of the culture channel is 100 μm, the depth of the air chamber 31 is 100 μm, the diameter of the glass tube is 8 mm, and the height is 15 mm.

[0034] Example 2 A cell stretching method using the circular organ-on-a-chip in Example 1 includes the following steps: Step 1: During the experiment, the cells (using human umbilical vein endothelial cells HUVEC as an example) should reach a confluence of approximately 70%–80%. Trypsin is used to change the cells from an adherent state to a floating state, and the process is terminated with a solution containing 20% ​​high glucose. After centrifugation, the supernatant is collected and added to the culture medium to prepare a cell density of 1×10⁻⁶ cells / mL. 6 Cell suspension of cells per ml.

[0035] Select an organ-on-a-chip that is not connected to an injection pump and sterilize it. Inject 10 μl of collagen into one well to modify the cell channels of the sterilized organ-on-a-chip until the collagen overflows from another well after passing through the serpentine tube 13, culture channel 14 and culture chamber 12. Then place the organ-on-a-chip in a cell culture incubator at 37°C and 5% carbon dioxide concentration for 10 min.

[0036] Step 2: The prepared cell suspension is injected into the cell channel through a through-hole. The cell suspension is evenly distributed within the upper flow channel layer 1, and the absence of fluorescence in the air cavity proves that there is no leakage of the cell suspension. After culturing the organ-on-a-chip in an incubator for 6 hours, cells are observed to grow tightly against the film layer 2. The organ-on-a-chip is then removed to prevent the injected culture medium from washing away the cells. 1200 μl and 200 μl of EGM-2 culture medium are then injected into the two glass tube reservoirs 11, respectively. The organ-on-a-chip is then placed in the incubator. Under the influence of gravity, the culture medium continuously perfuses the cell channel as follows: Figure 5 As shown.

[0037] Step 3, as follows Figure 6-7 As shown, insert the metal tube needle into the vent 113, place the 2ml syringe on the injection pump and clamp it, start the injection pump for continuous pumping, set the flow rate to 500μl, the unidirectional feeding time to 6s, the operation mode to continuous, and the operation direction to pump first and then push, to periodically stretch and stimulate the cells on the thin film layer 2, and observe the results; during observation, the organ-on-a-chip can be removed from the incubator and photographed under a microscope.

[0038] The injection pump draws and pushes air into the air chamber 31 through the syringe, needle, and vent 113, causing a change in air pressure within the air chamber 31. This causes deformation of the thin film layer 2 above the air chamber 31, stretching the cells on the thin film layer 2. Air is then injected through the vent 113 from the center of the air chamber 31, ensuring uniform deformation of the corresponding thin film layer 2 and providing uniform stimulation to the cells. When observing the results, cells on the thin film layer 2 corresponding to the area within the air chamber 31 are used as the experimental group, while cells on the thin film layer 2 corresponding to the area within the outer ring of the culture chamber outside the air chamber 31 are used as the control group.

[0039] Step 4: Change the culture medium in the glass tube reservoir 11 of the organ-on-a-chip every other day. Specifically, use a glass pipette to aspirate the original culture medium and then inject new culture medium, alternating the volume of culture medium injected into the two glass tubes. For example, if 1.2 ml of culture medium is added to the left reservoir 11 and 0.2 ml to the right reservoir 11 on the first day, then on the second day, add 1.2 ml of culture medium to the right reservoir 11 and 0.2 ml to the left reservoir 11.

[0040] Example 3 The rest of this embodiment is the same as that in Embodiment 2, except that the concentration of the injected cell suspension is 1.5 × 10⁻⁶. 6 Cells / ml. After 16 hours of cell adhesion, the syringe pump was started for continuous aspiration and dispensing, with a flow rate of 400 μl.

[0041] like Figure 8-9 As shown, after stretching for 9 hours, it is obvious that the number of cells has increased. The cells gather and grow towards the middle ring (the outline of the bottom air cavity 31), and the growth direction of the cells on the middle ring is mostly along the diameter direction, while the growth direction of the cells on the outer side is irregular.

[0042] Example 4 A method for preparing a ring-shaped organ-on-a-chip includes the following steps: Step 1, Fabrication and selection of thin film layer 2; Thin film layer 2 is preferably a PDMS film with a thickness of 50μm. Specifically, the PDMS prepolymer and curing agent are uniformly mixed at a ratio of 10:1, then vacuum-poured onto the lid of a petri dish, and dried by spin coating.

[0043] Step 2: Fabrication of the overall organ-on-a-chip structure; Using AutoCAD software, the designed flow channel layer 1 and bottom layer 3 structures are drawn as blueprints. After being processed into a mask, a silicon wafer with a microchannel structure (the selected culture channel depth is 100μm) is fabricated using soft photolithography. This wafer is then attached to a culture dish with double-sided adhesive to create a mold. Next, polydimethylsiloxane (PDMS) prepolymer and curing agent are uniformly mixed in a 10:1 ratio and poured into the mold. Through vacuuming, bubble blowing, drying, and peeling operations, a PDMS sheet with a microchannel structure is obtained, resulting in flow channel layer 1 and bottom layer 3. Subsequently, perforations are drilled at the through-hole positions of flow channel layer 1, and it is bonded to the PDMS film (or adhered with PDMS adhesive). After bonding (adhesion), perforations are drilled from the thin film side for pores 113, and then bottom layer 3 is bonded to it to obtain the chip (here, two through-holes penetrate the upper flow channel layer 1, and pores 113 penetrate both the upper flow channel layer 1 and the thin film layer 2). Finally, a small amount of PDMS was applied to the two through-holes using a glass tube. The chip was then placed in a drying oven at 65°C for 2 hours to obtain an organ-on-a-chip with a glass tube reservoir 11. Prior to the experiment, the chip underwent high-temperature sterilization and ultraviolet sterilization, and was stored in a clean bench.

[0044] Step 3: Mechanical stretching system connected to the infusion pump system; Place the completed organ-on-a-chip at the bottom of the culture dish and connect it to the infusion pump system using a metal needle and latex tubing. In use, insert the metal needle, which is attached to one side of the latex tubing, into the air cavity 31 through the pore 113 on the upper surface of the flow channel layer 1. Because the upper flow channel layer 1 is relatively thick and the metal needle is positioned at the very center of the chip, it will be supported and stand upright within it. A 2ml syringe is connected to the other side of the latex tubing.

[0045] Example 5 The rest of this embodiment is the same as that of embodiment 1, except that: Figure 10-11 As shown, the flow channel layer 1 is connected to the bottom layer 3, and there is no thin film layer 2; the diameter of the air cavity 31 is slightly smaller than the inner diameter of the culture cavity 12.

[0046] The organ-on-a-chip in this embodiment is used to observe cell growth morphology under negative pressure. In use, the needle of a 2ml syringe is connected to the vent 113, and the gas in the chamber is extracted using the syringe. Negative pressure is applied in the chamber, and the cells respond to the micro-changes in the environment. The culture chamber is stretched by the negative pressure, producing deformation towards the center, and the morphology of the cells in the culture chamber changes. Each negative pressure period is 6-8 hours. During observation, the organ-on-a-chip can be removed from the incubator and photographed under a microscope.

[0047] The specific procedure to confirm that the chamber is under negative pressure is to allow the piston inside the syringe to spring back after releasing the pressure. Therefore, after drawing gas from the chamber, clips need to be inserted into the piston and the syringe wall to prevent the piston from springing back.

[0048] Example 6 The rest of this embodiment is the same as that of embodiment 1, except that: Figure 12-13 As shown, no liquid reservoir 11 is provided; instead, a through-hole is used as the liquid reservoir 11. During chip fabrication, a 2mm punch is used to punch holes in the through-hole, and the resulting through-hole serves as its own liquid reservoir 11 to supply culture medium to the cells within the culture channel 14.

[0049] Example 7 The rest of this embodiment is the same as that of embodiment 1, except that: Figure 14-15 As shown, the serpentine tube can be modified into other channel configurations, such as a straight channel or a split channel.

[0050] Example 8 The rest of this embodiment is the same as that in embodiment 1, except that the thin film layer 2 is made directly from other biocompatible non-porous membranes, such as PC membranes, PET membranes, etc.

Claims

1. A ring organ chip comprising a middle thin film layer, further comprising a flow channel layer and a bottom layer arranged on both sides of the thin film layer, characterized in that, A culture chamber is provided on the lower surface of the flow channel layer, and the culture chamber is connected to a liquid storage tank through a cell channel; pores are provided on the flow channel layer and the film layer, and a metal tube needle is inserted into the pore to periodically stretch the cells on the film layer; an air cavity is provided on the upper surface of the bottom layer, which is connected to the pore; the projection of the edge of the air cavity on the flow channel layer is located between the inner edge and the outer edge of the culture chamber; the culture chamber is a circular groove structure, the air cavity is a circular groove structure, and the pore is a circular hole structure.

2. The ring organ chip of claim 1, wherein, The vent is connected to the center of the air cavity.

3. The ring organ chip of claim 1, wherein, The thin film layer is a PDMS thin film.

4. The ring organ chip of claim 1, wherein, The thickness of the flow channel layer is 4~5mm, the thickness of the bottom layer is 2~3mm, and the thickness of the thin film layer is 100μm~300μm.

5. The ring organ chip of any one of claims 1-4, wherein, The cell channel includes a pair of culture channels communicating with the culture chamber, and also includes two through holes opened on the flow channel layer communicating with the culture channels. Each culture channel and through hole is connected to a serpentine tube, which has a groove structure. Each through hole is connected to a liquid storage tank at the end away from the serpentine tube.

6. A cell stretching method using the ring-shaped organ chip according to claim 5, characterized by, Includes the following steps: Step 1: Inject collagen into one well to modify the cell channel until the collagen flows through the serpentine tube, culture channel and culture chamber and overflows from another well. Then place the organ-on-a-chip in an incubator. Step 2: Inject the prepared cell suspension through the through-hole, then place the organ-on-a-chip in an incubator for culture. After the cells grow tightly against the film layer, remove the organ-on-a-chip, add culture medium to the two reservoirs, and place the organ-on-a-chip in the incubator. Step 3: Connect the injection pump to the air vent, start the injection pump to continuously pump and push through the needle tube, needle tip and air vent to periodically stretch and stimulate the cells on the thin film layer, and observe the results. In step 3, when observing the results, the cells corresponding to the area inside the air cavity and the cells corresponding to the area outside the air cavity are divided into experimental group and control group.

7. The method of cell stretching of claim 6, wherein, In step 2, a cell suspension is prepared using human umbilical vein endothelial cells.

8. The method of claim 6, wherein the cell stretching is performed by a method comprising: In step 2, the ratio of culture medium added to the two storage tanks is 6:1.

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