Integrated fiber multilayer multi-channel microfluidic chip, preparation method and application

Abstract

The application provides a multi-layer multi-channel microfluidic chip capable of integrating fibers, comprising a bottom layer, a middle layer and a top layer; the bottom layer is provided with a bottom layer liquid inlet, a bottom layer incident channel, a material fixing channel, a bottom layer emission channel and a bottom layer liquid outlet, and a fiber limiting structure is arranged in the material fixing channel; the middle layer is provided with a middle layer liquid inlet, a middle layer incident channel, a middle layer emission channel and a middle layer liquid outlet; a plurality of through holes are arranged on the middle layer; and the top layer is provided with a top layer liquid inlet and a top layer liquid outlet. The application also provides a preparation method of the multi-layer multi-channel microfluidic chip, and the prepared microfluidic chip can be applied to the evaluation and screening of fiber materials for peripheral nerve repair. The microfluidic chip is integrated with the fiber material, the fibers are co-cultured with nerve cells, the proliferation and migration of the cells in different culture environments are monitored, and the implanted fiber material is screened and evaluated based on the results, thereby providing a reference for the selection of fiber materials for peripheral nerve repair.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidic chips, and particularly relates to a multi-layer multi-channel microfluidic chip capable of integrating fibers, a preparation method and application. BACKGROUND

[0002] Peripheral nerve injury brings a heavy burden to the daily life of patients, and thus effective intervention and treatment of peripheral nerve injury are needed. Fortunately, at present, peripheral nerve repair can be promoted by methods such as autologous nerve transplantation, allogeneic nerve transplantation, chemical or electrical stimulation, however, these methods still have many limitations, for example, the application is limited by the donor site, and there is a risk of inducing neuroma and secondary injury. At present, with the development of tissue engineering and material science, many emerging tissue engineering materials, such as fiber scaffolds or microtubes, have received extensive attention in the repair, regeneration and replacement of damaged nerves. Although these materials can guide the growth of axons in the nerve regeneration process, implantable nerve tissue engineering materials still lack a systematic in vitro comprehensive evaluation platform, and cannot compare and evaluate the nerve repair performance of the materials at the in vitro level, screen out materials with good nerve repair effect, and avoid causing unpredictable inflammation after implantation.

[0003] Organ chip technology is a kind of bionic chip organ system developed on the basis of microfluidic technology, which can simulate the cell growth environment in the human body environment, construct an in vitro organ model, and be applied to drug screening and the like. The microfluidic chip can flexibly design microchannels, and mix and redistribute the fluid in the chip by using specially designed fluid channels, so that the amount of reagents consumed can be greatly reduced, and thus the reaction and analysis speed can be improved. However, the current organ chip mainly focuses on cell culture on the surface of the chip channel, constructs an in vitro disease model, and evaluates the application of drug efficacy, and there is no example of using an organ chip to evaluate and screen the efficacy of tissue engineering materials.

[0004] Among various tissue engineering materials, fiber scaffolds show great properties in nerve regeneration due to their nerve fiber simulation structure. In order to prepare these microfibers, especially hollow microfibers, microfluidic spinning methods show their competitiveness due to the convenient adjustment of channel structure and flow parameters. However, the microstructure of some microfibers is quite different from that of natural fiber tissues, and cannot completely meet the stringent requirements of three-dimensional cell culture and in vitro nerve model reconstruction. Therefore, the evaluation and screening of the nerve regeneration performance of microfibers before their in vivo application is particularly important. However, there is currently no simple and efficient system suitable for the evaluation, comparison and screening of microfibers,

[0005] Therefore, the application designs a multi-layer multi-channel microfluidic chip capable of integrating fibers, which is used for evaluating the performance of materials to be implanted and screening fibers for peripheral nerve repair. SUMMARY

[0006] The application aims at solving the problems of the prior art, and provides a multi-layer multi-channel microfluidic chip capable of integrating fibers, a preparation method and application.

[0007] The application adopts the following technical scheme:

[0008] The application provides a multi-layer multi-channel microfluidic chip capable of integrating fibers, which comprises a bottom layer, a middle layer and a top layer which are bonded and packaged; the bottom layer is provided with a bottom layer liquid inlet, a bottom layer incident channel, a material fixing channel, a bottom layer outlet channel and a bottom layer liquid outlet which are sequentially connected; the material fixing channel is provided with a fiber limiting structure to fix the fiber; the middle layer is provided with a middle layer liquid inlet, a middle layer incident channel, a middle layer outlet channel and a middle layer liquid outlet which are sequentially connected; the middle layer is provided with a plurality of through holes corresponding to the positions of the material fixing channels, the upper part of the fiber is located in the middle layer, and the lower part of the fiber passes through the through holes and enters the bottom layer and is fixed by the fiber limiting structure; the top layer is provided with a top layer liquid inlet which is connected with the bottom layer liquid inlet and the middle layer liquid inlet respectively, and a top layer liquid outlet which is connected with the bottom layer liquid outlet and the middle layer liquid outlet respectively; the number of the top layer liquid inlet and the top layer liquid outlet is not less than two.

[0009] Further, the fiber limiting structure comprises a plurality of pairs of columns, and the fiber is fixed by being placed between each pair of columns.

[0010] Further, the bottom layer liquid inlet comprises a first bottom layer liquid inlet and a second bottom layer liquid inlet, the material fixing channel is provided with five groups which are distributed in parallel; the bottom layer incident channel is a split channel, and the bottom layer outlet channel is a confluence channel; liquid enters the bottom layer incident channel through the first bottom layer liquid inlet and the second bottom layer liquid inlet, is split into five channels and flows into the five groups of material fixing channels, is collected through the bottom layer outlet channel and is discharged from the bottom layer liquid outlet; the middle layer liquid inlet comprises a first middle layer liquid inlet and a second middle layer liquid inlet, the middle layer incident channel is a split channel, and the middle layer outlet channel is a confluence channel; liquid enters the middle layer incident channel through the first middle layer liquid inlet and the second middle layer liquid inlet, fully contacts with the fiber and is finally collected from the middle layer outlet channel and discharged from the middle layer liquid outlet.

[0011] Further, the top layer liquid inlet includes a first top layer liquid inlet, a second top layer liquid inlet, a third top layer liquid inlet and a fourth top layer liquid inlet; the top layer liquid outlet includes a first top layer liquid outlet and a second top layer liquid outlet; the first top layer liquid inlet and the third top layer liquid inlet are cell culture medium liquid inlets, the second top layer liquid inlet and the fourth top layer liquid inlet are cell suspension liquid inlets; the first top layer liquid outlet is a cell suspension liquid outlet, and the second top layer liquid outlet is a cell culture medium liquid outlet; the middle layer is further provided with a first conduit hole, a second conduit hole and a third conduit hole; the first top layer liquid inlet, the first conduit hole and the first bottom layer liquid inlet are in corresponding positions from top to bottom, the third top layer liquid inlet, the second conduit hole and the second bottom layer liquid inlet are in corresponding positions from top to bottom; the second top layer liquid inlet and the first middle layer liquid inlet are in corresponding positions from top to bottom; the fourth top layer liquid inlet and the second middle layer liquid inlet are in corresponding positions from top to bottom; the first top layer liquid outlet and the middle layer liquid outlet are in corresponding positions from top to bottom; and the second top layer liquid outlet, the third conduit hole and the bottom layer liquid outlet are in corresponding positions from top to bottom.

[0012] Further, the bottom layer inlet channel width is 40 μm, the depth is 800 μm; the material fixing channel width is 1.9 mm, the depth is 800 μm; the column height is 600 μm, the spacing is 800 μm; the bottom layer outlet channel width is 40 μm, the depth is 800 μm; the middle layer inlet channel width is 40 μm, the depth is 800 μm; the through hole diameter is 900 μm; and the middle layer outlet channel width is 40 μm, the depth is 800 μm.

[0013] Further, the fiber is a hollow structure containing an internal channel; and the preparation method of the fiber is as follows: the fiber prepolymer liquid is used as an external phase, the occupying liquid is used as an internal phase, and the fiber is prepared by being introduced into a capillary microfluidic chip, and the internal channel diameter of the fiber is adjusted by adjusting the flow rates of the internal phase and the external phase.

[0014] (II) The application further provides a preparation method of the multi-layer multi-channel microfluidic chip with integrable fibers, and the method comprises the following steps:

[0015] S1, preparing a bottom layer template, a middle layer template and a top layer template according to a design drawing;

[0016] S2, covering microfluidic chip prepolymer liquid on surfaces of the bottom layer template, the middle layer template and the top layer template, and preparing a bottom layer, a middle layer and a top layer through solidification, and punching holes at designed positions by using a puncher;

[0017] S3, passing the fiber through the through hole in the middle layer, and fixing the fiber through a fiber limiting structure, and finally bonding and packaging the bottom layer, the middle layer and the top layer.

[0018] Further, the materials of the bottom layer template, the middle layer template and the top layer template are polymethyl methacrylate, and the microfluidic chip prepolymer liquid is a mixed liquid of silica gel or polydimethylsiloxane and a curing agent.

[0019] Further, the microfluidic chip pre-polymer solution is a mixture of polydimethylsiloxane and a curing agent, wherein the mass ratio of polydimethylsiloxane to the curing agent is 10:1.

[0020] Further, in step S3, the upper surface of the bottom layer, the upper and lower surfaces of the middle layer, and the lower surface of the top layer are subjected to plasma treatment, and the bottom layer, the middle layer, and the top layer are stacked and aligned to realize bonding and packaging.

[0021] (Three) The application also provides the use of the above-mentioned multi-layer multi-channel microfluidic chip integrated with fibers in the evaluation and screening of fiber materials for peripheral nerve repair.

[0022] Further, the application method is as follows: integrating the fibers to be evaluated in the microfluidic chip, introducing the cell suspension into the fiber hollow channel from the upper part of the fiber, contacting the lower part of the fiber with the cell culture medium, monitoring the proliferation and migration of the peripheral nerve cells in the fiber hollow structure after a period of cultivation, and screening and evaluating the fiber materials based on the monitoring results.

[0023] The application has the following advantages:

[0024] (1) The multi-layer multi-channel microfluidic chip of the application comprises a three-layer structure, the fibers to be evaluated are fixed in the microfluidic chip through the stand of the bottom layer and the through hole of the middle layer, the cell suspension is introduced into the integrated fiber material channel through the middle layer, and the flow of the fiber external culture medium solution can be used to simultaneously evaluate and compare the performance of multiple fiber materials;

[0025] (2) The bottom layer shunt type liquid inlet channel of the application can mix and redistribute the cell culture solution containing different concentrations of active substances into cell culture solution with a concentration gradient, and can investigate the growth, proliferation, and migration of cells in the fiber under different culture environments;

[0026] (3) The multi-layer multi-channel microfluidic chip integrated with fibers is formed by stacking and bonding the top layer, the middle layer integrated with fibers, and the bottom layer in order, which can comprehensively screen and evaluate the fiber materials for peripheral nerve repair, has high integration, and is simple and efficient. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a template cross-section design structure diagram for copying the bottom layer chip;

[0028] Figure 2 It is a template cross-section design structure diagram for copying the middle layer chip;

[0029] Figure 3 It is a structure schematic diagram of the top layer, the middle layer, and the bottom layer chips of the application;

[0030] Figure 4 Schematic diagram for preparing integrated fiber of the embodiment of the present application;

[0031] Figure 5 Actual photos of the bottom layer, the middle layer, the top layer and the bonded chip prepared in the embodiment of the present application (a), microscope photos of the fiber materials 1, 2, 3 after being co-cultured with cells in the chip channel for 5 days (b) and microscope photos of the nerve cell fibers formed in the fiber materials 1, 2, 3 (c).

[0032] The signs in the drawings are:

[0033] 1, bottom layer design drawing; 2, middle layer design drawing; 3-1, bottom layer; 3-11, bottom layer incident channel; 3-12, material fixing channel; 3-121, stand column; 3-13, bottom layer exit channel; 3-14, first bottom layer liquid inlet; 3-15, second bottom layer liquid inlet; 3-16, bottom layer liquid outlet; 3-2, middle layer; 3-21, middle layer incident channel; 3-22, through hole; 3-23, middle layer exit channel; 3-24, first middle layer liquid inlet; 3-25, second middle layer liquid inlet; 3-26, middle layer liquid outlet; 3-27, first conduit hole; 3-28, second conduit hole; 3-29, third conduit hole; 3-3, top layer; 3-31, first top layer liquid inlet; 3-32, second top layer liquid inlet; 3-33, third top layer liquid inlet; 3-34, fourth top layer liquid inlet; 3-35, first top layer liquid outlet; 3-36, second top layer liquid outlet. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the protection scope of the present application. The implementation conditions not mentioned in the embodiments are usually the conditions in the conventional experiments.

[0035] Embodiment 1

[0036] Reference Figures 1-3 The present embodiment provides a multi-layer multi-channel microfluidic chip that can integrate fibers, which comprises a bonded and packaged bottom layer 3-1, a middle layer 3-2 and a top layer 3-3.

[0037] The bottom layer 3-1 is provided with a bottom layer liquid inlet, a bottom layer incident channel 3-11, a material fixing channel 3-12, a bottom layer emission channel 3-13 and a bottom layer liquid outlet 3-16 which are sequentially connected; the material fixing channel 3-12 is provided with a fiber limiting structure to fix the fiber. Specifically, in the embodiment, the fiber limiting structure includes a plurality of pairs of columns 3-121 arranged, and the fiber is fixed by being placed between each pair of columns 3-121.

[0038] The middle layer 3-2 is provided with a middle layer liquid inlet, a middle layer incident channel 3-21, a middle layer emission channel 3-23 and a middle layer liquid outlet 3-26 which are sequentially connected; the middle layer 3-2 is provided with a plurality of through holes 3-22 corresponding to the positions of the material fixing channel 3-12, the upper part of the fiber is located in the middle layer 3-2, and the lower part passes through the through hole 3-22 into the bottom layer 3-1 and is fixed by the fiber limiting structure.

[0039] The top layer 3-3 is provided with a top layer liquid inlet which is connected with the bottom layer liquid inlet and the middle layer liquid inlet respectively, and a top layer liquid outlet which is connected with the bottom layer liquid outlet 3-16 and the middle layer liquid outlet 3-26 respectively; the number of the top layer liquid inlet and the top layer liquid outlet is not less than two.

[0040] In application, the peripheral nerve cell culture medium is input through the bottom layer liquid inlet, the cell culture medium enters the material fixing channel 3-12 through the bottom layer incident channel 3-11, contacts with the fiber material to be evaluated, the cell culture medium flows outside the fiber to simulate the in-vivo cell environment, and finally is discharged through the bottom layer emission channel 3-13 and the bottom layer liquid outlet 3-16. The peripheral nerve cell suspension is input through the middle layer liquid inlet, the peripheral nerve cell suspension enters the inside of the middle layer chip through the middle layer incident channel 3-21, contacts with the fiber material to be evaluated, the cell suspension flows in the fiber hollow channel, and the peripheral nerve cells grow and adhere to the inside of the fiber hollow channel. The growth, proliferation and migration of the peripheral nerve cells in the fiber material hollow channel are monitored, and the fiber material is selected and evaluated based on the results to select the fiber material suitable for peripheral nerve repair.

[0041] Embodiment 2

[0042] The embodiment provides a multi-layer multi-channel microfluidic chip which can integrate fibers, and the structure of the chip is substantially the same as that of embodiment 1, and the difference lies in that the number, structure and positional relationship of the liquid inlet, the liquid outlet, the incident channel and the emission channel are limited.

[0043] Specifically, referring to Figures 1-3In the embodiment, the bottom layer liquid inlet includes a first bottom layer liquid inlet 3-14 and a second bottom layer liquid inlet 3-15, and the material fixing channels 3-12 are arranged in five groups and distributed in parallel. The bottom layer incident channel 3-11 is a split channel, and the bottom layer emission channel 3-13 is a converging channel. The liquid enters the bottom layer incident channel 3-11 through the first bottom layer liquid inlet 3-14 and the second bottom layer liquid inlet 3-15, is split into five paths, and flows into the five groups of material fixing channels 3-12, respectively, and is collected through the bottom layer emission channel 3-13 to the bottom layer liquid outlet 3-16 for discharge. The middle layer liquid inlet includes a first middle layer liquid inlet 3-24 and a second middle layer liquid inlet 3-25. The middle layer incident channel 3-21 is a split channel, and the middle layer emission channel 3-23 is a converging channel. The liquid enters the middle layer incident channel 3-21 through the first middle layer liquid inlet 3-24 and the second middle layer liquid inlet 3-25, fully contacts the fiber, and is finally collected to the middle layer liquid outlet 3-26 through the middle layer emission channel 3-23 for discharge.

[0044] In the embodiment, the top layer liquid inlet includes a first top layer liquid inlet 3-31, a second top layer liquid inlet 3-32, a third top layer liquid inlet 3-33, and a fourth top layer liquid inlet 3-34. The top layer liquid outlet includes a first top layer liquid outlet 3-35 and a second top layer liquid outlet 3-36. The first top layer liquid inlet 3-31 and the third top layer liquid inlet 3-33 are cell culture medium liquid inlets, and the second top layer liquid inlet 3-32 and the fourth top layer liquid inlet 3-34 are cell suspension liquid inlets. The first top layer liquid outlet 3-35 is a cell suspension liquid outlet, and the second top layer liquid outlet 3-36 is a cell culture medium liquid outlet. The middle layer 3-2 is further provided with a first conduit hole 3-27, a second conduit hole 3-28, and a third conduit hole 3-29. The first top layer liquid inlet 3-31, the first conduit hole 3-27, and the first bottom layer liquid inlet 3-14 correspond in position from top to bottom. The third top layer liquid inlet 3-33, the second conduit hole 3-28, and the second bottom layer liquid inlet 3-15 correspond in position from top to bottom. The second top layer liquid inlet 3-32 and the first middle layer liquid inlet 3-24 correspond in position from top to bottom. The fourth top layer liquid inlet 3-34 and the second middle layer liquid inlet 3-25 correspond in position from top to bottom. The first top layer liquid outlet 3-35 and the middle layer liquid outlet 3-26 correspond in position from top to bottom. The second top layer liquid outlet 3-36, the third conduit hole 3-29, and the bottom layer liquid outlet 3-16 correspond in position from top to bottom.

[0045] Embodiment 3

[0046] The embodiment provides a multi-layer multi-channel microfluidic chip capable of integrating fibers. The structure of the multi-layer multi-channel microfluidic chip is substantially the same as that of the embodiment 2, and the difference lies in that the specifications of the incident channel, the emission channel, the material fixing channel, and the stand are limited.

[0047] Specifically, in the embodiment, the bottom layer incident channel 3-11 has a width of 40 μm and a depth of 800 μm; the material fixing channel 3-12 has a width of 1.9 mm and a depth of 800 μm; the column 3-121 has a height of 600 μm and a spacing of 800 μm; the bottom layer exit channel 3-13 has a width of 40 μm and a depth of 800 μm; the middle layer incident channel 3-21 has a width of 40 μm and a depth of 800 μm; the through hole 3-22 has a diameter of 900 μm; and the middle layer exit channel 3-23 has a width of 40 μm and a depth of 800 μm.

[0048] Embodiment 4

[0049] The embodiment provides a preparation method of a multi-layer multi-channel microfluidic chip capable of integrating fibers.

[0050] (I) According to the bottom layer design Figure 1 The bottom layer template with protruding channels and groove columns is prepared by photolithography, and the middle layer template with protruding channels is prepared according to the middle layer design Figure 2 The bottom layer template, the middle layer template and the top layer template are made of polymethyl methacrylate.

[0051] (II) The microfluidic chip pre-polymer liquid is covered on the surfaces of the bottom layer template, the middle layer template and the top layer template, the bubbles in the container are extracted, and the bottom layer 3-1, the middle layer 3-2 and the top layer 3-3 are obtained by heat curing at 75 ℃ for 3 h, and the punching is performed at the designed position by using a puncher; wherein the microfluidic chip pre-polymer liquid is a mixture of polydimethylsiloxane and a curing agent, and the mass ratio of polydimethylsiloxane to the curing agent is 10:1.

[0052] (III) The upper surface of the bottom layer 3-1, the upper and lower surfaces of the middle layer 3-2 and the lower surface of the top layer 3-3 are subjected to plasma treatment, the fiber containing channels in the interior is passed through the through hole 3-22 in the middle layer 3-2 and is fixed by the fiber limiting structure, and finally the bottom layer 3-1, the middle layer 3-2 and the top layer 3-3 are stacked and aligned to realize bonding and packaging.

[0053] The fiber in the embodiment is a hollow structure containing channels in the interior, and the preparation method is as follows: the fiber pre-polymer liquid is used as an external phase, the occupying liquid is used as an internal phase, and the capillary microfluidic chip is used for preparation. The diameter of the internal channel of the fiber can be adjusted by adjusting the flow rates of the internal and external phases. Specifically, the fiber with a larger hollow channel can be obtained by increasing the flow rate of the channel occupying liquid, and the fiber with a smaller hollow channel can be obtained by decreasing the flow rate of the channel occupying liquid.

[0054] Embodiment 5

[0055] The embodiment provides an application of the multi-layer multi-channel microfluidic chip capable of integrating fibers in the evaluation and screening of fiber materials for peripheral nerve repair.

[0056] As an example, three kinds of fiber materials to be evaluated are integrated into the channels of the multilayer multichannel microfluidic chip of Example 3 to verify the feasibility of chip construction.

[0057] The preparation methods of the three kinds of fiber materials to be evaluated are as follows:

[0058] Fiber material 1: the fiber pre-polymer solution (containing 1.5wt% sodium alginate, 10wt% methacrylate gelatin and 1% v / v photoinitiator aqueous solution) as the outer phase, the channel occupying liquid (10wt% polyvinyl alcohol aqueous solution) as the inner phase, and the inner and outer channels of the capillary microfluidic chip as shown in Figure 4 , the flow rate of the channel occupying liquid is adjusted, and the two solutions are simultaneously introduced into the calcium alginate solution to obtain fibers with larger hollow channels.

[0059] Fiber material 2: the fiber pre-polymer solution (containing 1.5wt% sodium alginate, 10wt% methacrylate gelatin and 1% v / v photoinitiator aqueous solution) as the outer phase, the channel occupying liquid (10wt% polyvinyl alcohol aqueous solution) as the inner phase, and the inner and outer channels of the capillary microfluidic chip as shown in Figure 4 , the flow rate of the channel occupying liquid is adjusted, and the two solutions are simultaneously introduced into the calcium alginate solution to obtain fibers with smaller hollow channels.

[0060] Fiber material 3: the fiber pre-polymer solution (containing 1.5wt% sodium alginate, 10wt% methacrylate gelatin, 1% v / v photoinitiator and 10mg / ml graphene oxide aqueous dispersion) as the outer phase, the channel occupying liquid (10wt% polyvinyl alcohol aqueous solution) as the inner phase, and the inner and outer channels of the capillary microfluidic chip as shown in Figure 4 , the flow rate of the channel occupying liquid is adjusted, and the two solutions are simultaneously introduced into the calcium alginate solution to obtain fibers with the same size of hollow channels as in fiber material 2.

[0061] The fiber material 1, the fiber material 2 and the fiber material 3 are respectively integrated into the microfluidic chip described in Example 3, the (peripheral nerve cell) Schwann cell suspension is introduced into the internal channel of the integrated fiber through the liquid inlet 3-32, 3-34, and the cell culture solution is introduced into the bottom layer of the chip through the liquid inlet 3-31, 3-33. After co-culturing the cells with the fibers for 5 days, the formation of nerve cell fibers can be observed inside the fibers.

[0062] Figure 5In the figure, a is the bottom layer, middle layer, top layer and the bonded chip of the multi-layer multi-channel microfluidic chip of the integrable fiber, b is the microscope photo of the fiber material 1, 2, 3 after co-culturing with cells in the chip channel for 5 days, and c is the microscope picture of the nerve cell fiber formed in the fiber material 1, 2, 3.

[0063] By Figure 5 It can be seen that, in the same fiber material, the hollow fiber with smaller channel (fiber material 2) is better than the hollow fiber with larger channel (fiber material 1); in different fiber materials with the same hollow channel size, the hollow fiber containing graphene oxide (fiber material 3) is better than the hollow fiber without graphene oxide (fiber material 2).

[0064] Therefore, the multi-layer multi-channel microfluidic chip of the integrable fiber prepared by the application can systematically analyze the performance of the fiber in promoting the proliferation and migration of nerve cells, and provides a reference for the selection of fiber materials for peripheral nerve repair.

[0065] The above is only the preferred embodiment of the application, and the protection scope of the application is not limited to the above-mentioned embodiments, and any technical scheme falling within the idea of the application belongs to the protection scope of the application. It should be pointed out that, for ordinary skilled persons in the art, some improvements and decorations without departing from the principle of the application should be regarded as the protection scope of the application.

Claims

1. A multi-layer multi-channel microfluidic chip with integrated fibers, characterized in that, a bottom layer (3-1), a middle layer (3-2) and a top layer (3-3) are bonded together; a bottom layer inlet, a bottom layer incident channel (3-11), a material fixing channel (3-12), a bottom layer outlet channel (3-13) and a bottom layer outlet (3-16) are sequentially arranged on the bottom layer (3-1); a fiber limiting structure is arranged in the material fixing channel (3-12) to fix the fiber; a middle layer inlet, a middle layer incident channel (3-21), a middle layer outlet channel (3-23) and a middle layer outlet (3-26) are sequentially arranged on the middle layer (3-2); a plurality of through holes (3-22) corresponding to the positions of the material fixing channels (3-12) are arranged on the middle layer (3-2); one end of the fiber is connected to the middle layer incident channel (3-21), the other end passes through the through hole (3-22) into the bottom layer (3-1), is fixed by the fiber limiting structure, and is finally connected to the middle layer outlet channel (3-23); a top layer inlet is arranged on the top layer (3-3) and communicates with the bottom layer inlet and the middle layer inlet respectively; a top layer outlet is arranged on the top layer (3-3) and communicates with the bottom layer outlet (3-16) and the middle layer outlet (3-26) respectively; the number of the top layer inlet and the top layer outlet is not less than two. 2.The multi-layer multi-channel microfluidic chip with integrated fibers according to claim 1, characterized in that, the fiber limiting structure includes a plurality of pairs of arranged columns (3-121); the fiber is fixed by being placed between each pair of columns (3-121). 3.The multi-layer multi-channel microfluidic chip with integrated fibers according to claim 2, characterized in that, the bottom layer inlet includes a first bottom layer inlet (3-14) and a second bottom layer inlet (3-15); the material fixing channel (3-12) is arranged in five groups and is distributed in parallel; the bottom layer incident channel (3-11) is a split channel, and the bottom layer outlet channel (3-13) is a converging channel; liquid enters the bottom layer incident channel (3-11) through the first bottom layer inlet (3-14) and the second bottom layer inlet (3-15), is split into five paths, flows into the five groups of material fixing channels (3-12) respectively, and is collected through the bottom layer outlet channel (3-13) to the bottom layer outlet (3-16) for discharge; the middle layer inlet includes a first middle layer inlet (3-24) and a second middle layer inlet (3-25); the middle layer incident channel (3-21) is a split channel, and the middle layer outlet channel (3-23) is a converging channel; liquid enters the middle layer incident channel (3-21) through the first middle layer inlet (3-24) and the second middle layer inlet (3-25), fully contacts with the fiber, and is finally collected to the middle layer outlet (3-26) through the middle layer outlet channel (3-23) for discharge. 4.The multi-layer multi-channel microfluidic chip with integrated fibers according to claim 3, characterized in that, The top layer liquid inlet includes a first top layer liquid inlet (3-31), a second top layer liquid inlet (3-32), a third top layer liquid inlet (3-33) and a fourth top layer liquid inlet (3-34); the top layer liquid outlet includes a first top layer liquid outlet (3-35) and a second top layer liquid outlet (3-36); the first top layer liquid inlet (3-31) and the third top layer liquid inlet (3-33) are cell culture medium liquid inlets, the second top layer liquid inlet (3-32) and the fourth top layer liquid inlet (3-34) are cell suspension liquid inlets; the first top layer liquid outlet (3-35) is a cell suspension liquid outlet, and the second top layer liquid outlet (3-36) is a cell culture medium liquid outlet; the middle layer (3-2) is further provided with a first conduit hole (3-27), a second conduit hole (3-28) and a third conduit hole (3-29); The first top layer liquid inlet (3-31), the first conduit hole (3-27) and the first bottom layer liquid inlet (3-14) are vertically corresponding; the third top layer liquid inlet (3-33), the second conduit hole (3-28) and the second bottom layer liquid inlet (3-15) are vertically corresponding; the second top layer liquid inlet (3-32) and the first middle layer liquid inlet (3-24) are vertically corresponding; the fourth top layer liquid inlet (3-34) and the second middle layer liquid inlet (3-25) are vertically corresponding; the first top layer liquid outlet (3-35) and the middle layer liquid outlet (3-26) are vertically corresponding; and the second top layer liquid outlet (3-36), the third conduit hole (3-29) and the bottom layer liquid outlet (3-16) are vertically corresponding.

5. The multi-layer multi-channel microfluidic chip of the integrable fiber according to claim 4, wherein, The bottom layer incident channel (3-11) has a width of 40 μm and a depth of 800 μm; the material fixing channel (3-12) has a width of 1.9 mm and a depth of 800 μm; the column (3-121) has a height of 600 μm and a spacing of 800 μm; and the bottom layer emission channel (3-13) has a width of 40 μm and a depth of 800 μm. The middle layer incident channel (3-21) has a width of 40 μm and a depth of 800 μm; the through hole (3-22) has a diameter of 900 μm; and the middle layer emission channel (3-23) has a width of 40 μm and a depth of 800 μm.

6. The multi-layer multi-channel microfluidic chip of the integrable fiber according to claim 1, wherein, The fiber is a hollow structure containing an internal channel.

7. The method of claim 1-6, wherein the method further comprises: The method comprises the following steps: S1, preparing a bottom layer template, a middle layer template and a top layer template according to a design drawing; S2, covering a microfluidic chip pre-polymer liquid on the surfaces of the bottom layer template, the middle layer template and the top layer template, and preparing a bottom layer (3-1), a middle layer (3-2) and a top layer (3-3) through solidification, and punching holes at designed positions by using a puncher. S3, connect the fiber with the middle layer incident channel (3-21), pass through the through hole (3-22) in the middle layer (3-2) into the bottom layer (3-1), and fix it through the fiber limiting structure, then connect with the middle layer exit channel (3-23), make the cell culture medium of the bottom layer (3-1) flow outside the fiber, the cell suspension of the middle layer (3-2) flow in the fiber hollow channel, and finally bond and package the bottom layer (3-1), the middle layer (3-2) and the top layer (3-3).

8. The preparation method of the multi-layer multi-channel microfluidic chip integrated with fibers according to claim 7, wherein the materials of the bottom layer template, the middle layer template and the top layer template are polymethyl methacrylate, and the microfluidic chip pre-polymer solution is a mixture of silica gel or polydimethylsiloxane and a curing agent.

9. The preparation method of the multi-layer multi-channel microfluidic chip integrated with fibers according to claim 8, wherein in step S3, the upper surface of the bottom layer (3-1), the upper and lower surfaces of the middle layer (3-2) and the lower surface of the top layer (3-3) are subjected to plasma treatment, and the bottom layer (3-1), the middle layer (3-2) and the top layer (3-3) are bonded and packaged after being stacked and aligned.

10. The use of the multi-layer multi-channel microfluidic chip integrated with fibers according to any one of claims 1-6 in the evaluation and screening of fiber materials for peripheral nerve repair. ​ ​

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