CN111996100B

CN111996100B - Microfluidic chip for collecting and quantitatively analyzing nerve cell axon

Info

Publication number: CN111996100B
Application number: CN201910969823.8A
Authority: CN
Inventors: 朴宰源; 宋惠雪; 姚俊伊; 姬生健; 张建; 刘建辉
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2023-05-16
Anticipated expiration: 2039-10-12
Also published as: CN111996100A

Abstract

The invention relates to a microfluidic chip. The microfluidic chip comprises a plurality of cell bodies and a plurality of axon chambers, wherein the cell bodies and the axon chambers are alternately arranged at intervals, intermediate chambers are alternately arranged between the adjacent cell bodies and the axon chambers, and the adjacent cell bodies and the intermediate chambers, and the adjacent intermediate chambers and the axon chambers are respectively communicated through micro-channels. The microfluidic chip can collect a large amount of axon tissue materials with higher purity, can efficiently collect axon RNA for quantitative analysis or separation when being used for researches such as the culture of nerve cell axons, provides possibility for the subsequent researches of the axons, and has far-reaching scientific research and application significance.

Description

Microfluidic chip for collecting and quantitatively analyzing nerve cell axon

Technical Field

The invention relates to the field of biotechnology/biomedical engineering, in particular to a microfluidic chip.

Background

The micro-fluidic Chip is also called as Lab-on-a-Chip, and is a technology with micro-channel to form network for controllable fluid to pass through the whole system, and has the features of miniature, automation, integration, high flux, etc.

However, the amount of neurite tissue material collected by conventional microfluidic chips is far from satisfactory for subsequent transcriptomic and proteomic analysis of the neurites. Therefore, development of a new microfluidic chip is highly required to meet the requirements of the axon correlation study.

Disclosure of Invention

Based on this, it is necessary to provide a microfluidic chip that can significantly increase the collection amount of axon tissue.

The microfluidic chip comprises a plurality of cell bodies and a plurality of axon chambers, wherein the cell bodies and the axon chambers are alternately arranged at intervals, intermediate chambers are arranged between the adjacent cell bodies and the axon chambers at intervals, and the adjacent cell bodies and the intermediate chambers, and the adjacent intermediate chambers and the axon chambers are respectively communicated through micro-channels.

In one embodiment, the plurality of cell bodies, the plurality of axon chambers and the intermediate chamber form a chamber string, and both ends of the chamber string are the axon chambers.

In one embodiment, the opposite ends of the axon chamber have an axon chamber inlet and an axon chamber outlet, respectively, the axon chamber inlet or the axon chamber outlet being disposed on the same side of the chamber string.

In one embodiment, the opposite ends of the cell have a cell inlet and a cell outlet, respectively, the cell inlet or the cell outlet being disposed on the same side of the chamber string.

In one embodiment, the opposite ends of the intermediate chamber have an intermediate chamber inlet and an intermediate chamber outlet, respectively, the intermediate chamber inlet or the intermediate chamber outlet being provided on the same side of the chamber train.

In one embodiment, the axon chamber inlet and the axon chamber outlet are respectively circular and equal in diameter, the cell chamber inlet and the cell chamber outlet are respectively circular and equal in diameter, the intermediate chamber inlet and the intermediate chamber outlet are respectively circular and equal in diameter, and the axon chamber inlet is unequal in diameter to the cell chamber inlet.

In one embodiment, the number of the cells is 2 to 20, the number of the axon chambers is 3 to 21, and the number of the intermediate chambers is 4 to 40.

In one embodiment, the distance between the middle chamber and the adjacent cell is 0.1 mm-1 mm.

In one embodiment, the distance between the intermediate chamber and the adjacent axon chamber is 0.1mm to 1mm.

In one embodiment, the ratio of the width of the axon compartment to the width of the cell compartment is (1-5): 1.

in one embodiment, the axon chamber is rectangular, and the width of the axon chamber is 1 mm-10 mm, and the length of the axon chamber is 10 mm-60 mm.

In one embodiment, the cell body is rectangular, and the width of the cell body is 1 mm-10 mm, and the length of the cell body is 10 mm-60 mm.

In one embodiment, the middle chamber is rectangular, and the width of the middle chamber is 0.1 mm-1 mm, and the length of the middle chamber is 10 mm-60 mm.

In one embodiment, the adjacent cell body chambers are communicated with the middle chamber through a plurality of micro flow channels, and the distance between the two adjacent micro flow channels is 3-20 μm.

In one embodiment, the adjacent intermediate chambers are communicated with the axon chamber through a plurality of micro flow channels, and the distance between the two adjacent micro flow channels is 3-20 μm.

The microfluidic chip is provided with a plurality of cell bodies and a plurality of axon chambers, an intermediate chamber is arranged between each adjacent cell body and axon chamber, and nerve cell axons inoculated in the cell bodies can extend to the intermediate chamber through a micro-channel and continuously extend to the plurality of axon chambers for growth, and the existence of the intermediate chamber can effectively prevent the cell bodies or other tissues from entering the axon chambers and ensure that only axon tissues enter the axon chambers, so that a large amount of axon tissue materials with higher purity can be collected in each axon chamber, and axon RNA can be efficiently collected for quantitative analysis or separation when being used for the research of nerve cell axons and the like, thereby providing possibility for the subsequent research of axons, and having far-reaching scientific research and application significance.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic chip according to an embodiment;

fig. 2 is a microscopic photograph of a sixth day of growth of mouse embryonic dorsal root ganglion cells (DRGs) of embryonic stage E13.5 in a microfluidic chip in example 1.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It will be understood that when an element is referred to as being "fixed to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like are used herein for illustrative purposes only.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1, in an embodiment of a microfluidic chip, the microfluidic chip includes a plurality of cell chambers 110 and a plurality of axon chambers 120, the cell chambers 110 and the axon chambers 120 are alternately arranged at intervals, an intermediate chamber 140 is arranged between adjacent cell chambers 110 and axon chambers 120 at intervals, and the adjacent cell chambers 110 and the intermediate chamber 140, and the adjacent intermediate chamber 140 and the axon chambers 120 are respectively communicated through a micro flow channel 130.

The cell 110 is used to seed neural cells. When the microfluidic chip is used for culturing the nerve cell axon, the nerve cell axon inoculated in the cell chamber 110 can extend to the middle chamber 140 through the micro-channel 130 and continue to extend to the axon chamber 120, the existence of the middle chamber 140 can effectively prevent cell bodies or other tissues from entering the axon chamber 120, ensure that only the axon tissues enter the axon chamber 120, and finally collect a large amount of high-purity axon tissue materials without other tissues mixed in a plurality of axon chambers 120.

The plurality of cells 110 and the axon cells 120 may be alternately arranged at intervals along a direction on a plane with respective centers substantially on a straight line, and in fig. 1, the cells 110 and the axon cells 120 are alternately arranged at intervals along a direction from top to bottom in the drawing to facilitate the extended growth of the axon. Since the intermediate chambers 140 are disposed between the pairs of adjacent cell chambers 110 and the axon chambers 120, the number of intermediate chambers 140 is also plural. Further, the plurality of cell bodies 110, the plurality of intermediate chambers 140 and the plurality of axon chambers 120 are arranged in parallel and at intervals to form a chamber string, and both ends of the chamber string are the axon chambers 120. That is, in the chamber string formed by the plurality of cell bodies 110, the plurality of intermediate chambers 140 and the plurality of axon chambers 120, it is ensured that the intermediate chambers 140 and the axon chambers 120 are disposed at both sides of each cell body 110, so that the nerve cell axons can grow and extend according to the growth rule thereof, which is beneficial to lifting the collection amount of the axon tissue material.

Opposite ends of the axon chamber 120 have an axon chamber inlet 121 and an axon chamber outlet 122, respectively, in a direction perpendicular to the alternating arrangement direction of the axon chamber 120 and the cell 110 (i.e., the left-right direction in fig. 1). The axon compartment inlet 121 is used for adding cell culture medium and biological agents, and the axon compartment outlet 122 is used for collecting axons. The axon compartment inlet 121 and the axon compartment outlet 122 may be disposed in the same direction in a direction perpendicular to the drawing plane of fig. 1. The axon chamber inlets 121 or the axon chamber outlets 122 of the plurality of axon chambers 120 are preferably provided at the same end of the plurality of axon chambers 120, i.e. the axon chamber inlets 121 or the axon chamber outlets 122 of the plurality of axon chambers 120 are provided at the same side of the chamber string. For example, in fig. 1, the left ends of the plurality of axon chambers 120 are each connected to an axon chamber inlet 121, and the right ends are each connected to an axon chamber outlet 122. Further, the axon compartment inlet 121 and the axon compartment outlet 122 may be circular and equal in diameter, respectively. Specifically, the diameter of the axon chamber inlet 121 may be 1mm to 8mm, and the diameter of the axon chamber outlet 122 may be 1mm to 8mm.

Opposite ends of the cell 110 have a cell inlet 111 and a cell outlet 112, respectively, in a direction perpendicular to the alternating arrangement direction of the axon cells 120 and the cell 110 (i.e., the left-right direction in fig. 1). The cell inlet 111 is used for seeding neural cells and adding cell culture medium, and the cell outlet 112 is used for exchanging cell culture medium. The cell inlet 111 and the cell outlet 112 may be disposed in the same direction in a direction perpendicular to the drawing plane of fig. 1. The cell inlets 111 or cell outlets 112 of the plurality of cells 110 are preferably arranged at the same end of the plurality of cells 110, i.e. the cell inlets 111 or cell outlets 112 of the plurality of cells 110 are arranged at the same side of the chamber string. For example, in fig. 1, the plurality of cells 110 are each connected to a cell inlet 111 at the left end and to a cell outlet 112 at the right end. Further, the cell inlet 111 and the cell outlet 112 may be circular and have equal diameters, respectively. Specifically, the cell inlet 111 may have a diameter of 1mm to 8mm, and the cell outlet 112 may have a diameter of 1mm to 8mm.

The intermediate chamber 140 has an intermediate chamber inlet 141 and an intermediate chamber outlet 142 at opposite ends thereof in a direction perpendicular to the alternating arrangement direction of the axon chambers 120 and the cell body chambers 110 (i.e., left-right direction in fig. 1), respectively. The intermediate chamber inlet 141 is used for adding cell culture medium and the intermediate chamber outlet 142 is used for exchanging cell culture medium. The intermediate chamber inlet 141 and the intermediate chamber outlet 142 may be disposed in the same direction in a direction perpendicular to the drawing plane of fig. 1. The intermediate chamber inlets 141 or the intermediate chamber outlets 142 of the plurality of intermediate chambers 140 are preferably provided at the same end of the plurality of intermediate chambers 140, i.e. the intermediate chamber inlets 141 or the intermediate chamber outlets 142 of the plurality of intermediate chambers 140 are provided at the same side of the chamber train. For example, in fig. 1, the plurality of intermediate chambers 140 are each connected at a left end to an intermediate chamber inlet 141 and at a right end to an intermediate chamber outlet 142. Further, the intermediate chamber inlet 141 and the intermediate chamber outlet 142 may be circular and of equal diameter, respectively. Specifically, the diameter of the intermediate chamber inlet 141 may be 1mm to 5mm, and the diameter of the intermediate chamber outlet 112 may be 1mm to 5mm.

In order to distinguish between the cell inlet 111 and the cell outlet 112, the diameter of the axon chamber inlet 121 is preferably not equal to the diameter of the cell inlet 111, i.e. the diameter of the axon chamber outlet 122 is not equal to the diameter of the cell outlet 112. Further, the diameter of the axon chamber inlet 121 is smaller than the diameter of the cell chamber inlet 111, i.e. the diameter of the axon chamber outlet 122 is smaller than the diameter of the cell chamber outlet 112.

The number of cells 110, intermediate chambers 140, and axon chambers 120 may be adjusted as desired. For example, the number of cells 110 may be 2 to 20; the number of axon compartments 120 may be 3 to 21; the number of intermediate chambers 140 may be 4 to 40.

The distance between the intermediate chamber 140 and the adjacent cell 110 and axon 120 has an effect on the extended growth of the axon. In one embodiment, the distance between the middle chamber 140 and the adjacent cell 110 may be 0.1mm to 1mm; the distance between the intermediate chamber 140 and the adjacent axon compartment 120 may be 0.1 mm-1 mm. The above distance range is desirable to facilitate the growth of axons extending from the cell chamber 110 to the axon chamber 120, and to prevent excess cell or other tissue material from entering the axon chamber 120.

Since there is a correlation between the length of the axon and the size of the cell body, in order to provide a space suitable for the growth of the axon, the ratio of the width of the axon chamber 120 to the width of the cell body chamber 110 may be (1 to 5): 1. the widths of the axon cells 120 and the cell 110 refer to the dimensions of the axon cells 120 and the cell 110 in the direction in which they are alternately arranged (i.e., the up-down direction in fig. 1). When the widths of the axon compartment 120 and the cell compartment 110 are in the above-mentioned ratio range, the growth of the axon in the axon compartment 120 is better, so that more axon tissue material can be finally collected.

In one embodiment, the axonal compartment 120 may be rectangular as shown in fig. 1. Such a shape is more suitable for the growth of axons, facilitating the collection of the maximum amount of axonal tissue material. Further, the width of the axon compartment 120 may be 1mm to 10mm and the length may be 10mm to 60mm. Wherein the length of the axial chamber 120 refers to its largest dimension in the left-right direction. In other embodiments, the axonal compartment 120 may also have other shapes, such as kidney-shaped, oval-shaped, etc. The plurality of axon chambers 120 may be the same or different in shape and size, and preferably the plurality of axon chambers 120 have the same shape and size. The height of the axonal chamber 120 may be 50 μm to 150 μm, wherein the height of the axonal chamber 120 refers to its dimension in a direction perpendicular to the drawing plane of fig. 1.

In one embodiment, the cells 110 may be rectangular. Further, the width of the cell 110 may be 1mm to 10mm and the length may be 10mm to 60mm. Where the length of the cell 110 refers to its dimension in the left-right direction. The lengths and widths of the plurality of cells 110 may be the same or different, and may be designed according to practical needs, and in the embodiment shown in fig. 1, the lengths of the plurality of cells 110 are not exactly the same. The height of the cell 110 may be 50 μm to 150 μm, wherein the height of the cell 110 refers to its dimension in a direction perpendicular to the plane of the drawing of fig. 1.

In one embodiment, the intermediate chamber 140 may be rectangular. Further, the intermediate chamber 140 may have a width of 0.1mm to 1mm and a length of 10mm to 60mm. Wherein the length of the middle chamber 140 refers to its dimension in the left-right direction. The length and width of the plurality of intermediate chambers 140 may be the same or different, and may be designed according to actual needs. The height of the intermediate chamber 140 may be 50 μm to 150 μm, wherein the height of the intermediate chamber 140 refers to its dimension in a direction perpendicular to the plane of the drawing of fig. 1.

By rationally designing the number, shape, size, and spacing of the cells 110, the intermediate chambers 140, and the axon compartments 120, it is advantageous to collect as much axonal tissue material as possible in a limited space.

Adjacent cells 110 and intermediate chamber 140 may be in communication via a plurality of microchannels 130, thereby allowing the growth of nerve cell axons to more closely approximate the physiological environment. The plurality of micro flow channels 130 are uniformly spaced apart, and a distance between two adjacent micro flow channels 130 may be 3 μm to 20 μm. Here, the distance between the adjacent two micro flow channels 130 refers to the distance between the openings of the adjacent two micro flow channels 130 communicating with the cell 110 or the openings communicating with the intermediate chamber 140. The number of the micro flow channels 130 between the adjacent cells 110 and the middle chamber 140 may be adjusted according to the lengths of the cells 110 and the middle chamber 140, for example, the number of the micro flow channels 130 between the adjacent cells 110 and the middle chamber 140 may be 1000 to 2000.

The adjacent intermediate chambers 140 and the axon chambers 120 can be communicated through the plurality of micro-channels 130, so that the growth of the nerve cell axons is more similar to the operation under the physiological environment. The plurality of micro flow channels 130 are uniformly spaced apart, and a distance between two adjacent micro flow channels 130 may be 3 μm to 20 μm. Here, the distance between the adjacent two micro flow channels 130 refers to the distance between the openings of the adjacent two micro flow channels 130 communicating with the intermediate chamber 140 or the openings communicating with the axon chamber 120. The number of the micro flow channels 130 between the adjacent intermediate chambers 140 and the axon chambers 120 may be adjusted according to the lengths of the intermediate chambers 140 and the axon chambers 120, for example, the number of the micro flow channels 130 between the adjacent intermediate chambers 140 and the axon chambers 120 may be 1000 to 2000.

The micro flow channel 130 may have a rectangular shape. Further, the micro flow channel 130 may have a width of 3 μm to 10 μm and a length of 0.4mm to 1mm. Where the width of the micro flow channel 130 refers to its dimension in the left-right direction and the length refers to its dimension in the up-down direction. The height of the micro flow channel 130 may be 1 μm to 3 μm, wherein the height of the micro flow channel 130 refers to its dimension in a direction perpendicular to the drawing plane of fig. 1.

The material for preparing the microfluidic chip can be Polydimethylsiloxane (PDMS), has good air permeability and light transmittance, is favorable for the growth of nerve cells, and has low price.

The microfluidic chip may be fabricated by a conventional method in the art. For example, firstly, according to the design, the cell 110, the middle chamber 140 and the axon chamber 120 are manufactured as film masks, the micro flow channel 130 is manufactured as chrome masks, and the length of the micro flow channel 130 can be slightly longer than the interval distance between the cell 110 and the middle chamber 140, between the middle chamber 140 and the axon chamber 120, so as to ensure that the micro flow channel 130 can connect the cell 110 and the middle chamber 140, between the middle chamber 140 and the axon chamber 120; then, manufacturing a corresponding negative photoresist template by a soft photoetching method; and then, a Polydimethylsiloxane (PDMS) material is deposited on the negative photoresist template, and the PDMS is solidified and peeled and then sealed with a glass substrate, so that the microfluidic chip is obtained.

The microfluidic chip can be used for culturing neurites of nerve cells, can efficiently collect the RNA of the neurites for quantitative analysis or separation, provides possibility for subsequent research of the neurites, and has far-reaching scientific research and application significance in research fields such as guidance of the neurites, signal transduction of the neurites, reverse signal transduction, local synthesis of proteins in the neurites, dendritic cell nucleus signals and the like.

The invention is further illustrated by the following specific examples, which are not intended to limit the invention.

Example 1

The microfluidic chip of this embodiment is designed in AutoCAD software, and has a structure shown in fig. 1, and includes 3 axon chambers 120 and 2 cell chambers 110 alternately arranged at intervals, and intermediate chambers 140 (4 in total) are arranged between adjacent axon chambers 120 and cell chambers 110, and the adjacent axon chambers 120 are communicated with the intermediate chambers 140, and the intermediate chambers 140 and cell chambers 110 through a micro flow channel array 130, and the size of the microfluidic chip is 5.6cm×2.4cm. Each of the axon cells 120 is rectangular in shape, 3mm in width, 22mm or 48mm in length, and 100 μm in height, and has a circular axon cell inlet 121 of 2mm in diameter connected to the left end and a circular axon cell outlet 122 of 2mm in diameter connected to the right end. Each cell 110 is rectangular in shape, 1.5mm wide, 28mm long and 100 μm high, with a circular cell inlet 111 of 5mm diameter connected to the left end and a circular cell outlet 112 of 5mm diameter connected to the right end. Each intermediate chamber 140 is rectangular in shape, 0.2mm wide, 28mm long and 100 μm high, and has a circular intermediate chamber inlet 141 of 2mm diameter connected to the left end and a circular intermediate chamber outlet 142 of 2mm diameter connected to the right end. The distance between the intermediate chamber 140 and the adjacent axon chamber 120 is 300 μm and the distance between the adjacent cell chamber 110 is 300 μm. The number of micro flow channels 130 between the adjacent axon chambers 120 and the intermediate chamber 140, and between the adjacent intermediate chamber 140 and the cell body chamber 110 is 1830 (not shown in the figure), the shape is a rectangle with a width of 6 μm, a length of 450 μm, a distance of 6 μm and a height of 2.5 μm.

1. Manufacturing exposure mask plate

According to the structural design shown in fig. 1, the cell 110, the intermediate chamber 140 and the axon chamber 120 are manufactured as film masks, the micro flow channel 130 is manufactured as a chrome mask, and the corresponding negative photoresist SU-8 template is manufactured.

2. Manufacturing negative photoresist SU-8 template

Plating a layer of chromium with the thickness of 50nm on the cleaned 3-inch silicon wafer by utilizing electron beam evaporation; uniformly coating a layer of positive photoresist rzj-304 (Suzhou Ruihong) with the thickness of 1 mu m on the silicon wafer, pre-baking, ultraviolet lithography and development to obtain a pattern of an alignment mark; wet etching is carried out by using chromium etching liquid, and chromium which only remains the alignment mark pattern is obtained after positive photoresist is removed; uniformly coating a layer of negative photoresist SU-8 2005 (MicroChem) with the thickness of 2.5 mu m on a silicon wafer with an alignment mark, pre-baking with ultraviolet light (the alignment mark is needed), post-baking and developing to obtain a micro-channel array pattern; 5. uniformly coating a layer of negative photoresist SU-8 2075 (MicroChem) with the thickness of 100 μm on the silicon wafer with the micro-channel array structure, pre-baking, ultraviolet light (alignment mark is needed), post-baking and developing to obtain a cell, a middle chamber and an axon chamber structure; and placing the template on a glue baking table at 150 ℃ for hardening for 10 minutes, so that the adhesiveness between the photoresist and the silicon wafer is enhanced, and the durability of the chip is improved.

3. Microfluidic chip fabricated using Polydimethylsiloxane (PDMS) material and glass substrate

Uniformly stirring and mixing the solution A and the solution B of PDMS according to the mass ratio of 10:1; the template is subjected to silanization treatment, namely the template and a small amount of trichlorosilane (trichlorosilane) are placed in a vacuum dryer for about 15 minutes, isopropanol is used for flushing the template to remove residual trichlorosilane, and then nitrogen is used for drying; fixing the SU-8 template subjected to silanization treatment on a flat-bottom culture dish, pouring PDMS solution with the thickness of about 2-3 mm, removing bubbles in vacuum, and then placing the solution in an oven at 80 ℃ for baking for 1 hour; peeling the cured PDMS from the SU-8 template and perforating at the entrance and exit of all chambers; and (3) simultaneously placing the PDMS chip with the holes and the glass substrate with the thickness of 24mm multiplied by 60mm into a plasma cleaning machine for processing for 1min, and aligning and sealing to obtain the microfluidic chip.

4. Performing nerve cell culture and axon quantitative analysis on a microfluidic chip

Preprocessing a microfluidic chip: polylysine (PDL) is added at the inlet of each chamber of the microfluidic chip, the mixture is placed in a carbon dioxide cell incubator overnight, the mixture is washed three times with deionized water of a cell culture grade, a culture medium containing 3.3 mug/mL of fibronectin is added after the mixture is dried in the air, the mixture is placed in the carbon dioxide cell incubator for incubation for 2 hours, and the culture medium is sucked dry before inoculating cells.

Acquisition of dorsal root ganglion cells (DRGs): taking out embryo stage E13.5, placing in L-15 culture medium containing blue chain mycin, placing on ice, separating back spinal column, taking dorsal root ganglion cells (DRG) on the spinal column with fine forceps, sucking into 15mL centrifuge tube with glass pipette, and placing on ice. After completion of the collection, the mixture was centrifuged at 800rpm/min for 3min. After discarding the L-15 medium, 2mL of TrypLE was added, and the mixture was digested in a 37℃water bath for 15min (shaking every 2 min) and centrifuged at 800rpm/min for 5min. TrypLE was discarded, 2mL of Neurobasal Medium containing 10% FBS was added, the cells were blown into a cloud with a wetted glass tube, and then centrifuged at 800rpm/min for 5min. Neurobasal Medium containing 10% FBS was discarded, and an appropriate amount of complete medium (Neurobasal Medium +B-27+Glutamax was added ^TM -I+Pen-strep+NGF+5-Fluoro-2' -deoxyuridine) to blow off cells. A small amount of cells were pipetted into the cell chamber 110 in the microfluidic chip and after waiting for 30min for complete attachment of the cells to the coverslip, complete medium was replenished. Half of the complete medium was changed every two days.

Extraction of RNA: culturing until day six, washing twice with Phosphate Buffer (PBS), first blotting off the PBS of the axonal compartment, adding 50uL TRIzol reagent from the inlet, and aspirating from the axonal compartment outlet. After collection of the axons, the RNA of the axons was extracted by TRIzol method. Fig. 2 is a microscopic photograph of a mouse embryonic dorsal root ganglion cell (DRG) of embryonic stage E13.5 grown on a microfluidic chip on the sixth day, and a large amount of axon tissue with good growth state was seen in the axon compartment 120.

Quantitative detection of RNA: 1uL of axonal RNA was analyzed on an Agilent 2100Bioanalyzer machine using RNA 6000 Pico Kit&Reagents to give a final RNA concentration of 10680pg/uL.

Comparative example 1

The microfluidic chip of this comparative example 1 was a microfluidic chip available from U.S. Xona Microfluidics company under the model XC-T500. The microfluidic chip has a cell, an intermediate chamber and an axon chamber, and has dimensions of 3.5cm by 2.4cm.

The neural cells were cultured and analyzed according to the axon quantitative analysis procedure in example 1, and the final RNA concentration was measured to be 336.67pg/uL.

As can be seen from comparison of example 1 and comparative example 1, compared with the traditional microfluidic chip, the RNA amount of mouse embryonic dorsal root ganglion cell (DRG) axon of embryonic period E13.5 collected in the unit area of the axon chamber of the microfluidic chip is greatly improved and can reach 9.3 times of that of the traditional microfluidic chip.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The microfluidic chip is characterized by comprising a plurality of cell bodies and a plurality of axon chambers, wherein the cell bodies and the axon chambers are alternately arranged at intervals, an intermediate chamber is arranged between the adjacent cell bodies and the axon chambers at intervals, the adjacent cell bodies and the intermediate chamber, the adjacent intermediate chamber and the axon chambers are respectively communicated through micro-channels, the cell bodies, the axon chambers and the intermediate chamber form a chamber string, the axon chambers are arranged at two ends of the chamber string, the adjacent cell bodies and the intermediate chamber are communicated through a plurality of micro-channels, the distance between the adjacent two micro-channels is 3-20 mu m, and the distance between the adjacent intermediate chamber and the axon chamber is 3-20 mu m; wherein the distance between the middle chamber and the adjacent cell body chamber is 0.1 mm-1 mm; the distance between the middle chamber and the adjacent axon chamber is 0.1 mm-1 mm; the ratio of the width of the axon compartment to the width of the cell compartment is (1-5): 1, a step of; the axon chamber is rectangular, the width of the axon chamber is 1-10 mm, and the length of the axon chamber is 10-60 mm; the cell body chamber is rectangular, the width of the cell body chamber is 1 mm-10 mm, and the length of the cell body chamber is 10 mm-60 mm; the middle chamber is rectangular, the width of the middle chamber is 0.1 mm-1 mm, and the length of the middle chamber is 10 mm-60 mm.

2. The microfluidic chip according to claim 1, wherein opposite ends of the axon chamber have an axon chamber inlet and an axon chamber outlet, respectively, the axon chamber inlet or the axon chamber outlet being disposed on the same side of the chamber string;

the opposite ends of the cell are respectively provided with a cell inlet and a cell outlet, and the cell inlet or the cell outlet is arranged on the same side of the chamber string;

the opposite ends of the intermediate chamber are respectively provided with an intermediate chamber inlet and an intermediate chamber outlet, and the intermediate chamber inlet or the intermediate chamber outlet is arranged on the same side of the chamber string.

3. The microfluidic chip of claim 2, wherein the axon chamber inlet and the axon chamber outlet are each circular and equal in diameter, the cell inlet and the cell outlet are each circular and equal in diameter, the intermediate chamber inlet and the intermediate chamber outlet are each circular and equal in diameter, and the axon chamber inlet is not equal in diameter to the cell inlet.

4. A microfluidic chip according to any one of claims 1 to 3, wherein the number of the cell bodies is 2 to 20, the number of the axon chambers is 3 to 21, and the number of the intermediate chambers is 4 to 40.