A microfluidic chip for fixing a clamping organoid pellet and a preparation method thereof

Abstract

The application provides a microfluidic chip for fixing organoid microspheres, comprising: a microchannel structure; the microchannel structure comprises: an air cavity structure located at an upper layer and a clamping channel located at a lower layer; the air cavity structure forms a closed cavity for applying pressure to the clamping channel; the clamping channel is provided with a clamping structure, and the clamping structure is a claw-shaped structure composed of a plurality of conical bodies. The microfluidic chip can stably fix the organoid microspheres, prevent the organoid microspheres from drifting away from a detection surface, ensure the stability of the cell position in the signal acquisition process, thereby ensuring the continuous and stable acquisition of the signal in time and space, improving the continuity and accuracy of the signal acquisition; moreover, the fresh culture solution can flow, nutrients are provided for the organoid microspheres, and the long-term activity and function of the organoid microspheres are maintained; in addition, no chemical molecular pollution and interference are introduced, and the organoid microspheres can be released and recovered at any required time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, in particular to a microfluidic chip for fixing and clamping organoid spheroids and a preparation method thereof. BACKGROUND

[0002] Organoids have developed rapidly as a research hotspot in recent years, and they have important significance in basic research and clinical research. Organoids have more advantages than traditional 2D culture and can show physiological cell composition and behavior. The emergence of organoid research no longer confines medical research. By studying the response of organoids to external stimuli, the function and physiological mechanism of real organs can be better understood, the development process of diseases can be simulated, and new ideas and methods for disease treatment can be provided. This has important significance for understanding cell processes, disease mechanisms, and drug development.

[0003] Microelectrode array (MEA) is a commonly used electrical detection technology for recording the electrical activity of organoid spheroids. It can be used to monitor the electrical activity of neurons in organoids. MEA is a method that uses an array of electrodes to record the electrical signals of firing cells. These electrodes can capture the weak electrical signals generated by cells during activity, allowing real-time monitoring of cell activity. By analyzing these electrical signals, researchers can understand the electrical response of organoids to external stimuli.

[0004] In the step of obtaining electrical signals of organoid cells in traditional MEA technology, the MEA needs to be pre-treated before the organoid cells are placed on it. The MEA is coated with an adhesive layer for 30 minutes to ensure that its surface is suitable for cell growth and adhesion. The organoid cells are placed above the electrode array in an appropriate manner, and the cell's own adhesion and adhesion molecules are used to promote the adhesion of the cells to the MEA. The main purpose of pre-treating the MEA is to form a more intimate adhesion between the cells and the electrodes to obtain biological signals from the cells. The size and shape of the recorded signals depend on several factors: the nature of the contact between the cells and the MEA electrodes (such as contact area and tightness); the voltage amplitude detected by the electrode is inversely proportional to the distance of cell depolarization, so it is necessary to culture cells on the electrode to place cells as close to the electrode as possible.

[0005] The following problems exist in the process of obtaining electrical signals by treating cells using the MEA method: 1. The adhesion effect between the organoid ball and the electrode array is not obvious, and the organoid cells cannot be stably attached to the electrode surface, which results in leakage current, introduces noise, reduces the accuracy and reliability of measurement, and seriously interferes with the measurement of the biological electrical characteristics of the cells; 2. The cell position is not fixed and deviates from the detection position, and when the cell culture solution is perfused, the organoid ball may drift away from the detection surface, the cell position cannot be fixed, the detection is interrupted, and long-term effective detection cannot be achieved.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a microfluidic chip for fixing and clamping an organoid ball and a preparation method thereof, which can stably fix the organoid ball on the detection surface of a biosensor and realize perfusion culture of cells, so as to improve the stability of signal acquisition and the efficiency of cell culture.

[0008] In a first aspect, the present application provides a microfluidic chip for fixing and clamping an organoid ball, comprising: a microchannel structure; the microchannel structure comprises: an air cavity structure located on the upper layer and a clamping channel located on the lower layer; the air cavity structure forms a closed cavity for applying pressure to the clamping channel; the clamping channel is provided with a clamping structure, and the clamping structure is a claw-shaped structure composed of a plurality of conical bodies.

[0009] Preferably, the microchannel structure is provided with a sample inlet hole and a sample outlet hole at both ends, and the sample inlet hole and the sample outlet hole are respectively in communication with the clamping channel.

[0010] Preferably, the clamping structure is located in the middle of the clamping channel, and the air cavity structure is located above the clamping structure.

[0011] Preferably, the plurality of conical bodies form a space matched with the size of the organoid ball, so as to ensure that the organoid ball can be fixed in the clamping channel.

[0012] Preferably, the number of conical bodies is four.

[0013] Preferably, the microfluidic chip further comprises a glass electrode bonded with the microchannel structure.

[0014] Preferably, the clamping channel is a straight channel. The straight channel structure with one inlet and one outlet is used, and when in use, the liquid level difference between the sample inlet hole and the sample outlet hole drives the organoid ball to deform and enter the clamping area and be stably clamped, cultured and detected.

[0015] Preferably, the material of the micro-channel structure is formed by mixing PDMS monomer and curing cross-linking agent; more preferably, the PDMS monomer and the curing cross-linking agent are mixed in a mass ratio of (8-12):1. PDMS is selected as the main material of the micro-fluidic chip mainly based on the following advantages: first, it has high transparency, which is convenient for observation; second, the PDMS treated by oxygen plasma can form irreversible and firm chemical bonds on the glass surface, and the bonding process is fast, which can be completed in only 3 minutes; more importantly, the PDMS has good air permeability, which can effectively guarantee the exchange of gas and nutrients during cell culture, and improve the cell culture efficiency.

[0016] In a second aspect of the present application, a preparation method of a micro-fluidic chip for fixing and clamping organoid beads is provided, comprising the following steps:

[0017] S1, preparing a 3D printed micro-channel structure mold;

[0018] S2, mixing PDMS monomer and curing cross-linking agent, placing them in a vacuum pumping device to exhaust gas, pouring them on the 3D printed micro-channel structure mold, and placing them in an oven to cure the PDMS;

[0019] S3, peeling the cured PDMS from the mold to obtain a micro-channel structure, and punching sample inlets and outlets at both ends of the micro-channel structure.

[0020] Preferably, the method further comprises the following steps:

[0021] S4, placing the micro-channel structure and the glass electrode in a plasma processor, and bonding the surfaces of the micro-channel structure and the glass electrode treated by oxygen plasma, wherein the Si-OH groups on the surfaces of the two will undergo dehydration reaction to form Si-O-Si bonds, the formation of chemical bonds is the key to the firm combination between the PDMS and the glass electrode, which makes the bonding of the chip more compact, creates a micro-flow channel for the close contact of cells and sensors, and completes the preparation of the micro-fluidic chip.

[0022] In a third aspect of the present application, a micro-fluidic system is provided, comprising a perfusion system and the micro-fluidic chip for fixing and clamping organoid beads, wherein the perfusion system is connected with the micro-fluidic chip.

[0023] Preferably, the perfusion system comprises an injection pump and a control system, the injection pump is connected with the micro-fluidic chip through a pipeline, and the control system is used for controlling the working of the injection pump.

[0024] The present application has at least the following beneficial effects:

[0025] (1) The microfluidic chip of the present application is used for a biosensor, the clamping structure and the air cavity structure in the clamping channel can stably fix the organoid ball on the detection surface of the base in the microfluidic chip, ensure the stability of the cell position in the signal acquisition process, prevent the organoid ball from drifting away from the detection surface, and also prevent the cell from drifting and rotating to cause the change of the corresponding detected position of the organoid ball in contact with the sensor, so that it is possible to stably observe the change of the spatial characteristics of the organoid ball surface at multiple points over time, thereby ensuring the continuous and stable acquisition of the signal in the time and spatial dimensions, and improving the continuity and accuracy of the signal acquisition.

[0026] (2) The microfluidic chip of the present application considers the perfusion culture requirement of cells, the fresh culture solution is allowed to flow in the clamping channel to maintain the cell activity; the clamping structure clamps the organoid ball in the center of the clamping channel, occupies the middle part in the cross section, and the perfusion culture solution can flow from both sides and above the organoid ball to provide nutrients for the organoid ball, discharge waste liquid, and maintain the long-term activity and function of the organoid ball.

[0027] (3) The microfluidic chip of the present application adopts physical clamping, which will not introduce chemical molecular pollution and interference, and can release and recover the organoid cell ball at any time.

[0028] (4) The microfluidic chip of the present application is prepared by using conventional PDMS and the like, can be reused, and the microfluidic system is simple in structure and does not need external peripherals, and has the advantages of low cost. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 The microfluidic chip structure diagram for fixing and clamping the organoid ball provided by the present application is shown in the figure.

[0031] Figure 2 The top view of the microfluidic chip for fixing and clamping the organoid ball provided by the present application is shown in the figure.

[0032] Figure 3 The cross-sectional view of the microfluidic chip for fixing and clamping the organoid ball provided by the present application is shown in the figure.

[0033] Figure 4 The microfluidic system structure diagram provided by the present application is shown in the figure.

[0034] Explanation of reference numerals in the attached figures: 1. Microchannel structure; 2. Glass electrode; 3. Gas cavity structure; 4. Clamping channel; 5. Sample inlet; 6. Sample outlet; 7. Clamping structure; 8. Myocardial organoid sphere; 9. Injection pump. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0037] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly; for example, they may refer to a fixed connection, a detachable connection, or an integral connection; they may refer to a mechanical connection or an electrical connection; they may refer to a direct connection or an indirect connection through an intermediate medium; and they may refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0038] Example 1

[0039] like Figures 1 to 3 As shown, this embodiment provides a microfluidic chip for fixing and clamping organoid spheres, including: a microchannel structure 1 and a glass electrode 2 bonded to the microchannel structure; the microchannel structure 1 is divided into upper and lower layers, including: an air cavity structure 3 in the upper layer and a clamping channel 4 in the lower layer; the air cavity structure 3 forms a closed cavity for applying pressure to the clamping channel 4; a clamping structure 7 is provided in the clamping channel 4, and the clamping structure 7 is a claw-shaped structure composed of several cones.

[0040] In this embodiment, the microchannel structure 1 is provided with a sample inlet hole 5 and a sample outlet hole 6 at both ends, which are respectively communicated with the clamping channel 4. The clamping channel 4 is used in one-in and one-out, and in use, the liquid level difference between the sample inlet hole 5 and the sample outlet hole 6 drives the organoid ball to deform into the clamping area and be stably clamped for culture and detection.

[0041] In this embodiment, the clamping structure 7 is located in the middle of the clamping channel 4, and the air cavity structure 3 is located above the clamping structure 7. By applying additional pressure to the air cavity structure 3, the position of the clamping channel 4 can be controlled to be pressed downward, so as to exert a more stable and larger clamping force on the organoid ball. This "downward pressing" effect not only enhances the stability of clamping, but also significantly improves the capture ability of the organoid ball, ensuring higher clamping efficiency of the clamping channel 4 and more reliable experimental results.

[0042] In this embodiment, the several conical bodies form a space suitable for the size of the organoid ball, so as to ensure that the organoid ball can be fixed in the clamping channel 4.

[0043] In this embodiment, the number of conical bodies is four, and the spacing of the four conical bodies is determined by the diameter of the organoid ball, which is slightly smaller than the diameter of the organoid ball. The shape of the conical body is subtracted by the arc of the spherical shape in the middle, so as to ensure that the organoid ball can be stably embedded therein and not easily fall off.

[0044] In this embodiment, the clamping channel 4 is a straight channel, and in operation, only the flow rate needs to be controlled to make the organoid ball enter the clamping area. By changing the flow rate, the organoid ball can be released from the clamping position. The size of the clamping channel 4 is adjusted according to the size of the organoid ball, so as to ensure that the ball can be stably clamped in the channel.

[0045] In this embodiment, the material of the microchannel structure 1 is formed by mixing PDMS monomer and solidified crosslinking agent.

[0046] The embodiment also provides a preparation method of the microfluidic chip for fixing and clamping the organoid ball, which comprises the following steps:

[0047] S1, preparing a 3D printed microchannel structure mold;

[0048] Specifically, a channel with a claw-shaped clamping structure can be designed by using three-dimensional design technology, and is accurately manufactured by 3D printing technology. The clamping structure 7 is designed by using three-dimensional modeling software, and the height and spacing of the clamping structure 7 are related to the size and diameter of the organoid ball.

[0049] S2, mix the PDMS monomer with the curing crosslinking agent at a mass ratio of 10:1, stir evenly, and then place in a vacuum device to exhaust gas and pour on a 3D printed microchannel structure mold, and then place in an oven to cure the PDMS;

[0050] S3, the cured PDMS is peeled off from the mold to obtain a microchannel structure 1, and sample inlets 5 and sample outlets 6 are punched at both ends of the microchannel structure 1;

[0051] S4, the microchannel structure 1 and the glass electrode 2 are placed in a plasma processor, and the surface of the microchannel structure 1 and the glass electrode 2 after oxygen plasma treatment are attached to each other, so that the microfluidic chip is manufactured.

[0052] In use, the microfluidic chip is connected to a perfusion system, and an organoid ball is transported into the clamping channel 4 through the injection pump 9 and the pipeline of the perfusion system; the injection pump 9 transports the culture solution at a set flow rate and flow rate for cell perfusion culture; in the cell clamping area, the organoid ball is clamped in the clamping structure 7 according to the pressure difference between the sample inlet 5 and the sample outlet 6, and the contact area between the cell and the glass electrode 2 is stably increased, and in this process, the sensor electrode array located below the clamping position can accurately collect the weak signal generated by the cell.

[0053] Embodiment 2

[0054] As shown in Figure 4 , the embodiment provides a microfluidic system, which comprises a perfusion system and the microfluidic chip for clamping an organoid ball of embodiment 1, and the perfusion system is connected to the microfluidic chip.

[0055] In the embodiment, the perfusion system comprises an injection pump 9 and a control system, the injection pump 9 is connected to the microfluidic chip through a pipeline, and the control system is used for controlling the working of the injection pump 9.

[0056] Specifically, the perfusion system can be integrated by an injection pump 9, a transmission device, a control system and a pipeline, the injection pump 9 is connected to the sample inlet 5 of the microfluidic chip to ensure the continuous flow of fresh culture solution; fresh culture solution is added to the syringe, and the injection pump 9 transports the culture solution to the cell culture area at a set flow rate and flow rate through the pump and the pipeline of the perfusion system, and the perfusion rate and the perfusion time can be adjusted as needed to meet the growth needs of the cells; at the sample outlet 6, a liquid storage tank is inserted to realize continuous perfusion culture.

[0057] Detection example

[0058] The detection example adopts the microfluidic chip of example 1 and the microfluidic system of example 2 to realize stable detection of the electrophysiological signal of the myocardial organoid ball 8. First, sterilize the chip, instrument and reagent used, and then use the sterilized equipment to build the flow channel circuit of the chip.

[0059] In the perfusion system, the injection pump 9 is used to provide power to pump the culture solution into the cell culture chamber and discharge the waste liquid through the microchannel structure 1 made in the chip. The myocardial cells used in the experiment are injected into the chip chamber from the cell injection hole 5 of the chip using a syringe, and the injection pump 9 is used to continuously perfuse and culture the myocardial cells in the chip. The culture solution is continuously and stably input into the chip at a fixed flow rate from the flow channel injection hole 5, which ensures that the organoid ball can be stably clamped.

[0060] In the acquisition system, the MEA with 100 electrodes is used to constitute a sensor for acquiring cell signals. Myocardial cells are cells with automaticity and excitability, and can spontaneously generate electrical signals. The microelectrode array acquires the potential change generated by the myocardial cells on the electrode, acquires the weak signal generated by the myocardial cells, inputs the acquisition card, and converts the digital signal into a digital signal through the biosensor platform and wirelessly transmits it to the computer. Filter and display on the computer. The detection result shows that the stable biological signal of myocardial cell beating can be clearly and obviously observed.

[0061] In summary, the microfluidic chip of the present application is used for a biosensor, and the clamping structure 4 and air cavity structure 3 in the clamping channel 4 can stably fix the organoid ball on the detection surface of the microfluidic chip, ensuring the stability of the cell position during signal acquisition, preventing the organoid ball from drifting away from the detection surface, and also preventing the cell from drifting and rotating to cause changes in the corresponding detected position of the organoid ball in contact with the sensor. It is possible to stably observe the surface multi-point spatial characteristics of the organoid ball over time, thereby ensuring the continuous and stable acquisition of signals in the time and spatial dimensions, improving the continuity and accuracy of signal acquisition. The microfluidic chip of the present application considers the perfusion culture requirements of cells, and the clamping channel 4 allows fresh culture solution to flow, maintaining cell activity; the clamping structure clamps the organoid ball in the center of the clamping channel, occupying the middle part in the cross section, and the perfusion culture solution can flow from both sides and above the organoid ball, providing nutrients for the organoid ball, discharging waste, and maintaining the long-term activity and function of the organoid ball. The microfluidic chip of the present application uses physical clamping, which does not introduce chemical molecular pollution and interference, and can release and recover the organoid cell ball at any time. The microfluidic chip of the present application is made of conventional PDMS and other materials, and can be reused. Moreover, the microfluidic system is simple in structure and does not require external peripherals, having the advantage of low cost.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A microfluidic chip for fixing and holding organoid spheres, characterized in that, The microchannel structure comprises: a gas cavity structure on the upper layer and a clamping channel on the lower layer. The gas cavity structure forms a closed cavity for applying pressure to the clamping channel; the clamping channel is provided with a clamping structure, which is a claw-shaped structure composed of a plurality of conical bodies; and the microchannel structure is bonded with a glass electrode. The microchannel structure is provided with an inlet hole and an outlet hole at both ends, which respectively communicate with the clamping channel.

2. The microfluidic chip for immobilizing and holding the organoid pellet according to claim 1, wherein, The clamping structure is located in the middle of the clamping channel, and the gas cavity structure is located above the clamping structure; the clamping channel is a straight channel.

3. The microfluidic chip for immobilizing a clamped organoid pellet according to claim 1, wherein, The plurality of conical bodies form a space suitable for the size of the organoid spheroid, ensuring that the organoid spheroid can be fixed in the clamping channel.

4. The microfluidic chip for immobilizing a clamped organoid pellet according to claim 1, wherein, The number of conical bodies is four.

5. The microfluidic chip for immobilizing a clamped organoid pellet according to claim 1, wherein, The method comprises the following steps:

6. The method of claim 1-5, wherein the microfluidic chip for immobilizing the spheroids of the organoids is prepared by, S1, preparing a 3D printed microchannel structure mold; S2, mixing and stirring the PDMS monomer with the curing crosslinking agent, then placing it in a vacuum device to exhaust gas, pouring it on the 3D printed microchannel structure mold, and placing it in an oven to cure the PDMS; S3, peeling the cured PDMS from the mold to obtain the microchannel structure, and punching the inlet hole and outlet hole at both ends of the microchannel structure. The method further comprises the following steps:

7. The method for fabricating a microfluidic chip for fixing and holding organoid spheres according to claim 6, characterized in that, S4, placing the microchannel structure and the glass electrode in a plasma processor, and bonding the surfaces of the microchannel structure and the glass electrode after oxygen plasma treatment. The method comprises:

8. A microfluidic system, characterized in that, A perfusion system and a microfluidic chip for fixing and clamping organoid spheroids according to any one of claims 1-5, wherein the perfusion system is connected with the microfluidic chip. The perfusion system comprises: an injection pump connected with the microfluidic chip through a pipeline, and a control system for controlling the operation of the injection pump.

9. The microfluidic system of claim 8, wherein, ​

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