CN106282016B

CN106282016B - Two-dimensional cell scratch chip and preparation method and application thereof

Info

Publication number: CN106282016B
Application number: CN201610649492.6A
Authority: CN
Inventors: 陈健; 卫元晨; 郝锐; 栾韶亮; 张韬; 陈德勇; 贾鑫; 王军波; 郭伟
Original assignee: Institute of Electronics of CAS; Chinese PLA General Hospital; Peking University Peoples Hospital
Current assignee: Institute of Electronics of CAS; Chinese PLA General Hospital; Peking University Peoples Hospital
Priority date: 2016-08-10
Filing date: 2016-08-10
Publication date: 2020-08-07
Anticipated expiration: 2036-08-10
Also published as: CN106282016A

Abstract

The invention provides a two-dimensional cell scratch chip and a preparation method and application thereof. According to the invention, a micro-processing technology and a scratch experiment are combined, and a scratch chip which can realize high flux and can observe two-dimensional cell migration in real time is designed. The chip is structurally compatible with the traditional porous plate technology, and can be completely applied to the existing porous plate platform.

Description

Two-dimensional cell scratch chip and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and further relates to a two-dimensional cell scratch chip, a preparation method of the chip, and a cell migration experiment performed by using the chip.

Background

Cell migration participates in important physiological processes such as tissue organ formation, tissue injury repair and the like; dysfunction of cell migration can lead to tumor cell invasion and metastasis. The occurrence, development and prognosis of certain diseases (ischemic cardiovascular diseases and tumors) can be influenced by promoting or inhibiting cell migration, so that the research on the cell migration has important theoretical significance and practical value.

Currently, the conventional methods for measuring cell migration are most commonly used as a cell scratch Chamber (Boyden Chamber) and a scratch method. The former consists of an upper chamber and a lower chamber, and the middle is separated by a microporous filter membrane; chemotactic factor is added into the lower chamber to form a concentration gradient, and cells are inoculated into the upper chamber, are influenced by the concentration gradient of the chemotactic factor and move to the other side through the micropores on the membrane; thereafter, the cells were fixed, stained and counted to determine their migratory ability. In the latter, a scratch is formed on the fused cell by using a pipette tip, and the migration of the cell to the scratched area is observed to determine the migration ability of the cell. The scratching method has the advantages of simplicity and visualization of the cell migration process, so that the scratching method becomes a more common method in cell migration research.

Although the scratch method has been widely used in migration research, it is difficult to control the scratch speed and the shape and boundary of the wound area by the conventional scratch method, so that it is difficult to compare different experiments, resulting in poor repeatability of the experiments. Since microfabrication technology can be used to fabricate various biomimetic structures that can mimic the cellular microenvironment, this technology has been widely used in recent years to study cell migration. One prior art technique utilizes microfabrication to fabricate the cell-scratch chip and laminar flow to achieve cell migration. Another prior art is a method of achieving cell migration by direct current in combination with chemical surface modification. Compared with the traditional scratch method, the two-dimensional scratch method based on the micro-processing technology has the advantages of good controllability, high precision, capability of simulating a cell microenvironment and the like, thereby demonstrating the feasibility of researching cell migration through the processing technology.

However, the prior art has the following technical defects:

(1) the existing cell scratching method relies on manual scratching operation, so that the scratching quality among experimental groups in the method is not uniform, cells and extracellular matrix are damaged in the process, and migration results are possibly not accurate and credible.

(2) The existing two-dimensional scratch research based on the micromachining technology has generally low flux and high cost aiming at a scratch experiment, and is difficult to be applied in a large range.

Disclosure of Invention

Technical problem to be solved

In view of the above, the present invention is directed to a two-dimensional cell-scratch chip, a method for manufacturing the same, and an application of the same, so as to overcome at least one of the technical problems of the prior art.

(II) technical scheme

According to an aspect of the present invention, there is provided a two-dimensional cell-scratching chip comprising a substrate and an array of cells opened at upper and lower sides, wherein: the cell array is fixed on the substrate, so that the substrate closes one side opening of the cell array to form a plurality of cells with single-side openings; each of the chambers has a metal layer formed on a partial region on one side of the substrate, and a self-assembled molecular layer is further formed on the metal layer, and molecules in the self-assembled molecular layer have a first functional group that reacts with the metal and adsorbs, and have a second functional group that inhibits cell adsorption, at one end.

Preferably, the boundary of the partial region is circular, triangular, rectangular or diamond-shaped.

Preferably, the boundary of the cells on the substrate is circular or square.

Preferably, the first functional group is a hydrogen-sulfur bond.

Preferably, the second functional group is a long chain polyethylene glycol.

Preferably, the cell array is an array of N rows and M columns, N is a natural number between 1 and 20, and M is a natural number between 1 and 30.

According to another aspect of the present invention, there is also provided a method for preparing a two-dimensional cell-scratch chip, comprising the steps of:

s1: preparing a small chamber array with openings at the upper side and the lower side;

s2: depositing a metal layer array on a substrate;

s3: fixing the substrate and the cell array by adopting a bonding mode, so that the substrate closes one side opening of the cell array to form a plurality of three-dimensional cells with single-side openings, and the metal layer array is correspondingly positioned in each cell one by one;

s4: and forming a self-assembled molecular layer on the metal layer, wherein one end of molecules in the self-assembled molecular layer is provided with a first functional group which reacts with the metal and adsorbs the metal, and the other end of the molecules is provided with a second functional group which inhibits cell adsorption.

Preferably, step S4 includes the sub-steps of: cleaning the substrate to remove impurities on the surface of the metal; and (3) dropwise adding a diluted mercaptan solution into each chamber, sealing and soaking, and forming a self-assembled molecular layer on the surface.

According to another aspect of the present invention, there is provided a two-dimensional cell migration assay using any one of the above chips, comprising:

modifying extracellular matrix on the surface of the region of the chamber without the metal layer, so that the cells can easily grow adherent to the surface of the substrate after being inoculated;

digesting the cells to a suspension state, inoculating the cell suspension to each chamber, and then culturing the cells until the cells grow in the chamber in an adherent manner in the metal layer-free region;

after the cells are grown in an adherent way, the cells are limited in the metal layer-free area under the action of the self-assembled molecular layer;

when the cells are fused and grown into a monolayer, sucking out the culture medium in the small chamber, adding a collagen solution, and after the collagen solution is naturally spread, placing the chip in an incubator to completely solidify collagen;

after the collagen is solidified, cell culture media containing different types or concentrations of chemotactic factors are added into each chamber, then a two-dimensional cell monolayer can adhere to the collagen to migrate, and then the two-dimensional migration condition of the cells is recorded by photographing.

(III) advantageous effects

According to the technical scheme, the two-dimensional cell scratch chip and the preparation method and application thereof have the following beneficial effects:

(1) compared with the existing cell scratching method, the method controls the scratching boundary by using the structure, covers mercaptan by using a collagen adding method, can ensure that scratches among holes are consistent, and cells and extracellular matrix cannot be damaged in the process, so the migration result of the method is accurate and reliable;

(2) compared with the conventional scratch research based on the micro-machining technology, the invention can realize multiple groups (such as 384 groups) of parallel experiments on one chip and has high flux. In terms of cost reduction, on the one hand because of the high flux; on the other hand, the chip can be reused in theory. After the used chip is cleaned and subjected to aseptic treatment, thiol modification can be carried out again, and the chip is put into a scratch experiment, so that the cost of the technical method is greatly reduced again;

(3) the boundary of the deposited metal layer is not in contact with the boundary of the chamber on one side of the substrate, and cells can be controlled to realize two-dimensional migration.

Drawings

FIG. 1 is a general flowchart of a two-dimensional cell-scratching chip technology according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a two-dimensional cell-scratch chip according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a two-dimensional cell scratch chip according to an embodiment of the present invention;

FIG. 4 is a flow chart of a two-dimensional cell-scratch chip according to an embodiment of the present invention;

FIG. 5 is a schematic diagram of a two-dimensional cell scratch chip according to an embodiment of the present invention;

FIG. 6 is a flow chart of an on-chip experiment of a two-dimensional cell scratch chip according to an embodiment of the present invention;

FIG. 7 is a schematic diagram of an on-chip experiment of a two-dimensional cell scratch chip according to an embodiment of the present invention.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The invention combines the micro-processing technology with the scratch experiment and designs the scratch chip which can realize high flux and can observe the cell migration in real time. The chip is structurally compatible with the traditional porous plate technology, and can be completely applied to the existing porous plate platform. The technical method of the present invention will be described below by taking a 96-well plate as an example, but the present invention is not limited thereto, and may be any array of N rows and M columns, where N is a natural number between 1 and 20, and M is a natural number between 1 and 30. The specific implementation conditions of the technical method are as follows:

the general flow of the technical method comprises chip design (namely design of a two-dimensional cell scratch chip), chip manufacturing (namely preparation method of the two-dimensional cell scratch chip), on-chip experiment and data processing (namely application of the two-dimensional cell scratch chip), and the schematic diagram is shown in fig. 1:

fig. 1 is a general flowchart of a two-dimensional cell scratch chip technology method according to an embodiment of the present invention, and the flow includes: step 1, chip design:

the principle of chip design can be seen with reference to fig. 2. FIG. 2 is a schematic diagram of a two-dimensional cell-scratch chip according to an embodiment of the present invention. This example uses a self-assembled molecular layer containing a first functional group and a second functional group, such as a polyethylene glycol (PEG) long-chain thiol molecule (EG-terminated thiols, abbreviated as EG 6) of the formula HS (CH)₂)₁₁(OCH₂CH₂)₆OH). Such thiol molecules can react with metallic gold (Au) to form a gold-sulfur bond on one side of the sulfur atom (S). The other end of the thiol has the PEG, so that the protein adsorption resistance effect can be realized, and the cell adsorption on the Au surface can be further prevented.

It will be appreciated by those skilled in the art that the long chain of polyethylene glycol in the thiol molecule that inhibits cellular and protein adsorption is not limited to a particular chemical formula, but may also be [ -CH₂CH₂O-]₄OH、[-CH₂CH₂O-]₄CH₃And other chemical formulas. The thiol molecule is not limited to a polymer molecule, and may have a hydrogen-sulfur bond at one end which can be adsorbed on the gold surface and at the other end which can be adsorbed on the gold surfaceLong chains of polyethylene glycol (PEG) which prevent cell adhesion may be used herein.

FIG. 3 is a schematic diagram of a two-dimensional cell-scratch chip according to an embodiment of the present invention. Fig. 3, panel a is a side view of the two-dimensional cell scratch chip designed by the present invention, the chip is divided into three layers, which are a scratch cell layer made of Polydimethylsiloxane (PDMS), a gold structure layer, and a bottom glass substrate layer in sequence from top to bottom.

It will be appreciated by those skilled in the art that the scratch chamber can be made of any material that can be used to perform cell experiments and is not limited to polydimethylsiloxane, the metal of the metal array is not limited to gold, and the substrate material is not limited to glass, and the above materials are listed in this embodiment for the purpose of describing details and are not intended to limit the invention.

Panel B in fig. 3 is a top view of a two-dimensional cell scratch chip according to the present invention. The PDMS was 8 mm thick and formed a scratch cell layer with 96 circular scratch cells in 8 rows and 12 columns, with a diameter of 6 cm, and the center-to-center spacing between adjacent cells was 9 cm, consistent with the size of a standard 96-well plate. Each cell corresponds to a circular gold structure with a diameter of 1 mm. The thickness of the glass substrate of the lowermost layer was 1 mm. The above sizing is merely exemplary and one skilled in the art can design any chamber and Au circle size that will allow three-dimensional migration of cells.

An enlarged view of each scratch cell is given in panel C of fig. 3, which shows the inclusion of a gold surface and a glass surface within each scratch cell hole. Wherein the gold structure is a circle with a diameter of 1 mm, and the rest is a glass surface.

Step 2, chip manufacturing:

the manufacturing process comprises the steps of manufacturing a PDMS scratch cell array and an Au structure on the surface of a glass substrate, and carrying out thiolation on the Au structure after bonding the two, so that PEG is formed on the surface of the Au structure. The manufacturing method of the PDMS scratch cell array comprises the following steps: and 3D printing a PDMS male mold, pouring PDMS, and turning over the mold to obtain the scratch cell. The manufacturing method of the Au structure on the surface of the glass substrate comprises the following steps: and (3) manufacturing a photoresist male die (such as AZ1500), sputtering chromium and gold, and stripping to obtain a gold structure. Then, PDMS and glass are bonded, the gold structure is modified by mercaptan, and PEG is formed on the surface of the gold to complete the chip manufacturing. The whole chip manufacturing process is as shown in fig. 4: the process comprises the following steps:

s1: preparing an array of cells open at both upper and lower sides, the step comprising the substeps A2-C2:

substep a2, 3D printing PDMS master mold:

and drawing a three-dimensional graph of the PDMS male mold by using three-dimensional drawing software, and inputting the three-dimensional graph into 3D printer software, wherein the printer can automatically print the male mold shown in a subgraph A in the graph 5.

Substep B2, casting PDMS:

PDMS and curing agent mix according to certain proportion, stir, pour on 3D printed male mold, bake overnight at certain temperature and make it solidify. See panel B of fig. 5.

And a substep C2, performing die flipping to obtain scratch cells:

and (3) turning the cured PDMS to obtain a 96-hole array, namely the scratch cell array. See panel C in fig. 5.

Step S2: depositing an array of metal layers on a substrate, the step comprising substeps D2-F2

Substep D2, AZ1500 male mold making:

and sequentially cleaning the glass sheet in acetone, ethanol and deionized water, drying, uniformly and rotationally coating a layer of AZ1500 on the surface, and placing a mask plate for exposure. After development in the developing solution, the glass surface is exposed at the position where the sputtering structure is needed, and the sacrificial layer formed by AZ1500 is left at the position where the sputtering structure is not needed. See sub-graph D in fig. 5. Of course, the AZ1500 is only an exemplary material, and those skilled in the art may alternatively select any material from which a model can be made.

Substep E2, sputtering of chromium, gold:

and sputtering a layer of chromium on the glass sheet obtained in the last step to enhance the adsorbability of the Au structure, and then sputtering a layer of Au. See panel E of figure 5.

Substep F2, lift-off to obtain a gold structure:

and (3) soaking the glass sheet in acetone and carrying out ultrasonic treatment until the metal layer is completely stripped, thereby finishing the manufacture of the Au structure. See panel F in fig. 5.

Substep G2, bonding PDMS to glass (i.e. step S3):

referring to fig. 5, panel G, the scratched cell layer made of PDMS and the glass substrate layer made of Au structure are bonded to obtain a complete chip.

Step S4: and forming a self-assembled molecular layer on the metal layer, wherein one end of molecules in the self-assembled molecular layer is provided with a first functional group which reacts with the metal and adsorbs the metal, and the other end of the molecules is provided with a second functional group which inhibits cell adsorption. This step includes sub-steps H2 and I2.

Substep H2, thiol-modified gold structure:

before thiol modification, the complete chip is cleaned to remove impurities on the Au surface, so that the thiol modification effect is enhanced. Then, referring to panel H of fig. 5, a dilute thiol solution is added dropwise to each scratch cell and soaked for a period of time, typically 24 hours, to react the Au structure with the thiol.

Substep I2, PEG formation on the gold surface:

referring to fig. 5, panel I, the Au structure forms a layer of PEG on its surface after being soaked in a thiol solution for a period of time. So far, the fabrication of the chip is completed.

Step 3, on-chip experiment:

the above-mentioned steps for fabricating the completed two-dimensional cell-scratch chip will be described in detail herein. Mainly comprises the steps of modifying extracellular matrix, inoculating and attaching cells, covering thiol with collagen, curing collagen and migrating cells, and is shown in a flow chart of figure 6, wherein the flow comprises the following steps:

substep a3, modifying the extracellular matrix:

before cell seeding, it is necessary to modify the extracellular matrix on the glass surface of the scratch chamber, where the extracellular matrix is usually fibronectin, to enhance the adhesion of cells on the glass surface, so that the cells can grow adherent to the glass surface more easily after seeding, see fig. 7, panel a

Substep B3, cell seeding and adherence:

the cells were digested to suspension and seeded in individual scratch chambers using a pipette gun. Cells were cultured until adherent growth of cells was obtained outside the Au surface, see panel B in fig. 7. Usually, the culture medium is replaced once after the cells are attached to the wall, and the cells which are not attached to the wall are removed, wherein the specific liquid replacement time depends on the specific condition of the cells. The culture precautions are the same as those of a common 96-well plate.

Substep C3, collagen coating thiol and collagen curing:

generally, before performing the scratch test, the whole glass surface is covered with cells to form a monolayer of cells. First, the medium in the scratch cell was aspirated, exposing the thiol-modified gold structure. A concentration of collagen solution (this concentration may be 1-10mg/ml) is added, the volume of solution being such that it covers the entire bottom surface of the scratch chamber and does not spill out (typically 30-150. mu.l is added).

The chip with the collagen added is put into a large culture dish, and sterile pure water or PBS is added into the culture dish to ensure the air humidity in the culture dish, and the liquid level can not exceed the height of the chip. The whole dish with the chip was placed in a 37 ℃ incubator and left for a period of time (typically 1h) to fully solidify the collagen, see panel C in FIG. 7. The PEG on the surface of the Au structure is completely covered, and the cells can grow adherently or migrate to the surface of the Au.

Substep D3, cell migration:

after the step of adding collagen to cover thiol is completed, culture media with different types or concentrations of chemokines are added into different scratch chambers according to specific experimental requirements. The photograph records the migration of the cells to the Au surface after a certain time, see Panel D in FIG. 7.

And 4, data processing:

and (3) carrying out data processing on the micrographs of the migration condition of the cells to the Au surface so as to obtain specific parameters of the cell migration. The analysis method can use image recognition software to recognize the area of Au surface not occupied by cells, and then the experimental conclusion can be obtained through the comparison between the migration results of different types or concentrations of chemokines.

It is worth mentioning that during the above experiments on a chip, cells can be seeded in a corresponding number of wells according to the actual need, without having to use 96 wells completely in one experiment. In addition, in the step of adding the collagen to cover the mercaptan, the mercaptan in the pores can not be covered according to experimental needs, so that the mercaptan in the pores still functions, scratches still exist, and the method is flexible and controllable.

Furthermore, the above definitions of the various elements and methods are not limited to the particular structures, shapes or arrangements of parts mentioned in the examples, which may be easily modified or substituted by one of ordinary skill in the art, for example:

(1) the shape of the scratch cell is not limited to a circle, and can be other regular or irregular figures such as a square, a triangle and the like;

(2) the number of the scratch cells is not limited to 96, and can be 384, 24, 12 or 6, and other standard multi-well plate holes;

(3) in each scratch cell, the pattern of the gold structure is not limited to the shape shown in sub-graph B in fig. 3, and can be other regular or irregular shapes such as a long strip, a square, a triangle and the like according to the shape of the needed scratch;

(4) the male die for pouring the PDMS is not only a 3D printing method, but also the used material is not limited to a certain material, and any method and material capable of forming a columnar array can be used for manufacturing the PDMS male die;

(5) the long polyethylene glycol chains in the thiol molecule that inhibit cellular and protein adsorption are not limited to a particular chemical formula, including [ -CH ]₂CH₂O-]₄OH、[-CH₂CH₂O-]₆OH、[-CH₂CH₂O-]₄CH₃、[-CH₂CH₂O-]₆CH₃And other similar chemical formulas;

(6) the extracellular matrix for enhancing the cell adhesion effect is not limited to fibronectin, and other extracellular matrices such as polylysine (Poly-L-L ysine) and the like can be used;

(7) the reagent for constructing the three-dimensional cell migration environment is not limited to collagen, and can also be sodium alginate, hydrogel and other reagents;

(8) directional phrases used in this disclosure, such as "upper," "lower," "front," "rear," "left," and "right," etc., refer only to the direction of reference and are not intended to limit the invention.

Up to this point, the present embodiment has been described in detail with reference to the accompanying drawings. From the above description, those skilled in the art should clearly understand the two-dimensional cell-scratch chip and the method for preparing and using the same.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for carrying out two-dimensional cell migration experiment by using a two-dimensional cell scratch chip is not applied to the treatment and diagnosis of diseases; the two-dimensional cell scratch chip comprises a substrate and a cell array with openings at the upper side and the lower side, wherein the cell array is fixed on the substrate to enable the substrate to close one side of the cell array with the openings, so that a plurality of cells with openings at one side are formed; each chamber is provided with a metal layer on a partial area of one side of the substrate, a self-assembly molecular layer is further formed on the metal layer, and molecules in the self-assembly molecular layer have a first functional group which reacts and adsorbs with the metal at one end and a second functional group which inhibits cell adsorption at the other end; the boundary of the partial area is in a circle shape, a triangle shape, a rectangle shape or a diamond shape; the method comprises the following steps:

modifying extracellular matrix on the surface of the metal layer-free region of the scratch chamber, so that cells can easily adhere to the surface of the substrate after being inoculated and grow;

2. The method of claim 1, wherein the cells are bounded on the substrate by a circle or a square.

3. The method of claim 1, wherein the first functional group is a hydrogen sulfide bond.

4. The method of claim 1, wherein the second functional group is a long chain of polyethylene glycol.

5. The method of claim 1, wherein the array of cells is an array of N rows and M columns, N being a natural number between 1-20, and M being a natural number between 1-30.