CN117567820A - Device containing topological appearance array surface and preparation method and application thereof - Google Patents

Device containing topological appearance array surface and preparation method and application thereof Download PDF

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Publication number
CN117567820A
CN117567820A CN202311527931.2A CN202311527931A CN117567820A CN 117567820 A CN117567820 A CN 117567820A CN 202311527931 A CN202311527931 A CN 202311527931A CN 117567820 A CN117567820 A CN 117567820A
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array
micro
cell
topological
cell adhesion
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丁建东
王弘宇
刘瑞丽
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Fudan University
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Fudan University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00023—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems without movable or flexible elements
    • B81C1/00103—Structures having a predefined profile, e.g. sloped or rounded grooves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00436—Shaping materials, i.e. techniques for structuring the substrate or the layers on the substrate
    • B81C1/00555—Achieving a desired geometry, i.e. controlling etch rates, anisotropy or selectivity
    • B81C1/00626—Processes for achieving a desired geometry not provided for in groups B81C1/00563 - B81C1/00619
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C08J7/123—Treatment by wave energy or particle radiation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
    • C08J2325/02—Homopolymers or copolymers of hydrocarbons
    • C08J2325/04—Homopolymers or copolymers of styrene
    • C08J2325/06—Polystyrene
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention belongs to the technical field of material surface modification, and particularly relates to a device containing a topological appearance array surface, and a preparation method and application thereof. The device comprises a substrate and cell adhesion contrast modification positioned on the surface of the substrate, wherein the topological morphology array comprises a non-embedded topological morphology array and an embedded topological morphology array; the non-embedded topological appearance array is a micro-pit array, micro-column arrays in the embedded topological appearance array are distributed in the micro-pits, and the height of each micro-column is lower than the upper edge of each micro-pit; the cell adhesion contrast is modified such that the exterior of the micro-pits have cell adhesion-resisting properties and the interior has cell adhesion-promoting properties. The invention prepares the device containing the topological appearance array surface by using a solvent volatilization method of a soft template, and realizes the application of the device containing the topological appearance array in single cell positioning, independent regulation and control of cell appearance and cell nucleus shape by micro-pit inside-outside cell adhesion contrast treatment of a plasma technology and a micro-contact printing technology.

Description

Device containing topological appearance array surface and preparation method and application thereof
Technical Field
The invention belongs to the technical field of material surface modification, and particularly relates to a device containing a topological appearance array surface, and a preparation method and application thereof.
Background
The shape and the shape of the cell nucleus of the cell can be changed under the mechanical stimulation of physiological microenvironments such as peripheral cells, extracellular matrixes and the like, so that the cell behavior such as proliferation, migration, invasion, differentiation and the like is obviously changed, and a plurality of important physiological and pathological processes such as development, wound repair, cancer progression and the like are further influenced. In order to study the influence of cell shape change and cell nucleus deformation on cell behavior and fate, researchers develop a series of artificial extracellular matrixes with surface topological structures or cell adhesion contrast patterns based on micromachining technology, induce the active change of cell shape and cell nucleus shape by space geometric limitation, and bring great convenience to in vitro study, so that the method is widely applied to the basic study field of cell biology.
In vivo, changes in cell morphology and nuclear deformation often alter transduction processes and gene expression of intracellular signaling pathways through different pathways. Therefore, it is necessary to independently control two processes, namely, to be able to change the cell shape under the condition that the degree of cell nuclear deformation is equivalent and to be able to change the degree of cell nuclear deformation under the condition that the cell shape is the same, in order to explore the influence of each of the cell shape change and the cell nuclear deformation on the cell behavior such as phenotype transformation. However, the current surface topology and cell adhesion contrast pattern always inevitably change both cell shape and cell nucleus shape, and thus independent regulation of these two factors cannot be achieved, and their respective effects on cell behavior such as transformation of cancer cell phenotype cannot be explored.
Therefore, developing an artificial extracellular matrix capable of independently controlling the cell shape and the cell nucleus shape of single cells would have great significance in the fields of cell biology, tissue regeneration research, cancer treatment, and the like.
Disclosure of Invention
In view of the above, a first object of the present invention is to provide a device with a topological shape array surface capable of independently controlling the cell shape and the cell nucleus shape of individual cells, aiming at the problems existing in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a device comprising a topographic array surface comprising a substrate and a cell adhesion contrast modification on the substrate surface, the substrate surface having a topographic array comprising a non-chimeric topographic array and a chimeric topographic array.
The topological appearance array is a precise and controllable material surface appearance which can cause space limitation on cell adhesion, the shape, the size and the spacing of the topological patterns can be flexibly adjusted, and the topological appearance array can be used for researching cell biology such as cell adhesion limitation, migration, cell nucleus deformation and the like. The cell adhesion contrast modification technology is a technology for limiting the cell adhesion area and the spatial distribution of the cell adhesion area on the surface of a material, and can be used in the fields of single cell arrays, cell shape regulation, mechanism research of cell adhesion and the like. The topological appearance array surface disclosed by the invention can combine the topological appearance array with a cell adhesion contrast modification technology, so that independent regulation and control on the appearance and the nuclear shape of cells are realized.
Further, the base material is a non-biotoxic polymeric material; the cell adhesion contrast on the surface of the substrate is modified into a polymer molecular layer with cell adhesion promoting surface inside the micro pits and cell adhesion resisting property outside the micro pits.
Still further, the non-biotoxic polymer material comprises polystyrene PS, polypropylene PP, polyglycolide-co-lactide PLGA, poly-l-polylactic acid PLLA, silicone rubber, natural rubber or plexiglass; the cell adhesion resistant polymer is polyethylene oxide-polypropylene oxide-polyethylene oxide PEO-PPO-PEO triblock polymer or polyethylene oxide-polypropylene oxide PEO-PPO diblock polymer.
The invention uses polymer material without biological toxicity as a substrate, is suitable for the application in the fields of cell biology and molecular biology research and medical treatment, and has wide potential application scenes. In particular, for polymers with poor surface cell adhesion such as PS, the surface hydrophilic treatment is a method for improving the surface cell adhesion of materials. PEO-PPO-PEO and PEO-PPO are taken as two block polymers, wherein a PEO segment has the property of resisting cell adhesion, and a hydrophobic PPO segment can self-assemble on a hydrophobic surface, so that the PEO segment is exposed to realize the modification of resisting cell adhesion on the hydrophobic surface.
Further, the non-chimeric topological morphology array is a micro-pit array; the micro-column arrays in the embedded topological appearance array are distributed in the micro-pits, and the height of the micro-columns is lower than the upper edge of the micro-pits.
It is worth noting that the micro-column array with proper height and interval is a topological shape capable of inducing cell nuclei to actively generate remarkable deformation. The micro-column array in the embedded topological morphology array is positioned in the micro-pit, the height, the size and the interval range of the micro-column array ensure that the nuclei of cells in the micro-pit can be obviously deformed, the height is not more than that of the micro-pit, and the surface modification state which is the same as that in the micro-pit is maintained.
Further, the aspect ratio of the micro-pits is 1-9, and the projection area is 600-1200 mu m 2 The distance between the micro pits is not less than 10 mu m, and the depth is 5-15 mu m; the side length of the microcolumn is 2-7 mu m, the height is 4-12 mu m, and the interval is 3-12 mu m.
It is worth noting that the range of dimple sizes described in the present invention can just accommodate single cell adhesion, i.e., not too small to cause good cell spreading, nor too large to cause accommodation of multiple cells; the spacing of the micro-pits is proper, so that the situation that single cells cross a plurality of micro-pits is avoided, and meanwhile, the density of the single cell array can be improved by reasonably reducing the spacing, and then the signal flux of cell detection is improved.
A second object of the present invention is to provide a method of manufacturing a device comprising a topologically topographical array surface as described above.
A method of making a device comprising a topologically topographical array surface as described above, comprising the steps of:
i: preparing a micro-pit array pattern on a micro-column array or a smooth surface silicon wafer by a micro-machining surface etching technology to obtain an initial template, pouring Polydimethylsiloxane (PDMS) prepolymer on the surface of the initial template, heating and curing, and demolding to obtain a PDMS soft template;
II: pouring a polymer solution or a prepolymer solution on the PDMS soft template obtained in the step I, and demolding after the solvent is fully volatilized or the prepolymer is fully crosslinked to obtain a device containing the topological appearance array surface;
III: performing plasma treatment on the device containing the topological appearance array surface obtained in the step II to obtain a device containing the topological appearance array surface with hydrophilic surface;
IV: pouring the PDMS prepolymer on the surface of a smooth surface silicon wafer, heating and curing, and demolding to obtain a PDMS stamp with a smooth surface;
v: the PDMS stamp obtained in the step IV is used, a solvent of the polymer solution in the step II or a methylene dichloride solution of PS is used as printing ink, and a micro-contact printing technology is adopted to destroy a hydrophilic layer of the device micro-pit external plasma treatment of the surface hydrophilic array surface containing the topological morphology, so that the device with the surface with the hydrophilic-hydrophobic contrast surface containing the topological morphology is obtained;
VI: performing plasma treatment on the PDMS stamp obtained in the step IV to obtain a PDMS stamp with a hydrophilic surface;
VII: and (3) preparing the device with the cell adhesion contrast modified topological morphology array surface on the device with the topological morphology array surface with the hydrophilic and hydrophobic contrast surface obtained in the step (V) by using the PDMS stamp with the hydrophilic surface obtained in the step (VI) and using the solution of the cell adhesion resistant polymer as printing ink through a microcontact printing technology.
It is worth to say that the soft seal micro-contact printing polymer good solvent with smooth surface can dissolve the hydrophilic layer on the surface of the external material of the micro-pit after plasma treatment, and the hydrophobic surface is re-exposed, so that the hydrophilic-hydrophobic contrast surface is obtained, only the external of the micro-pit is subjected to cell adhesion resistance modification, and finally the cell adhesion contrast modification is obtained.
Further, the solute of the anti-cell adhesion polymer solution in the step VII is PEO-PPO-PEO triblock polymer or PEO-PPO diblock polymer, and the solvent is ethanol or water.
It is a third object of the present invention to provide the use of a device comprising a topologically topographical array surface as described above.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the device is applied to the fields of cell and material interaction evaluation, genetics and proteomics, drug effect evaluation and new cancer drug screening.
Further, the device is applied to independent regulation and control of single cell positioning, cell appearance and cell nucleus shape.
It should be noted that, the topological morphology and the cell adhesion contrast pattern of the material surface are important tools and means for researching cell behaviors, and although surface modification strategies such as adhesion contrast patterns for inducing cell deformation and micro-column arrays for inducing significant cell nucleus deformation exist at present, the surface modification cannot realize the respective regulation and control of the cell appearance and the cell nucleus shape, so that the influence of the two factors on the cell behaviors cannot be decoupled. Therefore, it is of great importance to create an artificial extracellular matrix capable of independently controlling the shape of the cell and the shape of the nucleus. According to the invention, through a modification strategy with cell adhesion contrast modification and topology morphology, a single cell array platform capable of independently regulating and controlling cell appearance and cell nucleus shape is established for the first time, and the single cell array platform is applied to relevant researches of cell biology, tumor metastasis mechanism, drug effect evaluation and screening.
When the device is applied, the device containing the topological appearance array surface positions cell adhesion in the cell culture process to obtain a single cell array, and the change of the cell appearance and the cell nucleus shape is independently regulated and controlled.
Specifically, the application of transformation-inducing factor TGF- β1 to single cell arrays obtained using devices comprising topologically contoured array surfaces induces epithelial-mesenchymal transformation of cancer epithelial cells, and the extent of transformation is assessed for its effect and its mechanism by changes in cell morphology and nuclear shape, before and after epithelial-mesenchymal transformation occurs. In particular, after the occurrence of epithelial-mesenchymal transition, cells in a single-cell array are administered with an antitumor drug such as cisplatin, and the cellular response and effect of the drug are observed and evaluated.
Compared with the prior art, the invention has the advantages that:
1) The invention discloses a device containing a topological appearance array surface, which is used for positioning cell adhesion and obtaining a single cell array, and can independently regulate and control the appearance and the shape of a cell nucleus, which cannot be achieved by the prior surface modification method.
2) By means of the technical scheme provided by the invention, a single cell array for cancer cell epithelial-mesenchymal transition mechanism research is effectively established, compared with the traditional in-vitro model, the influence of cell deformation and cell nuclear deformation on epithelial-mesenchymal transition can be explored from the single cell level, and the method is more suitable for high-throughput screening and genomics research of various cancer drugs aiming at specific targets.
3) The device with the topological appearance array surface constructed by the invention has good stability, operability and repeatability, and is convenient for batch preparation and popularization; different mechanical stimulation conditions in the body can be simulated, a more effective in-vitro efficacy evaluation platform is established, and the cost problem and ethical dispute caused by taking animals as modeling objects are avoided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. The drawings in the following description are only examples of the present invention and other drawings may be obtained from the drawings provided without inventive effort for a person of ordinary skill in the art.
Fig. 1 is a view of a scanning electron microscope of a Polystyrene (PS) device with a topological morphology array surface prepared in example 2 of the present invention.
Fig. 2 is a schematic diagram of a preparation method of a Polystyrene (PS) device including a topological morphology array surface disclosed in example 2 of the present invention.
FIG. 3 is a schematic diagram of a cell adhesion contrast modification method according to example 3 of the present invention.
FIG. 4 shows the results of the water contact angle measurement of the device surface after each step of the treatment in example 3 of the present invention.
FIG. 5 is a photograph of single cell array cell staining (red for cytoskeleton, blue for nucleus) prepared in example 5 of the present invention.
FIG. 6 is a graph showing statistics of aspect ratio and nuclear solidity of cells in example 5 of the present invention.
Fig. 7 shows the results of the statistics of nuclear solidity in example 6 of the present invention (0.05 > p > 0.01; 0.01> p > 0.001; p > 0.001).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples herein, unless otherwise indicated, was performed using conventional testing methods in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically identified herein are those commonly employed by those of ordinary skill in the art.
In the description of the present invention, it should be understood that the terms "medium," "upper," "lower," "ascending," "descending," "vertical," "face," "top," "bottom," "inner," "outer," and the like indicate or are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the present invention and simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
Numerous specific details are set forth in the following examples in order to provide a better understanding of the present application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.
On the premise of no conflict, the technical features disclosed in the embodiments of the present application may be combined arbitrarily, and the obtained technical solution belongs to the disclosure of the embodiments of the present application.
The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.
EXAMPLE 1 preparation of initial templates
(1) Manufacturing of initial template of micro-pit array
Design and drawing of projected area 900 μm using computer drawing software (CAD) 2 A micro-pit array pattern with a pitch of 15 μm. Taking a 3-inch single-polished silicon wafer, and preprocessing by using oxygen plasma (power 100W, processing time length 90 s); and spin-coating positive photoresist on the treated silicon wafer to form a glue layer with the thickness of about 10 mu m, and heating to 100 ℃ and baking the glue layer for 80s. And (3) immersing the silicon wafer subjected to exposure and photoresist baking by a laser direct-writing photoetching machine with positive photoresist developer to remove positive photoresist in an exposed area, completely removing the positive photoresist, eluting with ultrapure water, drying the surface by nitrogen, and developing to obtain the initial template of the micro-pit array.
(2) Manufacturing of embedded topological appearance array initial template
The same pattern design as the micro-pit array was used. Taking a micro-column array surface silicon wafer with the micro-column side length of 3 mu m, the interval of 6 mu m and the height of 8 mu m, and preprocessing by using oxygen plasma (the power is 100W and the processing time is 90 s); and spin-coating positive photoresist on the treated silicon wafer to form a glue layer with the thickness of about 10 mu m, and heating to 100 ℃ and baking the glue layer for 80s. And (3) immersing the silicon wafer subjected to exposure and photoresist baking by a laser direct-writing photoetching machine with positive photoresist developer to remove positive photoresist in an exposed area, completely removing the positive photoresist, eluting with ultrapure water, drying the surface by nitrogen, and developing to obtain the embedded topological appearance array initial template.
Example 2 preparation of Polystyrene (PS) devices containing topologically topographical array surfaces
The specific method and the process are the same except that the initial templates used in the method for manufacturing the PS device on the surface of the micro-pit array and the PS device on the surface of the embedded topological morphology array are different, so that the description is combined below, and the two initial templates are collectively called as an initial template; the two PS devices are collectively referred to as "PS devices with topological feature arrays".
(1) The same initial template as in example 1 was made.
(2) Preparation of Polydimethylsiloxane (PDMS) soft template
And mixing a proper amount of PDMS prepolymer and a crosslinking agent (Dow coating 184) in a mass ratio of 10:1, pouring the mixture on an initial template, removing bubbles, and heating and curing the mixture on a glue baking table at 70 ℃ for 4 hours. And stripping the cured PDMS from the initial template to obtain the PDMS soft template.
(3) Fabrication of PS device containing topological morphology array surface
A methylene chloride solution of PS was prepared at a concentration of 0.1g/mL. And placing the PDMS soft template in a flat-bottom glass dish, pouring the PS solution on the PDMS soft template, and cleaning to remove bubbles generated after pouring. And covering the glass dish cover, fully volatilizing the solvent, and then uncovering the film to obtain the PS device containing the topological appearance array.
The image of a scanning electron microscope of the PS device with the embedded topological appearance array surface is shown in figure 1, and the schematic diagram of the preparation method of the PS device with the topological appearance array is shown in figure 2.
EXAMPLE 3 cell adhesion contrast modification
(1) Fabrication of the same PS device containing topological morphology array of example 2
(2) Fabrication of PS device with surface hydrophilic and topological morphology array
And (3) treating the PS device containing the topological appearance array by using oxygen plasma, wherein the treatment power is 100W, the treatment time is 90s, and the PS device containing the topological appearance array with the hydrophilic surface is obtained after the treatment.
(3) Preparation of PDMS seal
And mixing a proper amount of PDMS prepolymer and a crosslinking agent (Dow coating 184) in a mass ratio of 10:1, pouring the mixture into a plastic dish, removing bubbles, and heating and curing the mixture on a glue baking table at 70 ℃ for 4 hours. And stripping the cured PDMS from the plastic dish to obtain the PDMS stamp.
(4) Fabrication of PS devices with topological morphology arrays having hydrophilic-hydrophobic contrast modification
And soaking the PDMS stamp in Dichloromethane (DCM) until the PDMS stamp is fully swelled, taking out the PDMS stamp and slightly volatilizing the PDMS stamp until the surface of the PDMS stamp is free of obvious liquid, stamping the PDMS stamp on a PS device containing the topological morphology array for 3-5s, and observing the printing effect through an optical microscope. And (3) fully volatilizing the printed dichloromethane to obtain the PS device with the topological morphology array and the hydrophilic-hydrophobic contrast modification.
(5) Preparation of PDMS seal with hydrophilic surface
And (3) treating the PDMS stamp by using oxygen plasma, wherein the treatment power is 100W, the treatment time is 90s, and the PDMS stamp with hydrophilic surface is obtained after the treatment.
(6) Cell adhesion contrast modification
Preparing an ethanol saturated solution of PEO-PPO-PEO in a water bath at 60 ℃, soaking a PDMS stamp with hydrophilic surface in the solution for 30min, taking out and volatilizing the stamp to a state that the surface is free of obvious liquid but not completely dried, and stamping the surface of the PS device with the topological morphology array modified by hydrophilic-hydrophobic contrast for about 30s, and then lifting the stamp. And drying to obtain the PS device with the cell adhesion contrast modification and the topological morphology array.
The cell adhesion contrast modification method is shown in FIG. 3, and the change of the water contact angle of the device surface after each step of treatment is shown in FIG. 4.
EXAMPLE 4 preparation of human lung cancer epithelial cells (A549) single cell arrays with identical cell profiles
(1) Fabrication of the same PS device with topologically topographic array with cell adhesion contrast modification of example 3
(2) Sterilization
Cutting the device in the step (1) to the size just enough to be placed into a cell culture pore plate, soaking the device in 75% ethanol water solution for 15min for sterilization, washing the device in sterilized ultrapure water for 3 times, and airing the device for standby.
(3) Cell culture pretreatment
The sterilized devices were placed into cell culture well plates and polytetrafluoroethylene clamping rings were placed over the devices to secure the devices. Complete medium (high sugar DMEM medium+10% fbs+1% p/S) for culturing a549 cells was added and placed in a constant temperature incubator at 37 ℃ for soaking overnight to remove bubbles in the micro pits.
(4) Cell seeding
After aspiration of the complete medium of the pre-soak device, the medium was removed at 1X 10 4 The A549 cells were seeded at a suspension concentration of individual/mL such that the cell density on the device surface was approximately 5X 10 3 Individual/cm 2 Culturing in a constant temperature incubator at 37 ℃ for 24 hours to obtain the A549 single-cell array with the same cell shape.
EXAMPLE 5 independent control of cell appearance on Single cell arrays
(1) A plurality of groups of PS devices having cell adhesion contrast modified micro-pit arrays with the same micro-pit area and aspect ratios of 1, 2, and 4, respectively, were prepared in the same manner as in example 3.
(2) Human lung cancer epithelial cells A549 are inoculated on the surface of the device in the step (1) according to the same method in the example 4, and after culturing for 24 hours, the single cell array with different cell shapes and cell nucleus shapes without obvious change is obtained.
(3) Fluorescent staining and observation
After the incubation time period was reached, the medium was aspirated from the well plate and washed 3 times with PBS. After fixing the cells by adding 4% paraformaldehyde solution for 5min, the cells were aspirated and washed 3 times with ultrapure water. After washing, cells were permeabilized with 0.1% Triton solution for 5min and then aspirated, and washed 3 times with ultrapure water. After permeabilization, the cytoskeletal microfilaments were marked with red fluorescence by adding rhodamine-phalloidin solution for 1h incubation, the staining solution was aspirated and washed 3 times with ultrapure water. The nuclei were labeled with blue fluorescence by incubation with DAPI solution for 10min, and after aspiration of the staining solution and 3 washes with ultrapure water, they were observed under an inverted fluorescence microscope and photographs were taken.
(4) Cell and nucleus shape parameter assessment
For the taken fluorescence photographs, the shape of cells and nuclei was analyzed by ImageJ software. The deformation of cells and nuclei was assessed by aspect ratio, projected area, shape factor (shape index) and solidity (solidity). The shape factor and solidity are calculated according to the following equation:
where S is the projected area, l is the projected perimeter, and convex area is the minimum convex hull area containing the selected region. The smaller the shape factor and solidity, the greater the degree of deformation of the cells and nuclei. Through the above parameters, it can be verified that the PS device with cell adhesion contrast modification and micro-pit array independently adjusts the change of cell appearance under the condition of maintaining the shape of cell nucleus unchanged.
The cell staining photograph is shown in FIG. 5. The statistics of aspect ratio and nuclear solidity are shown in FIG. 6.
EXAMPLE 6 independent modulation of Nuclear shape on Single cell arrays
(1) A PS device with a cell adhesion contrast modified micro-pit array and a PS device with a chimeric topological morphology array were prepared with the same micro-pit area and aspect ratio of 1 according to the same method as in example 3.
(2) Human lung cancer epithelial cells a549 were inoculated on the surfaces of the two devices described in (1) and cultured for 24 hours in the same manner as in example 4.
(3) Single cell arrays with the same cell morphology and varying degrees of nuclear deformation were fluorescent stained and observed following the same procedure as in example 4.
(4) Cell and nucleus shape parameter assessment
For the obtained fluorescence photographs, shape parameters of cells and nuclei were analyzed by ImageJ software. The micro-pit array regulates the length-diameter ratio of cells to be 1, and the cell nucleus is maintained in a form with a round bias and a large solidity; the chimeric topological morphology array regulates the length-diameter ratio of the cell to be 1, and the cell nucleus is maintained in a form with smaller solidity. Both patterns regulate nuclei to have different shapes while controlling cell appearance.
The results of the cell nucleus solidity statistics are shown in FIG. 7.
EXAMPLE 7 Induction of cell epithelial-mesenchymal transition on Single cell array
(1) PS devices with topologically topographical arrays with cell adhesion contrast modifications were prepared in the same manner as in example 3.
(2) Human lung cancer epithelial cells a549 were seeded on the surface of the device described in (1) in the same manner as in example 4.
(3) Epithelial-mesenchymal transition induction
When the cells are inoculated, the transformation induction factor TGF-beta 1 is added at the concentration of 10-20ng/mL, and then the cells are placed into a constant temperature incubator at 37 ℃ for culturing for 48 hours, so that the induction of the cell epithelial-mesenchymal transformation on the single cell array with specific cell shape and specific cell nucleus shape is realized.
Example 8 influence of cell shape and Nuclear shape on epithelial-mesenchymal transition
(1) A549 cells were seeded and induced epithelial-mesenchymal transition on PS devices containing micropin arrays and PS devices containing chimeric topological morphology arrays with cell adhesion contrast modifications of the same micropin area and aspect ratios of 1, 2, 4, respectively, following the same procedure as in example 7.
(2) After the incubation time period, the cells were labeled with vimentin and E-cadherin by immunofluorescent staining, and observed and photographed by an inverted fluorescent microscope.
(3) The shape parameters of cells and cell nuclei in the fluorescent photograph, the fluorescence intensity of cell vimentin and E-cadherin are measured by using imageJ software, and the expression change of the two markers is analyzed, so that the influence of the cell nuclei shape on the epithelial-transformation can be verified by independently changing the cell shape when the cell nuclei are identical in shape and independently changing the cell nuclei shape when the cell nuclei are identical in shape.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. A device comprising a topographic array surface, comprising a substrate and a cell adhesion contrast modification on the substrate surface, the substrate surface having a topographic array comprising a non-chimeric topographic array and a chimeric topographic array.
2. The device of claim 1, wherein the base material is a non-bio-toxic polymeric material; the cell adhesion contrast on the surface of the substrate is modified into a polymer molecular layer with cell adhesion promoting surface inside the micro pits and cell adhesion resisting property outside the micro pits.
3. The device of claim 2, wherein the non-bio-toxic polymeric material comprises polystyrene PS, polypropylene PP, polyglycolide-co-lactide PLGA, poly-l-polylactic acid PLLA, silicone rubber, natural rubber, or plexiglass; the cell adhesion resistant polymer is polyethylene oxide-polypropylene oxide-polyethylene oxide PEO-PPO-PEO triblock polymer or polyethylene oxide-polypropylene oxide PEO-PPO diblock polymer.
4. The device of claim 1, wherein the non-chimeric topological feature array is a micro-pit array;
the micro-column arrays in the embedded topological appearance array are distributed in the micro-pits, and the height of the micro-columns is lower than the upper edge of the micro-pits.
5. The device of claim 4, wherein the micro-pits have an aspect ratio of between 1 and 9 and a projected area of 600 to 1200 μm 2 The distance between the micro pits is not less than 10 mu m, and the depth is 5-15 mu m; the side length of the microcolumn is 2-7 mu m, the height is 4-12 mu m, and the interval is 3-12 mu m.
6. A method of making a device comprising a topologically topographical array surface as claimed in any one of claims 1 to 5, comprising the steps of:
i: preparing a micro-pit array pattern on a micro-column array or a smooth surface silicon wafer by a micro-machining surface etching technology to obtain an initial template, pouring Polydimethylsiloxane (PDMS) prepolymer on the surface of the initial template, heating and curing, and demolding to obtain a PDMS soft template;
II: pouring a polymer solution or a prepolymer solution on the PDMS soft template obtained in the step I, and demolding after the solvent is fully volatilized or the prepolymer is fully crosslinked to obtain a device containing the topological appearance array surface;
III: performing plasma treatment on the device containing the topological appearance array surface obtained in the step II to obtain a device containing the topological appearance array surface with hydrophilic surface;
IV: pouring the PDMS prepolymer on the surface of a smooth surface silicon wafer, heating and curing, and demolding to obtain a PDMS stamp with a smooth surface;
v: the PDMS stamp obtained in the step IV is used, a solvent of the polymer solution in the step II or a methylene dichloride solution of PS is used as printing ink, and a micro-contact printing technology is adopted to destroy a hydrophilic layer of the device micro-pit external plasma treatment of the surface hydrophilic array surface containing the topological morphology, so that the device with the surface with the hydrophilic-hydrophobic contrast surface containing the topological morphology is obtained;
VI: performing plasma treatment on the PDMS stamp obtained in the step IV to obtain a PDMS stamp with a hydrophilic surface;
VII: and (3) preparing the device with the cell adhesion contrast modified topological morphology array surface on the device with the topological morphology array surface with the hydrophilic and hydrophobic contrast surface obtained in the step (V) by using the PDMS stamp with the hydrophilic surface obtained in the step (VI) and using the cell adhesion resistant polymer solution as printing ink through a microcontact printing technology.
7. The method of manufacturing a device according to claim 6, wherein the solute of the anti-cell adhesion polymer solution in step VII is PEO-PPO-PEO triblock polymer or PEO-PPO diblock polymer, and the solvent is ethanol or water.
8. Use of a device comprising a topologically topographical array surface as claimed in any one of claims 1 to 5, in the fields of cell-to-material interaction assessment, genetics and proteomics, drug efficacy assessment and cancer new drug screening.
9. The use of a device comprising a topologically topographical array surface as claimed in claim 8, wherein said device is used for independent regulation of single cell localization, cell topography and cell nucleus shape.
CN202311527931.2A 2023-11-16 2023-11-16 Device containing topological appearance array surface and preparation method and application thereof Pending CN117567820A (en)

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