Method for regulating cell morphology

Abstract

The invention discloses a method for regulating and controlling the morphology of cells, and the method comprises the following steps: etching a cell chip structure on the surface of a substrate by using a maskless digital projection exposure photoetching technology to obtain a programmable cell chip which simultaneously has one or more topological array structures with different morphologies; and inoculating the cells to the programmable cell chip to realize the regulation and control of the morphology of the single cells. According to the method, a high-precision programmable cell chip structure with one or more different morphologies is prepared by utilizing a maskless digital projection exposure photoetching technology, so that the spreading morphologies of cells on different morphological structures are regulated and controlled; and a theoretical basis with a reference value is provided for basic biological research in the fields of high-throughput cell screening and tissue engineering.

Description

Technical Field

[0001] The present invention relates to the technical fields of laser micro-structure processing and cell culture technology. More specifically, it relates to a method for regulating the morphology of cells. Background Art

[0002] As the place for cells to survive and activities in the body, the cell microenvironment is mainly composed of extracellular matrix, homotypic or heterotypic cells, growth factors, metabolites and other complex components, which can not only provide structural support for cells, but also play a role in dynamically regulating cell functions to maintain homeostasis. Current biomaterials aim to simulate the structure and function of the natural extracellular matrix of tissues in the body, so as to provide a cell microenvironment closer to that in biological tissues for cells. Research shows that the surface morphology of biomaterials, such as surface roughness, substrate stiffness and surface topological structure, has a significant regulatory effect on cell proliferation, adhesion and differentiation. To simulate the real cell microenvironment in the body and further explore the influence of material surface morphology on cell morphology, it is necessary to prepare a programmable cell chip with arbitrarily designed morphological structures to realize the simultaneous observation of the spreading morphology of cells on different topological structures on the same surface. Although traditional ultraviolet exposure lithography technology can quickly prepare large-scale microstructures, it is necessary to replace expensive photomasks when preparing different structures and it is difficult to prepare high-precision structures. Therefore, limited by the processing range, flexibility and the ability to prepare high-precision microstructures, the influence of material surface morphology on the spreading morphology of cells has not been deeply explored. Summary of the Invention

[0003] Based on the above facts, the purpose of the present invention is to provide a method for regulating the morphology of cells. This method uses maskless digital projection exposure lithography technology to prepare a programmable cell chip structure with high precision and one or more different morphologies, so as to realize the regulation of the spreading morphology of cells on different morphological structures, and provide a valuable theoretical basis for basic biological research in the fields of high-throughput cell screening and tissue engineering.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] On the one hand, the present invention provides a method for regulating the morphology of cells, and the method comprises the following steps:

[0006] Using maskless digital projection exposure lithography technology to etch a cell chip structure on the surface of a substrate to obtain a programmable cell chip, and the programmable cell chip has a topological array structure with one or more different morphologies at the same time;

[0007] Inoculating cells onto the programmable cell chip to realize the regulation of the morphology of single cells.

[0008] Further, the thickness of the programmable cell chip is 800 - 1200 nm.

[0009] Further, there is no requirement for the shape of the programmable cell chip in the technical solution of the present invention. Generally, it is circular, and can also be changed to geometric shapes such as rectangles according to requirements.

[0010] Further, the area of the topological array structure is 1% - 99% of the area of the programmable cell chip.

[0011] Further, the area of the topological array structure is 0.01 - 10 mm 2 。

[0012] Further, the structure of the programmable cell chip includes a first region and a second region. The first region has one or more topological array structures with different morphologies, and the second region is a planar structure or has at least one topological array structure with a morphology different from that of the first region.

[0013] Further, the topological array structure in the first region is one or several of the array structures of columns, holes, dots, tubes, and islands.

[0014] Further, the topological array structure in the second region is one or several of the array structures of straight lines, curves, and grooves.

[0015] Further, the topological array structure in the first region is an array structure of cylinders or round holes; the second region is a planar structure or an array structure of straight lines.

[0016] Further, in the array structure of cylinders or round holes, the diameter of the cylinder or round hole is greater than the induction threshold of a single cell to the cylinder or round hole.

[0017] Further, in the array structure of straight lines, the line width of the straight line is greater than the induction threshold of a single cell to the line width of the straight line, and the line spacing between adjacent straight lines is equal to or greater than the width of the ridge line and less than or greater than the size of a single cell.

[0018] Further, the area of the programmable cell chip is not less than 5 times the area of the contact between the cell and the programmable cell chip.

[0019] Further, the size of the cell is 5 - 100 μm.

[0020] Further, in the array structure of cylinders or round holes of the cell chip, the ratio of the diameter of a single cylinder or round hole to the size of a single cell is (0.01 - 10):1, preferably (0.01 - 3):1.

[0021] Further, in the array structure of the cylinders or round holes, the ratio of the height of a single cylinder or the depth of a single round hole to the size of a single cell is (0.01 - 1):1.

[0022] Further, in the array structure of the straight lines of the cell chip, the ratio of the line width of the straight lines to the size of a single cell is (0.01 - 1):1; the ratio of the line spacing between adjacent straight lines to the size of the cell is (0.01 - 1):1.

[0023] Further, in the array structure of the straight lines of the cell chip, the ratio of the height of the straight lines to the size of a single cell is (0.01 - 1):1.

[0024] Further, the cells are 786 - O human renal clear cell adenocarcinoma cells, L929 mouse connective tissue fibroblasts, or A549 human non - small cell lung cancer cells.

[0025] Further, the number of the inoculated cells is more than 10.

[0026] Further, the method further includes: after etching the cell chip structure on the surface of the substrate, treating the cell chip structure in O 2 plasma for 3 - 15 min, ultraviolet irradiation for 30 - 90 min, and soaking in a cell adhesion - promoting solution for 4 - 12 h and then drying.

[0027] Further, the active ingredient of the cell adhesion - promoting solution is one or more of poly - D - lysine, poly - L - lysine, collagen, fibronectin, vitronectin, and laminin.

[0028] Further, the material of the substrate is one of soft materials (such as polymers and hydrogels, etc.), glass, metal, ceramic, and silicon wafer.

[0029] In addition, unless otherwise specified, the raw materials used in the present invention can be obtained through commercial purchase. Any range described in the present invention includes the end values and any numerical value between the end values, as well as any sub - range formed by any numerical value between the end values or the end values.

[0030] The beneficial effects of the present invention are as follows:

[0031] In the method for regulating the cell morphology provided by the present invention, a programmable cell chip structure with a topological array structure having different morphologies is obtained by means of a maskless digital projection exposure lithography system. Mature cell seeds (preferably at least ten cell seeds) are inoculated on the programmable cell chip structure. After the cells come into contact with the cell chip structure of the substrate, adhesion and spreading occur, realizing the regulation of the spreading morphology of the cells. The spreading direction is random in the cylindrical or circular hole array structure and in the plane direction, while in the linear array structure, the spreading direction conforms to the linear direction, presenting a single-oriented arrangement. In addition, different sizes and different types of cell chip array structures can also regulate the cell morphology, which can provide a reference for exploring the response mechanism of cells to surface morphology and the research of the cell microenvironment. Further, in this method, the unit size and the spacing between each unit in the topological array structure are adjusted by using a programmable digital mask template to realize the regulation of the cell morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following further elaborates on the specific embodiments of the present invention with reference to the accompanying drawings.

[0033] Figure 1 Figure a shows the cell chip structure composed of a cylindrical array and a linear array in Example 1. Figure 1 Figure b shows the cell chip structure composed of a cylindrical array and a planar substrate in Example 2. Figure 1 Figure c shows the cell chip structure composed of a circular hole array and a linear array in Example 3 and Figure 1 Figure d shows a schematic diagram of the cell chip structure composed of a circular hole array and a planar substrate in Example 4.

[0034] Figure 2 Shows a schematic diagram of the spreading morphology of cells on the surface of cell chip structures composed of different types of structural units. Among them, a is the cell spreading morphology of cells in Example 1 on the cell chip structure composed of a cylindrical array and a linear array, b is the cell spreading morphology of cells on the cell chip structure composed of a cylindrical array and a planar substrate in Example 2, c is the cell spreading morphology of cells on the cell chip structure composed of a circular hole array and a linear array in Example 3, and d is the cell spreading morphology of cells on the cell chip structure composed of a circular hole array and a planar substrate in Example 4.

[0035] Figure 3 Shows the bright-field images of the cell spreading on the cell chip structures of Example 1 and Example 2 for 24 hours. Among them, a is the cell spreading situation on the cell chip structure composed of a cylindrical array and a linear array, and b is the cell spreading situation on the cell chip structure composed of a cylindrical array and a planar substrate.

[0036] Figure 4Show the bright-field images of cell spreading on the cell chip structures of Example 3 and Example 4 for 24 hours. Among them, a shows the cell spreading on the cell chip structure composed of a circular hole array and a straight line array, and b shows the cell spreading on the cell chip structure composed of a circular hole array and a planar substrate. Detailed implementation mode

[0037] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0038] In the present invention, unless otherwise specified, the methods mentioned are all conventional methods. The raw materials used can be obtained from public commercial channels unless otherwise specified, and the percentages are mass percentages unless otherwise specified.

[0039] In order to study the influence of surface topography on cell spreading morphology, the present invention uses a maskless digital projection exposure lithography system to prepare programmable cell chips with different types of structural units for auxiliary research, and specifically provides a method for regulating the morphology of cells. The method includes the following steps:

[0040] Use maskless digital projection exposure lithography technology to etch a cell chip structure on the substrate surface to obtain a programmable cell chip, and the programmable cell chip has one or more topological array structures with different topographies at the same time;

[0041] Inoculate cells onto the programmable cell chip to realize the regulation of the morphology of single cells.

[0042] In this technical solution, the maskless digital projection exposure lithography technology uses a digital micromirror device as a mask plate, without using a physical mask plate. The processed pattern can be programmed and flexibly designed, and large-area cross-scale processing and preparation of cell chips can be realized while having high processing accuracy. Cell chips with various types of structural units can be prepared to explore the spreading morphology of cells on different topographies on the same cell chip, so as to promote the understanding of the response mechanism between cells and different types of topological structures on the material surface by researchers.

[0043] In the technical solution of the present invention, the maskless digital projection exposure lithography technology is Femtosecond lasermaskless optical pro jection lithography (FS-MOPL).

[0044] Exemplarily, use maskless digital projection exposure lithography technology to etch a programmable cell chip structure with an arbitrary shape on the substrate surface, which specifically includes the following steps:

[0045] 1) Drop the photoresist on a clean substrate and spin-coat it. Then place the sample on a heating device for pre-baking.

[0046] 2) Place the spin-coated sample substrate on the three-dimensional moving stage of the maskless digital projection exposure lithography system and fix it. Turn on the program of the processing system to raise the moving stage, focus to find the appropriate processing focal plane, import the programmed pattern into the program of the processing system, set reasonable laser power, exposure time, exposure range, and processing cycle, open the shutter, and expose and process the sample.

[0047] 3) Immerse the sample processed in step 2) in the pre-prepared developer solution in a certain proportion for a certain time for development, and then rinse it with ultrapure water to complete the sample preparation.

[0048] Exemplarily, the thickness of the programmable cell chip is 800 - 1200 nm.

[0049] Exemplarily, according to the different unit heights of the required array structure, the concentration of the photoresist in step 1) can be diluted in a certain proportion using the diluent 1,2-propylene glycol monomethyl ether acetate, or the thickness of the photoresist film on the sample substrate can be changed by changing the rotation speed during spin-coating.

[0050] In addition, to promote the adhesion between the photoresist and the substrate, the substrate can be immersed in a mixed solution of KH-570 and toluene for 2 - 6 h and then spin-coated.

[0051] Exemplarily, in step 2), the maskless digital projection exposure lithography system uses a digital micromirror device containing hundreds of thousands of micromirrors as a mask. When the programmed pattern is loaded into the program of the processing system, each micromirror in the digital micromirror device will rotate to form a specific angle according to the color displayed by the pixel points on the pattern. Therefore, during the process of processing the sample, the laser emitted by the laser passes through the optical path system, passes through the digital micromirror device, and is focused on the corresponding processing focal plane of the photoresist under the high-power objective lens to achieve the exposure processing of the programmed pattern.

[0052] Exemplarily, the wavelength of the laser is 400 - 800 nm, the laser power in front of the lens is 0.060 - 0.500 mW, and the exposure time is 500 - 5000 ms. Preferably, the wavelength of the laser is 400 nm, the laser power is 0.110 - 0.130 mW, and the exposure time is 600 - 1400 ms.

[0053] Exemplarily, the developer in the developer solution is a special developer corresponding to the photoresist model. The developer needs to be diluted with ultrapure water before use to achieve an ideal development effect. Preferably, the mass ratio of the developer to the ultrapure water dilution is preferably 1:4 - 1:5.

[0054] Exemplarily, the developing time is 30 - 70 s.

[0055] Exemplarily, after etching the cell chip structure on the substrate surface, it further includes the steps of processing the cell chip structure in O 2 plasma for 3 - 15 min, ultraviolet irradiation for 30 - 90 min, soaking in a cell adhesion promoting solution for 4 - 12 h, and then drying.

[0056] Exemplarily, the soaking time in the cell adhesion promoting solution should be at least 4 h, and after soaking, the sample should be placed in a cool and ventilated place for drying.

[0057] Exemplarily, the ultraviolet irradiation should always be carried out in a laminar flow hood to reduce the risk of sample contamination.

[0058] Exemplarily, the structure of the programmable cell chip includes a first region and a second region. The first region has one or more topological array structures with different morphologies, and the second region is a planar structure or has at least one topological array structure with a morphology different from that of the first region.

[0059] Preferably, the structure of the programmable cell chip consists of a first region and a second region.

[0060] Further, when cells are inoculated on the programmable cell chip, both the first region and the second region are inoculated with cells.

[0061] Exemplarily, the topological array structure in the first region is one or several of the array structures of columns, holes, dots, tubes, and islands; the topological array structure in the second region is one or several of the array structures of straight lines, curves, and grooves. The columns, holes, dots, tubes, islands, etc. refer to the morphologies of each structural unit in the programmable cell chip structure.

[0062] Exemplarily, the topological array structure in the first region is an array structure of cylinders or round holes; the second region is a planar structure or an array structure of straight lines. Among them, the cells spreading on the straight line array structure tend to spread along the direction parallel to the ridge line, maintaining the straight line width. The smaller the line spacing between the straight lines, the stronger the orientation of cell adhesion and spreading, the greater the geometric and physical constraints on the cells, and the more obvious the deformation of the cells. The ratio of the line width of the straight line to the size of a single cell is (0.01 - 1):1; the ratio of the line spacing between adjacent straight lines to the size of the cell is (0.01 - 1):1.

[0063] Further, in the cell chip structure composed of the array structure of cylinders or round holes, the ratio of the diameter of a single cylinder or round hole to the size of a single cell is (0.01 - 10):1, preferably (0.01 - 3):1.

[0064] Among them, the "size" of the cells in the present invention refers to the approximate diameter of the cells after spreading in structure, usually referring to the length of the long axis of the cells. According to the specific embodiments of the present invention, by studying the spreading of cells on the programmable cell chips with different types of structural units, immunofluorescence staining and fluorescence imaging can also be performed on the cells: after adding a certain amount of culture medium to a cell culture dish containing the cell chip structure substrate, add a cell seed suspension with an appropriate ratio. After the cells adhere and spread on the cell chip structure for 24 h, remove the culture medium, fix, permeabilize and block the spread cells, and then perform fluorescence staining on the focal adhesion protein, actin and cell nucleus of the cells. Use a laser confocal fluorescence microscope to observe the fluorescence images of the cells. Among them, the focal adhesion protein is stained by immunobinding of the primary antibody and the secondary antibody, phalloidin is selected as the fluorescence probe for actin staining, and DAPI is selected as the fluorescence probe for cell nucleus staining. Among them, the incubation time of the primary antibody is 30 - 120 min, the incubation time of the secondary antibody is 30 - 60 min, the incubation time of phalloidin staining in the dark is 30 - 60 min, and the incubation time of DAPI staining is 5 - 20 min. Preferably, the incubation time of the primary antibody is 60 min, the secondary antibody is incubated for 30 min, phalloidin is incubated in the dark for 30 min, and DAPI is incubated for 10 min.

[0065] In the method of this technical solution, a programmable digital mask is used to adjust the unit size and the spacing between units in the topological array structure, so as to realize the regulation of cell morphology.

[0066] Exemplarily, when the programmable cell chip is composed of a topological array structure (the first region) and a planar substrate (the second region), cells are inoculated onto the programmable cell chip. The topological structure arrays in the two regions induce changes in cell morphology. By using a programmable digital mask to adjust the unit size and the spacing between units in the topological array structure, cell morphology is regulated.

[0067] Exemplarily, when the programmable cell chip is such that two topological array structures each account for a certain proportion (that is, both the first region and the second region are topological array structures, and the topological array structures in the two regions are different), cells are inoculated onto the cell chip on the substrate surface. The topological structure arrays in the two regions induce changes in cell morphology. By using a programmable digital mask to adjust the unit size and the spacing between units in the topological array structure, cell morphology is regulated.

[0068] Exemplarily, the size of the cell seeds is 5 - 100 μm; preferably 20 - 80 μm, and more preferably 50 μm.

[0069] Exemplarily, the area of the cell chip is not less than 5 times the area of the contact between the cells and the cell chip. The area of the cell chip is preferably 0.01 - 10 mm 2 , more preferably 0.5 - 1 mm 2 .

[0070] In the present invention, the area of the cell chip refers to the area of the cell chip covering the substrate.

[0071] In addition, when the cells spread randomly in the cylindrical or circular hole array structure and the plane direction, while in the linear array structure, the spreading direction conforms to the linear direction, presenting a single-oriented arrangement. By changing the unit size in the array, the geometric and physical constraints on the cells during migration are different, and the cells exhibit different spreading morphologies.

[0072] Exemplarily, the cells are 786 - O human renal clear cell adenocarcinoma cells, L929 mouse connective tissue fibroblasts, or A549 human non-small cell lung cancer cells.

[0073] The technical solutions of the present invention are described below in conjunction with some specific embodiments:

[0074] Example 1

[0075] A method for regulating the morphology of cells, comprising the following steps:

[0076] 1) Immerse a clean glass slide that has been cleaned 8 times in an ultrasonic bath in a mixed solution of KH570 and toluene for 3 h. After immersion, rinse the surface of the glass slide with ethanol and dry it for standby; Drop a mixed solution of AZ P4620 photoresist and 1,2-propylene glycol monomethyl ether acetate with a mass ratio of 1:1 on the treated glass substrate and spin-coat it at a speed of 4000 rpm for 60 s. Then transfer the sample to a hot plate and pre-bake it at 95 °C for 60 s;

[0077] 2) Place the spin-coated sample on the three-dimensional moving stage in a maskless projection lithography system, then fix both ends of the glass substrate with tape. Open the program of the processing system to raise the moving stage, focus to find the appropriate processing focal plane, and import the designed pattern (a cell chip composed of a cylindrical array with a diameter of 75 μm and a linear array with a line spacing of 7 μm, as shown in Figure 1 a) into the processing program. Set the laser wavelength to 400 nm, the laser power to 0.120 mW, the exposure time to 600 ms, and the exposure area to 0.5 mm × 0.5 mm; Click the stitching in the program, and the laser will start to expose and process the photoresist material at the processing focal plane;

[0078] 3) After processing is completed, remove the tape used to fix the sample, take down the sample, and wipe off the oil droplets on the back of the sample with a lens paper dipped in ethanol. Then, soak it in an AZ 400K developer solution prepared at a ratio of 1:4 for 40 s to obtain a glass substrate with a cell chip structure composed of a cylindrical array with a diameter of 75 μm and a linear array with a line spacing of 7 μm (both the column height and the line height are 800 nm, and the cylinder spacing is also 75 μm).

[0079] 4) Rinse the glass substrate in step 3) with sterile ultrapure water 3 - 8 times, and dry it with a hair dryer at low wind power from the back of the glass substrate or leave it to dry in a laminar flow hood.

[0080] 5) To increase the hydrophilicity of the substrate and promote cell adhesion to the substrate, treat the glass substrate in step 4) in O 2 plasma for 10 min, then sterilize it by ultraviolet irradiation for 30 min in a laminar flow hood. After that, soak it in a 0.1 mg / mL poly-D-lysine solution for 12 h, and then rinse the sample 3 times with a sterile phosphate buffer solution with a pH value of 7.4 - 7.6 to obtain a sterile glass substrate with a cell chip structure composed of a cylindrical array with a diameter of 75 μm and a linear array with a line spacing of 7 μm (i.e., obtain the programmable cell chip), and leave it to dry in a laminar flow hood for standby.

[0081] 6) Add 2 ml of RPMI medium (containing 10 wt% BSA and 1 wt% penicillin-streptomycin) to a cell culture dish (with a diameter of 3.5 cm) containing the substrate with the cell chip structure (the programmable cell chip as described above), and then add 100 μL of a cell seed suspension of 1000 cells / mL (786-O human renal clear cell adenocarcinoma cells). After the cells adhere and spread on the cell chip structure for 24 h, remove the medium, fix, permeabilize, and block the spread cells, and then perform fluorescence staining on the focal adhesion protein, actin, and cell nucleus of the cells. Among them, the focal adhesion protein is stained by the immunobinding of the primary antibody and the secondary antibody, the actin staining selects phalloidin as the fluorescent probe, and the cell nucleus staining selects DAPI as the fluorescent probe. Among them, the incubation time of the primary antibody is 60 min, the secondary antibody is incubated for 30 min, phalloidin is incubated in the dark for 30 min, and DAPI is incubated for 10 min. Observe the bright-field image of the cells using a laser confocal fluorescence microscope (as shown in Figure 3 a in

[0082] Analyzing the experimental results of step 6), it can be known that: the cells spreading on the cylindrical array structure of the cell chip can extend lamellipodia in different directions for migration, and the cell morphological orientations are not uniformly arranged. Moreover, since the diameter of the cylinders exceeds the size of the cells themselves, the cells can not only spread on the top of the cylindrical structure, but also migrate around in the gaps of the cylindrical array structure; while the cells spreading on the linear array structure of the cell chip extend sheet-like pseudopodia along the direction parallel to the lines, and the migration directions of the cells are consistent and the cells are oriented arranged ( Figure 3 as shown in a of

[0083] Example 2

[0084] A method for regulating the morphology of cells, comprising the following steps:

[0085] 1) Immerse the clean glass slides washed 8 times in ultrasonic in a mixed solution of KH570 and toluene for 3 h. After immersion, rinse the surface of the glass slides with ethanol and dry them for standby; Drop a mixed solution of AZ P4620 photoresist and 1,2-propylene glycol monomethyl ether acetate with a mass ratio of 1:1 on the treated glass substrate and spin-coat it at a speed of 4000 rpm for 60 s. Then transfer the sample to a hot plate and pre-bake it at 95 °C for 60 s;

[0086] 2) Place the spin-coated sample on the three-dimensional moving stage in the maskless projection lithography system, then fix both ends of the glass substrate with tape, turn on the program of the processing system to raise the moving stage, focus to find a suitable processing focal plane, and import the designed pattern (a cell chip composed of a cylindrical array with a diameter of 3 μm and a planar substrate, as Figure 1 shown in b of

[0087] into the processing program, set the laser wavelength to 400 nm, the laser power to 0.120 mW, the exposure time to 600 ms, and the exposure area to 0.5 mm × 0.5 mm; Click the stitching in the program, and the laser will start to expose and process the photoresist material at the processing focal plane;

[0088] 4) Rinse the glass substrate in step 3) with sterile ultrapure water 3 - 8 times, and dry it from the back of the glass substrate with a hair dryer at low wind speed or place it in a laminar flow hood to dry.

[0089] 5) To increase the hydrophilicity of the substrate and promote cell adhesion to the substrate, treat the glass substrate in step 4) in O 2 plasma for 10 min, then sterilize it by ultraviolet irradiation for 30 min in a laminar flow hood. After that, soak it in a 0.1 mg / mL poly-D-lysine solution for 12 h, and then rinse the sample 3 times with a sterile phosphate buffer solution with a pH value of 7.4 - 7.6 to obtain a sterile glass substrate with a cell chip structure composed of a cylindrical array with a diameter of 3 μm and a linear array with a line spacing of 5 μm (i.e., the programmable cell chip), and dry it in a laminar flow hood for standby.

[0090] 6) Add 2 ml of RPMI medium (containing 10 wt% BSA and 1 wt% penicillin-streptomycin) to a cell culture dish (with a diameter of 3.5 cm) containing the substrate with the cell chip structure (the programmable cell chip as described above), and then add 100 μL of a cell seed suspension (786 - O human renal clear cell adenocarcinoma cells) at a concentration of 1000 cells / mL. After the cells adhere and spread on the cell chip structure for 24 h, remove the medium, fix, permeabilize, and block the spread cells, and then perform fluorescence staining on the focal adhesion protein, actin, and cell nucleus of the cells. Among them, the focal adhesion protein is stained by immunobinding of the primary antibody and the secondary antibody, the actin staining selects phalloidin as the fluorescent probe, and the cell nucleus staining selects DAPI as the fluorescent probe. Among them, the incubation time of the primary antibody is 60 min, the incubation time of the secondary antibody is 30 min, the phalloidin is incubated in the dark for 30 min, and the DAPI is incubated for 10 min. Observe the bright-field image of the cells using a laser confocal fluorescence microscope (see Figure 3 shown in b in

[0091] According to the experimental results of step 6), the spreading situation of cells on the cell chip composed of the cylindrical array structure and the planar substrate can be obtained. The cells spread on the top of the cylindrical array structure and can extend lamellipodia in any direction for migration; the cell morphology on the planar substrate also has no unified orientation, but the number of cells on the planar substrate is more than that on the 3 μm cylindrical array structure ( Figure 3 shown in b in ). It is analyzed that since the diameter of a single cylinder is smaller than the cell's own size, the cylindrical array structure plays a certain supporting role for the cells spreading on its top; the cells on the planar substrate are not subject to the geometric and physical constraints brought by the substrate topology, so that more cells tend to migrate to the planar substrate part. It shows that the change of the structural unit type in the programmable cell chip can regulate the cell spreading situation, which provides a reference for studying the microenvironment of cells in vivo and the design of biomaterials in the field of tissue engineering.

[0092] Example 3

[0093] A method for regulating the morphology of cells, comprising the following steps:

[0094] 1) Immerse a clean glass slide that has been washed 8 times in an ultrasonic bath in a mixed solution of KH570 and toluene for 3 h. After immersion, rinse the surface of the glass slide with ethanol and blow it dry for standby; Drop a mixed solution of AZ P4620 photoresist and 1,2-propylene glycol monomethyl ether acetate with a mass ratio of 1:1 onto the treated glass substrate and spin-coat it at a speed of 4000 rpm for 60 s. Then transfer the sample to a hot plate and pre-bake it at 95 °C for 60 s;

[0095] 2) Place the spin-coated sample on a three-dimensional moving stage in a maskless projection lithography system, then fix both ends of the glass substrate with tape. Open the program of the processing system to raise the moving stage, focus to find a suitable processing focal plane, and import the designed pattern (a cell chip composed of a circular hole array structure with a diameter of 75 μm and a straight line array structure with a line pitch of 7 μm, as shown in Figure 1 c) into the processing program. Set the laser wavelength to 400 nm, the laser power to 0.120 mW, the exposure time to 600 ms, and the exposure area to 0.5 mm × 0.5 mm; Click the stitching in the program, and the laser will start to expose and process the photoresist material at the processing focal plane;

[0096] 3) After processing is completed, remove the tape used to fix the sample and take down the sample. Wipe the oil droplets on the back of the sample with a lens paper dipped in ethanol. Then soak it in an AZ 400K developer solution prepared according to a ratio of 1:4 for 40 s to obtain a glass substrate with a cell chip structure composed of a circular hole array with a diameter of 75 μm and a straight line array with a line pitch of 7 μm (the hole depth and line height are both 800 nm, and the cylinder pitch is also 75 μm);

[0097] 4) Rinse the glass substrate in step 3) with sterile ultrapure water 3 - 8 times, and dry it with a hair dryer at low wind power from the back of the glass substrate or place it in a laminar flow hood to dry;

[0098] 5) To increase the hydrophilicity of the substrate and promote cell adhesion to the substrate, treat the glass substrate in step 4) in O 2 plasma for 10 min, then sterilize it by ultraviolet irradiation for 30 min in a laminar flow hood. Then soak it in a 0.1 mg / mL poly-D-lysine solution for 12 h, and then rinse the sample 3 times with a sterile phosphate buffer solution with a pH value of 7.4 - 7.6 to obtain a sterile glass substrate with a cell chip structure composed of a circular hole array with a diameter of 75 μm and a straight line array with a line pitch of 7 μm (i.e., the programmable cell chip), and dry it in a laminar flow hood for standby;

[0099] 6) Add 2 ml of RPMI medium (containing 10 wt% BSA and 1 wt% penicillin - streptomycin) to a cell culture dish (with a diameter of 3.5 cm) containing a cell chip structure substrate (the programmable cell chip as described above), and then add 100 μL of a cell seed suspension of 1000 cells / mL (786 - O human renal clear cell adenocarcinoma cells). After the cells adhere and spread on the cell chip structure for 24 h, remove the medium. Fix, permeabilize, and block the spread cells, and then perform fluorescence staining on focal adhesion protein, actin, and cell nuclei. Among them, focal adhesion protein is stained by immunobinding of primary antibody and secondary antibody. Phalloidin is selected as the fluorescent probe for actin staining, and DAPI is selected as the fluorescent probe for cell nucleus staining. Among them, the incubation time for the primary antibody is 60 min, the incubation time for the secondary antibody is 30 min, the incubation of phalloidin is carried out in the dark for 30 min, and the incubation of DAPI is 10 min. Observe the bright - field image of the cells using a laser confocal fluorescence microscope (as shown in Figure 4 a in

[0100] Analyzing the experimental results of step 6), it can be known that: The cells spreading on the circular - hole array structure of the cell chip extend lamellipodia in random directions for migration. Since the diameter of the circular holes exceeds the size of the cells themselves, the cells can not only spread on the photoresist substrate in the gaps between the circular - hole structures, but also adhere inside the circular - hole structures; while the cells spreading on the linear - array structure of the cell chip extend sheet - like pseudopodia along the direction parallel to the lines and migrate in this direction showing an oriented arrangement ( Figure 4 as shown in a in

[0101] Example 4

[0102] A method for regulating the morphology of cells, comprising the following steps:

[0103] 1) Immerse a clean glass slide that has been ultrasonically cleaned 8 times in a mixed solution of KH570 and toluene for 3 h. After immersion, rinse the surface of the glass slide with ethanol and dry it for standby; Drop a mixed solution of AZ P4620 photoresist and 1,2 - propylene glycol monomethyl ether acetate with a mass ratio of 1:1 on the treated glass substrate and perform spin - coating at a speed of 4000 rpm for 60 s. Then transfer the sample to a hot plate and pre - bake at 95 °C for 60 s;

[0104] 2) After placing the spin-coated sample on the three-dimensional moving stage in the maskless projection lithography system, fix both ends of the glass substrate with tape. Then, turn on the program of the processing system to raise the moving stage, focus to find the appropriate processing focal plane, and import the designed pattern (a cell chip composed of a circular hole array with a diameter of 3 μm and a planar substrate, as shown in d in Figure 1 ) into the processing program. Set the laser wavelength to 400 nm, the laser power to 0.120 mW, the exposure time to 600 ms, and the exposure area to 0.5 mm × 0.5 mm. Click the stitching in the program, and the laser will start exposing and processing the photoresist material at the processing focal plane;

[0105] 3) After the processing is completed, remove the tape used to fix the sample, take down the sample, and wipe the oil droplets on the back of the sample with a lens paper dipped in ethanol. Then, soak it in an AZ 400K developer solution prepared at a ratio of 1:4 for 40 s to obtain a glass substrate with a cell chip structure (the hole depth is 800 nm) composed of a circular hole array with a diameter of 3 μm and a planar substrate;

[0106] 4) Rinse the glass substrate in step 3) with sterile ultrapure water 3 - 8 times, and dry it with a hair dryer at low wind speed from the back of the glass substrate or place it in a clean bench to dry;

[0107] 5) To increase the hydrophilicity of the substrate and promote cell adhesion to the substrate, treat the glass substrate in step 4) in O 2 plasma for 10 min, then irradiate it with ultraviolet light in a clean bench for 30 min for sterilization. Then, soak it in a 0.1 mg / mL poly-D-lysine solution for 12 h, and rinse the sample 3 times with a sterile phosphate buffer solution with a pH value of 7.4 - 7.6 to obtain a sterile glass substrate with a cell chip structure composed of a cylindrical array with a diameter of 3 μm and a planar substrate (i.e., the programmable cell chip), and dry it in a clean bench for standby;

[0108] 6) Add 2 ml of RPMI medium (containing 10 wt% BSA and 1 wt% penicillin-streptomycin) to a cell culture dish (with a diameter of 3.5 cm) containing a cell chip structure substrate (the programmable cell chip as described above), and then add 100 μL of a cell seed suspension (786-O human renal clear cell adenocarcinoma cells) at a concentration of 1000 cells / mL. After the cells adhere and spread on the cell chip structure for 24 h, remove the medium, fix, permeabilize, and block the spread cells, and then perform fluorescence staining on the focal adhesion protein, actin, and cell nucleus of the cells. Among them, the focal adhesion protein is stained by the immunobinding of the primary antibody and the secondary antibody, the actin staining selects phalloidin as the fluorescent probe, and the cell nucleus staining selects DAPI as the fluorescent probe. Among them, the incubation time of the primary antibody is 60 min, the incubation time of the secondary antibody is 30 min, the phalloidin is incubated in the dark for 30 min, and the DAPI is incubated for 10 min. Observe the bright-field image of the cells using a laser confocal fluorescence microscope (see Figure 4 as shown in b in

[0109] Figure 4 b is the bright-field image taken after the cells spread on the cell chip structure composed of a cylindrical array with a diameter of 3 μm and a planar substrate for 24 h in step 6). The cell morphologies spreading on the circular hole array structure and the planar substrate of the cell chip both show normal epithelial-like shapes without obvious deformation ( Figure 4 as shown in b in

[0110] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for regulating the morphology of cells, characterized in that: The method comprises the following steps: Using maskless digital projection exposure lithography technology to etch a cell chip structure on the surface of a substrate to obtain a programmable cell chip, wherein the programmable cell chip has one or more topological array structures with different morphologies; Cells are seeded onto the programmable cell chip to achieve regulation of the morphology of single cells.

2. The method according to claim 1, characterized in that The structure of the programmable cell chip includes a first region and a second region, the first region has one or more topological array structures with different morphologies, and the second region is a planar structure or has at least one topological array structure with a morphology different from that of the first region.

3. The method according to claim 2, characterized in that The topological array structure in the first region is one or more of an array structure of columns, holes, dots, tubes and islands; and / or The topological array structure in the second region is one or more of a straight line, a curve and a groove array structure.

4. The method according to claim 2 or 3, characterized in that: The topological array structure in the first region is an array structure of cylinders or circular holes; and the second region is a plane structure or a linear array structure.

5. The method according to claim 4, characterized in that In the array structure of the cylinders or holes, the diameter of the cylinders or holes is greater than the sensing threshold of the single cell to the cylinders or holes; and / or In the array structure of straight lines, the line width of the straight lines is greater than the sensing threshold of a single cell to the line width of the straight lines, and the line spacing between adjacent straight lines is equal to or greater than the width of the ridge line and is smaller than or greater than the size of a single cell.

6. The method according to claim 1, characterized in that The area of ​​the programmable cell chip is not less than 5 times the area of ​​the cells in contact with the programmable cell chip; Preferably, the size of the cells is 5-100 μm.

7. The method according to claim 4, characterized in that In the array structure of cylinders or holes in the cell chip, the ratio of the diameter of a single cylinder or hole to the size of a single cell is (0.01-10):1; Preferably, in the array structure of the cylinders or holes, the ratio of the height of a single cylinder or the depth of a single hole to the size of a single cell is (0.01-1):1; Preferably, in the linear array structure of the cell chip, the ratio of the line width of the line to the size of a single cell is (0.01-1):1; the ratio of the line spacing between adjacent lines to the size of the cell is (0.01-1):1; Preferably, in the linear array structure of the cell chip, the ratio of the height of the straight line to the size of a single cell is (0.01-1):

1.

8. The method according to claim 1, characterized in that The cells are 786-O human renal clear cell adenocarcinoma cells, L929 mouse connective tissue fibroblasts or A549 human non-small cell lung cancer cells.

9. The method according to claim 1, characterized in that: The number of cells inoculated is more than 10.

10. The method according to claim 1, characterized in that The method further comprises: after etching the cell chip structure on the surface of the substrate, treating the cell chip structure in O2 plasma for 3-15 minutes, irradiating with ultraviolet light for 30-90 minutes, soaking in a cell adhesion promoting solution for 4-12 hours, and then drying; Preferably, the active ingredient of the cell adhesion promoting solution is one or more of poly-D-lysine, poly-L-lysine, collagen, fibronectin, vitronectin and laminin.

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