Cell stretching method, micro-fluidic chip and organoid generation platform

Abstract

The invention discloses a cell stretching method, a micro-fluidic chip and an organoid generation platform. The cell stretching method comprises the following steps that a body is provided, the body is provided with a capillary channel, the capillary channel comprises a sample inlet and a sample outlet, and the diameter of the sample outlet is smaller than the maximum diameter of micro-tissue to be stretched; and injecting a fluid containing the microstructure from the sample inlet, and enabling the fluid to flow out from the sample outlet. In the prior art, mechanical force is applied to a micro-fluidic chip, so that a cell mechanical microenvironment is simulated for cells in the chip, and the experimental flux is low. According to the cell stretching method, the capillary channel of which the diameter of the sample outlet is smaller than the maximum diameter of the micro-tissue to be stretched is arranged, and the fluid with the micro-tissue is injected into the capillary channel, so that the micro-tissue is extruded and deformed by the inner wall in the capillary channel and flows out of the sample outlet, and cells do not need to stay in a micro-fluidic chip; and the cells can be continuously cultured through the continuous fluid, so that the experimental flux is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a cell stretching method, a microfluidic chip and an organoid generation platform. Background Art

[0002] Microfluidic chips are a type of science and technology that is mainly characterized by the manipulation of fluids in micrometer-scale space. Under normal physiological conditions in the body, almost all cells are subjected to various mechanical forces during development, such as tensile force, compressive force, and shear force, which are applied to cells through the extracellular matrix. The response of cells to these forces determines basic biological functions such as gene expression, morphology, proliferation, differentiation, and migration, and plays a key role in tissue function, organoid formation, immune response, wound healing, embryonic development, and cancer formation and metastasis.

[0003] In the technical field of in vitro cell culture, microfluidic chips are used to simulate mechanical stimulation during cell growth in an in vitro environment to achieve the same-direction arrangement and growth of cells in the extracellular matrix. In the prior art, the study of organoid cells often involves setting up a cell culture device, and then applying mechanical stimulation such as stretching and squeezing to the outside of the device to simulate the cell mechanical microenvironment, so that the cells in the device feel the external force stimulation and develop into the target type of morphology. However, the prior art has the problem of low experimental throughput. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a cell stretching method that can increase the experimental throughput.

[0005] The present invention also provides a microfluidic chip and an organoid generation platform having the microfluidic chip.

[0006] According to the first aspect of the present invention, the cell stretching method of the embodiment includes the following steps: providing a main body, the main body having a capillary channel, the capillary channel including an inlet and an outlet, the diameter of the outlet is smaller than the maximum diameter of the microtissue to be stretched, and the microtissue contains the cells; injecting a fluid containing the microtissue from the inlet, and allowing the fluid to flow out from the outlet.

[0007] According to the cell stretching method of the embodiment of the present invention, there are at least the following beneficial effects: in the prior art, for cell culture, the microtissue containing cells is set in a microfluidic chip, and then the microfluidic chip is stretched to simulate the cell mechanical microenvironment for the microtissue in the chip, but the size of the microfluidic chip is limited, and the cells that can be cultured in a single chip are also limited, and the experimental throughput is low. The cell stretching method of the present invention sets a capillary channel with a sample outlet having a diameter smaller than the maximum diameter of the microtissue to be stretched, and injects a fluid with microtissue into the capillary channel, so that the microtissue is squeezed and deformed in the thickness direction of the inner wall in the capillary channel, thereby being stretched along the extension direction of the capillary channel and flowing out from the sample outlet. The microtissue does not need to stay in the microfluidic chip, but can continuously culture cells through a continuous fluid, thereby improving the experimental throughput.

[0008] According to some embodiments of the present invention, the method further includes the following steps: the diameter of the capillary channel gradually decreases from the injection port to the outlet, the diameter of the injection port is not less than the maximum diameter of the microtissue to be stretched, the fluid is injected from the injection port, and the fluid flows out from the outlet.

[0009] According to some embodiments of the present invention, the method further comprises the following steps: from the injection port to the outlet port, the diameter of the capillary channel remains constant, the fluid is injected from the injection port, and the fluid flows out from the outlet port.

[0010] According to some embodiments of the present invention, the method also includes the following steps: the capillary channel includes a first section, a second section and a third section connected in sequence, the end of the first section away from the second section is the sample inlet, the end of the third section away from the second section is the sample outlet, the diameter of the first section is greater than the diameter of the third section, the diameter of the second section gradually decreases from the first section to the third section, the diameter of the first section is constant, the diameter of the third section is constant, the fluid is injected from the sample inlet, and the fluid flows out from the sample outlet.

[0011] According to some embodiments of the present invention, the method further includes the following steps: the body has a plurality of independent capillary channels, the fluid is injected from the injection port of each capillary channel respectively, and the fluid flows out from each outlet.

[0012] According to some embodiments of the present invention, the method further comprises the following step: adjusting the flow rate of the fluid so that the flow rate of the fluid in each capillary channel is different.

[0013] According to the microfluidic chip of the second aspect of the present invention, the microfluidic chip is used for cell stretching, and includes a body and a capillary channel. The capillary channel includes an inlet and an outlet, and the capillary channel is arranged in the body. The capillary channel includes a first section, a second section, and a third section connected in sequence, the end of the first section away from the second section is the inlet, the end of the third section away from the second section is the outlet, the diameter of the first section is greater than the diameter of the third section, and the diameter of the second section gradually decreases from the first section to the third section, the diameter of the first section is constant, the diameter of the third section is constant, and the diameter of the third section is smaller than the maximum diameter of the microtissue to be stretched.

[0014] The microfluidic chip according to the embodiment of the present invention has at least the following beneficial effects: the microfluidic chip of the present invention, by setting a capillary channel with inconsistent diameters at both ends in the chip, allows the fluid containing microtissues to enter from the inlet at the large diameter end, be squeezed and deformed by the inner wall of the channel in the capillary channel, and flow out from the outlet at the small diameter end. The microtissue does not need to stay in the microfluidic chip, but cells can be continuously cultured through continuous fluid, thereby improving the experimental throughput.

[0015] According to some embodiments of the present invention, the diameter of the first section is D1, the diameter of the third section is D3, 0.98 mm ≤ D1 ≤ 1.02 mm, 0.48 mm ≤ D3 ≤ 0.52 mm.

[0016] According to some embodiments of the present invention, the length of the first segment is L1, the length of the second segment is L2, the length of the third segment is L3, 29mm≤L1≤31mm, 39mm≤L2≤41mm, 85mm≤L3≤95mm.

[0017] According to some embodiments of the present invention, a plurality of the capillary channels are disposed in the body, and each of the capillary channels is independent of each other.

[0018] According to some embodiments of the present invention, the material of the body is any one of polymethyl methacrylate, polydimethylsiloxane, and polytetrafluoroethylene.

[0019] The organoid generation platform according to the third aspect of the present invention comprises the microfluidic chip described in any one of the second aspect of the present invention, and further comprises an injection pump and an organoid culture system. The organoid culture system is connected to the sample outlet, and the organoid culture system is used to receive and culture the microtissue; the injection pump is connected to the sample inlet, and the injection pump is used to inject the fluid with the microtissue into the capillary channel.

[0020] The organoid generation platform according to the embodiment of the present invention has at least the following beneficial effects: the microfluidic chip of the present invention, by setting capillary channels with inconsistent diameters at both ends in the chip, allows the fluid containing microtissues to enter from the inlet at the large diameter end, be squeezed and deformed by the inner wall of the channel in the capillary channel, and flow out from the outlet at the small diameter end. The microtissues do not need to stay in the microfluidic chip, but cells can be continuously cultured through continuous fluid. The organoid generation platform using the microfluidic chip can effectively improve the throughput of cultured cells.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 is a three-dimensional diagram of a microfluidic chip in one embodiment of the present invention; Figure 2 is a cross-sectional view of the microfluidic chip in the first embodiment of the present invention; Figure 3 is a cross-sectional view of a microfluidic chip in a second embodiment of the present invention; Figure 4 is a cross-sectional view of a microfluidic chip in a third embodiment of the present invention; Figure 5 This is a schematic diagram of the morphology of cells on different dates after stretching in one embodiment of the present invention; Figure 6 This is a comparison diagram of the orientation angles of cells before and after stretching in one embodiment of the present invention; Figure 7 This is a comparison diagram of the ellipticity of cells before and after stretching in one embodiment of the present invention.

[0023] Reference numerals: microfluidic chip 100 , body 101 , sample inlet 102 , capillary channel 201 , microtissue 202 , sample outlet 203 , first section 204 , second section 205 , third section 206 . DETAILED DESCRIPTION

[0024] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0025] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., and orientations or positional relationships indicated are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0026] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0027] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0028] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0029] It should be noted that the microtissue 202 described herein includes various cell forms such as organoids, spheroids, hydrogels carrying cells, cell clusters without extracellular matrix, tissue engineering, and cell models, and the principle of the cell stretching method is to stretch the above-mentioned various forms of microtissues 202, so that the cells in the microtissue 202 are stretched and deformed by external force. Figures 5 to 7 It can be seen that after stretching the microtissue 202, the cell morphology inside it can be subjected to the same deformation effect, so the article will directly discuss the cells themselves when describing the beneficial effects. In addition, it should be noted that, if the process allows, in some embodiments of the present invention, the size of the capillary channel 201 can also be reduced to the scale of a single cell, that is, the stretching deformation is performed on the scale of a single cell. In this embodiment, the diameter of the sample outlet 203 is set to be smaller than the maximum diameter of the cell to be stretched, and the subsequent morphologies of various capillary channels 201 are also designed based on the size of the cell.

[0030] refer to Figure 1 and Figure 2 According to the first aspect of the present invention, the cell stretching method comprises the following steps: providing a body 101, the body 101 has a capillary channel 201, the capillary channel 201 includes an inlet 102 and an outlet 203, the diameter of the outlet 203 is smaller than the maximum diameter of the microtissue 202 to be stretched; injecting a fluid containing the microtissue 202 from the inlet 102, and causing the fluid to flow out from the outlet 203. In the prior art, for cell culture, the microtissue 202 is arranged in a microfluidic chip 100, and the cell mechanical microenvironment is simulated for the microtissue 202 in the chip by stretching the microfluidic chip 100. However, the size of the microfluidic chip 100 is limited, and the microtissue 202 that can be cultured in a single chip is also limited, and the experimental throughput is low, or a cell stretching method using some non-microfluidic chips is used. However, whether using microfluidic technology or non-microfluidic technology, there is a technical problem of low experimental throughput. The cell stretching method of the present invention is to set a capillary channel 201 with a sample outlet 203 having a diameter smaller than the maximum diameter of the microtissue 202 to be stretched, and inject a fluid with the microtissue 202 into the capillary channel 201, so that the microtissue 202 is squeezed and deformed in the thickness direction of the inner wall in the capillary channel 201, thereby being stretched along the extension direction of the capillary channel 201 and flowing out from the sample outlet 203. Cells do not need to stay in the microfluidic chip 100, but can be continuously cultured through continuous fluid, thereby improving the experimental throughput. Moreover, it needs to be further described that in the prior art, the microtissue 202 is usually attached to the membrane surface or the bottom of the dish to produce a stretching effect, or is stretched by supporting a pillar. The cell stretching method of the embodiment of the present invention can continuously stretch the microtissue 202 and cells, thereby significantly improving the experimental throughput.

[0031] It should be noted that in some embodiments of the present invention, a flexible main body 101 material can also be used. When the microtissue 202 flows through the capillary channel 201, the shape of the channel can be dynamically adjusted by applying external force stimulation to the main body 101 to achieve mechanical stimulation of the microtissue 202, which can also further enhance the effect of stretching and deforming the microtissue 202 in the fluid.

[0032] It should be noted that, in some embodiments of the present invention, after the microtissue 202 flows out of the sample outlet 203, magnetic particles, optical tweezers technology or atomic force microscopy can also be used to apply a stretching force to the organoid to further stimulate individual cells and deform them into a desired shape.

[0033] refer to Figure 3In some embodiments of the present invention, the method further comprises the following steps: from the injection port 102 to the outlet port 203, the diameter of the capillary channel 201 gradually decreases, the diameter of the injection port 102 is not less than the maximum diameter of the microtissue 202 to be stretched, the fluid is injected from the injection port 102, and the fluid flows out from the outlet port 203. The capillary channel 201 designed in this way is more convenient to process in structure, and can gradually squeeze the cells to stretch and deform them, thereby improving the cell stretching effect.

[0034] refer to Figure 4 In some embodiments of the present invention, the method further comprises the following steps: from the injection port 102 to the outlet 203, the diameter of the capillary channel 201 remains constant, the fluid is injected from the injection port 102, and the fluid flows out from the outlet 203. Such a design allows the microtissue 202 to be in an extruded state for a longer period of time, so that it can be better shaped, and because the diameter remains constant, the outer diameter of the deformed microtissue 202 can be closer to the diameter of the outlet 203, and the stretching effect is more significant.

[0035] refer to Figure 1 and Figure 2 In some embodiments of the present invention, the method further includes the following steps: the capillary channel 201 includes a first section 204, a second section 205 and a third section 206 connected in sequence, the end of the first section 204 away from the second section 205 is the injection port 102, the end of the third section 206 away from the second section 205 is the outlet port 203, the diameter of the first section 204 is greater than the diameter of the third section 206, the diameter of the second section 205 gradually decreases from the first section 204 to the third section 206, the diameter of the first section 204 is constant, the diameter of the third section 206 is constant, the fluid is injected from the injection port 102, and the fluid flows out from the outlet port 203. In this way, the fluid carrying the microtissue 202 can reach a stable flow rate when passing through the first section 204 with a constant diameter. When each microtissue 202 reaches the second section 205, the diameter of the second section 205 gradually and evenly decreases, and is squeezed by the vertical wall. The squeezing force perpendicular to the flow direction causes the diameter of the microtissue 202 to decrease, and the component force in the opposite direction of the flow causes the microtissue 202 to be squeezed and deformed unidirectionally. After the deformed microtissue 202 is further solidified or its shape is maintained in the third section 206, it flows out of the capillary channel 201 for subsequent culture. In this way, the microtissue 202 can first stabilize the flow rate, then gradually deform, and finally solidify to maintain its shape, thereby improving the yield and quality of cell stretch deformation. It should be noted that, referring to Figure 2 Preferably, the diameter of the first section 204 is not less than the maximum diameter of the microtissue 202, so that the microtissue 202 can stabilize the flow rate without being squeezed, and then be squeezed, stretched and shaped in the second section 205 and the third section 206.

[0036] It should be noted that the above three-stage embodiment is a preferred embodiment of the cell stretching method of the present invention. Figures 5 to 7 The control test data is also the effect diagram produced when the structure of the capillary channel 201 of this embodiment is used. Figures 5 to 7 Elaborate on this. Figure 5 , Figure 5 The left picture in the figure is a microscopic picture of cells on the day after stretching. In the picture, some cells have undergone obvious morphological changes after stretching and are elliptical. The right picture is a microscopic picture of cells on the second day after stretching using the stretching method of an embodiment of the present invention. It can be seen that Figure 5 The cells pointed by the arrows in the right figure have begun to proliferate in the direction of stretching. Therefore, the stretching method of the embodiment of the present invention not only produces morphological changes in the stretched cells themselves, but also enables the cells to proliferate in the stretching direction. This helps to improve the alignment of the cells during the culture process and enables the dividing cells to proliferate and arrange in the stretching direction. Figure 6 , Figure 6 The left figure is the unstretched control group, and the right figure is the stretched experimental group. The cell orientation angle refers to the angle at which the endpoints of the deformed cells are offset from the vertical direction (the ideal cell is round, and the cell orientation angle is 0 degrees or 180 degrees). It can be seen that the cell orientation angles of the control group are evenly distributed along all angles, and most of the cells in the experimental group are oriented to 90 degrees after being stretched, that is, extended in the horizontal direction. Therefore, the cell stretching method of the embodiment of the present invention has a significant effect on the uniform deformation of cells. Figure 7 , Figure 7 The left figure is the unstretched control group, and the right figure is the stretched experimental group. The closer the circularity value is to 1, the more the shape of the cell is similar to a circle. More than 80% of the cells in the control group have a circularity of 0.9 or above, while the number of cells in the experimental group with a circularity of 0.9 or above is less than 60%, that is, most cells have undergone morphological changes after being stretched by the method of the embodiment of the present invention, and are close to an ellipse, with a significant effect.

[0037] In some embodiments of the present invention, the method further comprises the following steps: the body 101 has a plurality of mutually independent capillary channels 201, the fluid is injected from the injection port 102 of each capillary channel 201, and the fluid is made to flow out from each outlet 203. In this way, the plurality of capillary channels 201 can be used simultaneously to stretch and culture the microtissues 202, further improving the experimental throughput and obtaining more experimental samples and data.

[0038] In some embodiments of the present invention, the method further comprises the following steps: adjusting the flow velocity of the fluid so that the flow velocity of the fluid in each capillary channel 201 is different. The flow velocity variation can produce shear forces of different sizes to the cells sandwiched in the fluid, and when the cells are in the flowing liquid, the speed and viscosity of the fluid can control the forces acting on the cells. These forces can cause the cells to undergo hydrodynamic deformation, i.e., the shape of the cells in the fluid changes to adapt to the flow state of the fluid, thereby making the flow velocity of the fluid in each capillary channel 201 different, and the influence of different flow velocities on cell morphology can be more intuitively seen, increasing the experimental flux.

[0039] It should be noted that, in some embodiments of the present invention, the method further includes the following steps: for the same capillary channel 201 in the main body 101, the flow rate of the fluid is changed during the injection process, so as to observe the impact of different fluid flow rates on the formed cells during the shrinkage and stretching of the microtissue 202.

[0040] refer to Figure 1 and Figure 2 According to the microfluidic chip 100 of the second embodiment of the present invention, the microfluidic chip 100 is used for cell stretching, and includes a body 101 and a capillary channel 201. The capillary channel 201 includes an inlet 102 and an outlet 203. The capillary channel 201 is arranged in the body 101. The capillary channel 201 includes a first section 204, a second section 205 and a third section 206 connected in sequence. The end of the first section 204 away from the second section 205 is the inlet 102, and the end of the third section 206 away from the second section 205 is the outlet 203. The diameter of the first section 204 is greater than the diameter of the third section 206. From the first section 204 to the third section 206, the diameter of the second section 205 gradually decreases. The diameter of the first section 204 is constant, the diameter of the third section 206 is constant, and the diameter of the third section 206 is smaller than the maximum diameter of the microtissue 202 to be stretched. The microfluidic chip 100 of the present invention provides a capillary channel 201 with different diameters at both ends in the chip, so that the fluid containing the microtissue 202 enters from the inlet 102 at the large diameter end, is squeezed and deformed by the inner wall of the capillary channel 201, and flows out from the outlet 203 at the small diameter end. The microtissue 202 does not need to stay in the microfluidic chip 100, but cells can be continuously cultured through continuous fluid, thereby improving the experimental throughput.

[0041] refer to Figure 2In some embodiments of the present invention, the diameter of the first section 204 is D1, and the diameter of the third section 206 is D3, 0.98mm≤D1≤1.02mm, 0.48mm≤D3≤0.52mm. The first section 204 is mainly used to ensure the stable fluidity of the microtissue 202 and avoid flow rate fluctuations and turbulence. D1 can specifically be 0.92mm, 1mm, 1.02mm and other values, and the preferred value is 1mm. When D1=1mm, stable fluctuations can be achieved under most experimental conditions. The function of the third section 206 is to ensure that the cells are solidified in the deformed state after stretching and maintain the structural bendability. D3 can specifically be 0.48mm, 0.5mm, 0.52mm and other values, and the preferred value is 0.5mm. When D3=0.5mm, the structural integrity of the cells after stretching can be effectively maintained. It should be noted that the diameter of the second section 205 is D2, and the value of D2 smoothly transitions from D1 to D3 and gradually decreases. The second section 205 is the main section in the capillary channel 201 for achieving cell stretching, and is used to apply uniaxial tensile force to the cells to achieve deformation.

[0042] refer to Figure 2 In some embodiments of the present invention, the length of the first section 204 is L1, the length of the second section 205 is L2, and the length of the third section 206 is L3, 29mm≤L1≤31mm, 39mm≤L2≤41mm, 85mm≤L3≤95mm. Such a design can allow the microtissue 202 to have enough length and time to undergo a process of stable flow, gradual shrinkage and deformation, and post-deformation solidification. Furthermore, the preferred value of L1 is 30mm, the preferred value of L2 is 40mm, and the preferred value of L3 is 90mm. It should be noted that Figures 5 to 7 The experimental data is a schematic diagram of the experimental group obtained by using the preferred values ​​for the lengths and diameters of the three segments, namely D1=1mm, D3=0.5mm, L1=30mm, L2=40mm, and L3=90mm.

[0043] It should be noted that the length and diameter ranges of the first section 204, the second section 205 and the third section 206 are suitable for stretching the microtissue 202. When the capillary channel 201 in the embodiment of the present invention is suitable for the size of a single cell, the lengths and diameters of the three sections can be reduced proportionally. Specifically, the minimum range of the diameter can be 30 μm, and the length can be controlled between 0-10 cm, so as to achieve the stretching of a single cell. The maximum range of the diameter can be 5000 μm, so as to achieve the stretching of structures such as larger microtissues 202. The focus is on the core features that the first section 204 is used to stabilize the flow rate, the second section 205 is used to achieve the reduction of the microtissue 202 or the single cell, and the third section is used to solidify the microtissue 202 or the single cell after deformation, and the lengths and diameters of the three sections are kept proportionally enlarged or reduced. The specific sizes can be selected and adjusted according to the process means and experimental requirements.

[0044] It should be noted that the cross-sectional shape of the capillary channel 201 in the embodiment of the present invention can be circular. In some embodiments, it can also be set to a rectangular, triangular or other shape as required to meet the needs of diverse cell culture. When it is set to a non-circular shape, the above requirements for the diameter size refer to the diameter of the inscribed circle of the cross section of the capillary channel 201.

[0045] In some embodiments of the present invention, a plurality of capillary channels 201 are disposed in the body 101, and each capillary channel 201 is independent of each other. In this way, multiple capillary channels can be used simultaneously to stretch and culture cells, further improving the experimental throughput and obtaining more experimental samples and data.

[0046] In some embodiments of the present invention, the material of the body 101 is any one of polymethyl methacrylate, polydimethylsiloxane, and polytetrafluoroethylene. Polymethyl methacrylate (PMMA) has high transparency, is easy to observe, easy to process, and has good biocompatibility; polydimethylsiloxane (PDMS) has excellent flexibility, excellent biocompatibility, and high transparency; polytetrafluoroethylene (PTFE) has excellent tensile properties, strong chemical inertness, and is suitable for complex fluid applications. The three have their own advantages, and different materials can be used according to different needs. In addition, different materials can also be used to manufacture microfluidic chips 100 through different processing methods. Through efficient manufacturing methods, microfluidic chips 100 that meet experimental needs can be manufactured without overly complex process means.

[0047] Several embodiments of the manufacturing method are described below: When the material of the body 101 used is PMMA, a first component having a first surface and a second component having a second surface can be prepared, a first groove is machined on the first surface, and a second groove is machined on the second surface; the first surface and the second surface are bonded and spliced ​​so that the first component and the second component together form the body 101, and the first groove and the second groove together define the capillary channel 201. In this way, the size of the capillary channel 201 can be processed more accurately, and further, the surface roughness of the capillary channel 201 can be processed more finely, so that Ra20nm≤the surface roughness of the first groove≤Ra100nm, Ra20nm≤the surface roughness of the second groove≤Ra100nm, so that cells can flow smoothly on the surface of the capillary channel 201 without being broken. It should be noted that the above manufacturing method is also applicable to scenarios where the material of the main body 101 is PC (polycarbonate), PTFE (polytetrafluoroethylene), etc. Glass can also be used as the material of the main body 101. Glass has extremely high optical transparency, is suitable for microscopic observation, and has good chemical stability, and can be used to accurately measure chemical reactions or biological processes in microfluidic devices.

[0048] When the main body 101 material used is polydimethylsiloxane (PDMS), the material has good fluidity and curing properties, and an outer mold and an inner mold can be prepared so that the length of the inner mold is equal to the length of the capillary channel 201, and the outer diameter of the inner mold is equal to the diameter of the capillary channel 201. The outer mold has a cavity inside, and the inner mold is set in the cavity and the two ends of the inner mold are respectively abutted against two wall surfaces opposite to the cavity. The main material and the curing agent are mixed to form a mixture, and the mixture is poured into the cavity and the mixture is cured, and the mixture is taken out of the cavity together with the inner mold, and the cured mixture forms the main body 101, and the channel generated after taking out the inner mold forms the capillary channel 201. The inner wall of the capillary channel 201 formed by the inverted molding method also has good smoothness, and the processing difficulty is lower and the processing efficiency is high. The above manufacturing method is also applicable to the situation where the material of the main body 101 is PU (polyurethane), fluorosilicone rubber, and polypropylene. Fluorosilicone rubber has good high temperature resistance and chemical corrosion resistance, good biocompatibility, and is suitable for microfluidic experiments in high temperature environments. Polypropylene has a low cost and is suitable for manufacturing disposable equipment. It also has good chemical stability and is suitable for complex chemical experiment scenarios.

[0049] When the body 101 used is made of polytetrafluoroethylene (PTFE), the material can maintain uniform deformation under high pressure to avoid structural cracking or unevenness of the channel wall. Therefore, a hollow tube with an inner diameter equal to the diameter of the first section 204 can be prepared, the two ends of the hollow tube are clamped and the hollow tube is coaxially rotated, and part of the hollow tube is heated and stretched to form the second section 205 and the third section 206, and the stretched hollow tube is cooled and shaped to form the capillary channel 201. The stretch forming method can reduce processing costs, achieve lightweight, and reduce the space occupied by the microfluidic chip 100. The above manufacturing method is also applicable to the situation where the material of the main body 101 is PFA (perfluoroalkoxy resin) or FEP (fluorinated ethylene propylene). PEEK (polyetheretherketone), ceramics, and metals can also be used. PEEK (polyetheretherketone) has better mechanical properties than polytetrafluoroethylene, and is more chemically inert and machinable. Ceramics can be used for chips in high temperature or extreme chemical environments due to their high chemical inertness and thermal stability. Metal materials are suitable for industrial applications or microfluidic devices that require high strength.

[0050] It should be noted that the above-mentioned various manufacturing methods are suitable for the preparation of the microfluidic chip 100 required for the stretching of the microtissue 202 scale. When the cell stretching method of the embodiment of the present invention is used for a single cell, the required capillary channel 201 is smaller in size and the capillary channel 201 can be processed by photolithography technology, specifically, soft photolithography manufacturing technology that does not require a mold.

[0051] According to the third aspect of the present invention, the organoid generation platform includes the microfluidic chip 100 of any one of the second aspect of the present invention, and also includes an injection pump and an organoid culture system. The organoid culture system is connected to the sample outlet 203, and the organoid culture system is used to receive and culture the microtissue 202; the injection pump is connected to the sample inlet 102, and the injection pump is used to inject the fluid containing the microtissue 202 into the capillary channel 201. The microfluidic chip 100 of the present invention, by setting the capillary channel 201 with inconsistent diameters at both ends in the chip, allows the fluid containing the microtissue 202 to enter from the sample inlet 102 at the large diameter end, be squeezed and deformed by the inner wall of the channel in the capillary channel 201, and flow out from the sample outlet 203 at the small diameter end, the microtissue 202 does not need to stay in the microfluidic chip 100, but can continuously culture cells through continuous fluid, and the organoid generation platform using the microfluidic chip 100 can effectively improve the flux of cultured cells.

[0052] It should be noted that, in some embodiments of the present invention, a constant pump is used as the injection pump, so that the flow rate of the fluid injected into the capillary channel 201 can be constant, making the cell morphology after molding more stable.

[0053] It should be noted that in some embodiments of the present invention, in addition to being used in an organoid generation platform, the microfluidic chip 100 can also be used in a cell mechanics research device to provide a controllable experimental environment for cell mechanical stimulation, which is used to study the mechanical transduction mechanism, the response of cells to the mechanical environment, and related biological behaviors. Or it can be a drug screening and toxicological evaluation system, which uses the chip to simulate the in vivo environment and tests the effects of drugs on organoids and cells through dynamic mechanical stimulation, which is particularly suitable for the development of cardiovascular and muscle drugs, and has multiple application prospects.

[0054] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A cell stretching method, characterized in that: The method comprises the following steps: Providing a body, wherein the body has a capillary channel, wherein the capillary channel includes an inlet and an outlet, wherein the diameter of the outlet is smaller than the maximum diameter of the microtissue to be stretched, wherein the microtissue contains the cells; The fluid containing the microtissue is injected from the injection port, and the fluid is discharged from the outlet port.

2. The cell stretching method according to claim 1, characterized in that: The diameter of the capillary channel gradually decreases from the injection port to the injection port, and the diameter of the injection port is not less than the maximum diameter of the microtissue to be stretched.

3. The cell stretching method according to claim 1, characterized in that: From the sample inlet to the sample outlet, the diameter of the capillary channel is constant.

4. The cell stretching method according to claim 1, characterized in that: The capillary channel includes a first section, a second section and a third section connected in sequence, the end of the first section away from the second section is the sample inlet, the end of the third section away from the second section is the sample outlet, the diameter of the first section is greater than the diameter of the third section, the diameter of the second section gradually decreases from the first section to the third section, the diameter of the first section is constant, and the diameter of the third section is constant.

5. The cell stretching method according to claim 1, characterized in that: The body has a plurality of mutually independent capillary channels, and the method further comprises the following steps: The fluid is injected into the injection port of each capillary channel respectively, and the fluid is made to flow out from each outlet.

6. The cell stretching method according to claim 5, characterized in that: The method further comprises the following steps: The flow rate of the fluid is adjusted so that the flow rates of the fluid in each of the capillary channels are different.

7. A microfluidic chip for cell stretching, characterized in that: include: ontology; A capillary channel, wherein the capillary channel comprises an inlet and an outlet, the capillary channel is arranged in the body, the capillary channel comprises a first section, a second section and a third section connected in sequence, the end of the first section away from the second section is the inlet, the end of the third section away from the second section is the outlet, the diameter of the first section is greater than the diameter of the third section, the diameter of the second section gradually decreases from the first section to the third section, the diameter of the first section is constant, the diameter of the third section is constant, and the diameter of the third section is smaller than the maximum diameter of the microtissue to be stretched.

8. The microfluidic chip according to claim 7, characterized in that: The diameter of the first section is D1, the diameter of the third section is D3, 0.98 mm ≤ D1 ≤ 1.02 mm, 0.48 mm ≤ D3 ≤ 0.52 mm.

9. The microfluidic chip according to claim 7, characterized in that: The length of the first section is L1, the length of the second section is L2, and the length of the third section is L3, 29mm≤L1≤31mm, 39mm≤L2≤41mm, 85mm≤L3≤95mm.

10. The microfluidic chip according to claim 7, characterized in that: A plurality of capillary channels are arranged in the body, and each of the capillary channels is independent of each other.

11. The microfluidic chip according to claim 7, characterized in that: The material of the body is any one of polymethyl methacrylate, polydimethylsiloxane and polytetrafluoroethylene.

12. An organoid generation platform, characterized in that include: The microfluidic chip as claimed in any one of claims 7 to 11; An organoid culture system, the organoid culture system is connected to the sample outlet, and the organoid culture system is used to receive and culture the microtissue; An injection pump is connected to the injection port and is used to inject the fluid containing the micro-tissue into the capillary channel.

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