CN108641892B - Micro-contact printing system for cell patterning - Google Patents
Micro-contact printing system for cell patterning Download PDFInfo
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- CN108641892B CN108641892B CN201810345302.0A CN201810345302A CN108641892B CN 108641892 B CN108641892 B CN 108641892B CN 201810345302 A CN201810345302 A CN 201810345302A CN 108641892 B CN108641892 B CN 108641892B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
- C08G73/0666—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms
- C08G73/0672—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only one nitrogen atom in the ring
Abstract
The present invention provides a novel microcontact printing system for cell patterning. The system comprises oligomeric dopamine formed by polymerizing dopamine monomers, the molecular weight of the oligomeric dopamine is between 1000-10000D, and the structural formula of the oligomeric dopamine is shown as follows. The method comprises the steps of carrying out chemical modification treatment on a substrate to obtain an anti-bioadhesion inert substrate; and then preparing an aqueous solution of oligomeric dopamine, preparing a seal, soaking the seal into the aqueous solution of oligomeric dopamine, and then stamping the seal adsorbed with the oligomeric dopamine on an inert substrate, thereby obtaining a pattern on the seal on the surface of the substrate. The patterned substrate can also be used for patterned growth of cells. The system is simple to implement and simplifies existing techniques for cell patterning.
Description
Technical Field
The present invention relates to a novel microcontact printing system for cell patterning, and more particularly, to a novel microcontact printing system for cell patterning.
Background
Cell patterning is an in vitro cell culture technique, specifically, by preparing a patterned substrate, inducing the response mechanism of the cells themselves to adhere to the designed areas to form a pattern. This method is distinguished from the direct seeding of cells into culture dishes, which are commonly used in laboratories, where the latter usually forms a monolayer membrane of cells.
Cell patterning technology has become an important tool for studying and controlling cell biological behaviors, and is widely used for studying cell-cell and cell-substrate interactions (including cell adhesion and apoptosis, cell polarization behaviors, cell migration rate, cell differentiation behaviors, and the like); and the construct external model can be used in the fields of tissue engineering, cell sensing, drug screening, wound treatment and the like.
Currently, cell patterning techniques can be broadly divided into two categories: (1) the method for limiting the domain comprises the following steps: that is, by forming some barriers (such as microwells) by physical or chemical means, the cells are confined in the barriers and grow to form patterns; (2) surface modification method: the cell culture substrate is modified to form well-defined pro-and/or de-cellular regions, such that cells are selectively grown in the pro-cellular regions to form a pattern. Among the numerous cell patterning methods, the microcontact printing technique is widely used due to its simple operation, low cost, ability to prepare large area patterns, and its used materials (such as polydimethylsiloxane, PDMS) are non-toxic, transparent, and easy to form.
In early studies, microcontact printing was used to transfer thiols to gold surfaces to form patterned self-assembled monolayer films. The common process is that a PDMS stamp is soaked in mercaptan solution and is dried and then contacted with the surface of gold, mercaptan molecules are transferred from the convex part of the stamp to the surface of the gold to form a self-assembly layer; the concave part of the stamp is not in contact with the substrate and is therefore exposed. And soaking the gold substrate into another thiol molecule, wherein the exposed part of the gold substrate is covered by the second thiol molecule, so that two types of self-assembly layers are formed. For example, methyl-containing thiols can promote cell adhesion, polyethylene glycol-containing thiols can resist cell adhesion, and cells can grow in a patterned manner after being seeded on the gold surface. Later developments have been directed to the printing of silane molecules onto glass or silicon substrates. However, this printing method requires self-assembly of two molecules on the surface and has specific requirements for the substrate.
In view of the above limitations, a jiang xing topic group in the national nano-science center develops a poly-dopamine-based one-step printing method for patterning cells, which is suitable for various substrates, such as gold surfaces, glass, silicon surfaces, Polystyrene (PS) surfaces, and the like, and has the characteristics of simple operation, low cost, and the like, but also has the disadvantages of non-transparent patterns, long time consumption, and the like. Therefore, there is a need for a means and method for patterning cells that is simpler and less costly to manufacture.
Disclosure of Invention
It is a primary object of the present invention to provide a novel microcontact printing system for cell patterning.
The oligomeric dopamine prepared by the novel method realizes the patterning of cells by adopting a simpler and more convenient operation process.
The technical purpose is realized by the following technical scheme:
a novel microcontact printing system for cell patterning, said system comprising oligomeric dopamine polymerized from dopamine monomers having a molecular weight between 1000 and 10000D and having the formula:
preferably, the operation of implementing the patterning is: the seal with the pattern is soaked in an aqueous solution of oligomeric dopamine, then is transferred to a substrate modified by polyethylene glycol or amphoteric molecular substances for cell culture, and an observable pattern is obtained.
Preferably, the substrate for preparing the stamp with the pattern is made of a glass sheet, a silicon sheet, a titanium sheet, a gold sheet or a polystyrene sheet, and the polyethylene glycol substance is methoxy polyethylene glycol silane, sulfhydryl polyethylene glycol or polylysine-polyethylene glycol; chemical modification to combat bioadhesion the main substances used are polyethylene glycol-based substances or amphiphilic molecules.
Preferably, the polyethylene glycol substance is methoxy polyethylene glycol silane, sulfhydryl polyethylene glycol or polylysine-polyethylene glycol; the amphoteric molecule is mainly carboxylic acid betaine amphoteric ion polymer or sulfobetaine amphoteric ion polymer;
preferably, the specific method to achieve cell patterning is as follows:
s1, preparing an anti-bioadhesion modified substrate: cleaning and activating the substrate, and grafting polyethylene glycol substances or amphoteric molecules on the substrate to obtain the substrate modified by the biological adhesion resistance;
s2, preparing a seal, namely placing a liquid siloxane prepolymer in a template with a micron-scale structure, and curing to obtain the seal with a pattern;
s3, soaking the seal with the pattern in the oligomeric dopamine solution in the S2, drying, and then placing the seal on the substrate resistant to biological adhesion in the S1 to obtain the surface with the oligomeric dopamine pattern.
Among them, S1 is mainly used for obtaining an inert substrate resistant to adhesion of biomolecules; the inert substrate can be a self-assembled monolayer formed on the surface of the substrate through polyethylene glycol silane or amphoteric molecules; s3, obtaining a pattern on the inert surface, wherein the pattern is formed by a substance allowing protein and cell adhesion, and the inert surface is not completely covered by the pattern;
furthermore, the pattern formed on the inert surface of the substrate by the substance that allows cell adhesion is any pattern, including but not limited to an irregular pattern or array consisting of dots, lines, stripes, bands, blocks, irregular shapes, and the like.
Preferably, the specific steps of cleaning and activating the substrate in S1 are: the method comprises the steps of performing ultrasonic treatment on a substrate in acetone and ethanol solutions respectively, boiling the substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide for 0.5-1.5 h, cleaning the substrate with water, drying the substrate, performing low-temperature plasma treatment on the substrate, soaking the substrate in a solution of polyethylene glycol or amphoteric molecular substances, adding triethylamine, reacting for a certain time, cleaning and drying the substrate to obtain the catalyst.
Preferably, the pH of the oligomeric dopamine solution in S3 is 4.0-7.0, and the concentration is 0.02-2 mg/mL.
Preferably, a surface with an oligomeric dopamine pattern is obtained in S3 for selective adhesion of cells, resulting in patterned cells.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the invention, the transparent pattern is formed by oligomeric dopamine, the substrate can be used for inoculating cells without special treatment, and the substrate is nontoxic and harmless to the cells.
2. The method has simple steps, only needs to provide a substrate to which cells are not adhered, and then prints substances to which the cells can be adhered on the surface through microcontact printing, for example, oligomeric dopamine is adhered on the substrate through a physical contact method, the method is simple and easy to implement, expensive instruments are not needed, and the method is suitable for common laboratories; the oligomeric dopamine is simple to prepare and low in price; the oligomeric dopamine ink used in the invention has strong acting force with polyethylene glycol on the substrate, so that cell adhesion is guaranteed, and pattern falling is avoided.
Drawings
FIG. 1 is a schematic view of the preparation of a Polydimethylsiloxane (PDMS) stamp.
Fig. 2 is a schematic diagram of oligomeric dopamine adsorbed on the stamp surface to form a monolayer.
FIG. 3 is a pattern of murine fibroblasts (NIH.3T 3) after 1 hour incubation on a quartz plate surface containing a pattern of oligomeric dopamine.
Detailed Description
The present invention is further described below in conjunction with the following detailed description and the appended drawings, wherein examples are illustrated in the accompanying drawings and described below, and some detailed implementations and specific operations are given. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1:
1. preparation of oligomeric dopamine
And (3) putting the dopamine monomer molecules into a Tris-HCl buffer solution with the pH value of 8.5, and standing for 8 hours to obtain a substance with black precipitates. The solution was centrifuged at 15000 rpm to give a yellow supernatant solution containing oligodopamine.
2. Printing oligomeric dopamine pattern on surface of quartz plate modified by polyethylene glycol silane
(1) And oxidizing the quartz plate
Quartz plates (8 x 0.5 mm)3Purchased from dada quartz products ltd, japan) in acetone and absolute ethanol in sequence for 10 min, and then mixed with a mixture of concentrated sulfuric acid and hydrogen peroxide (volume ratio 3: 1) boiling for 1 h, washing with ultrapure water, and drying. And (3) placing the quartz plate in a low-temperature plasma processor for oxidation for 6 min.
(2) Inert modification of the quartz plate
The oxidized quartz plate is soaked in 6 g/L methoxy polyethylene glycol silane (MW 2000) in toluene for 24 h, and 1% triethylamine is added as a catalyst. After the reaction is finished, washing with absolute ethyl alcohol for 2 times, and washing with ultrapure water for 2 times, N2Blow-drying, and storing in N2In the atmosphere.
3. Preparation of polydimethylsiloxane seal
First, a matrix (1X 1 cm) of a circle (diameter 50 μm, pitch 100 μm), a rectangle (side length 50 μm, pitch 100 μm), a triangle (side length 50 μm, pitch 100 μm), etc. was designed by Auto-CAD software2) And printed on a chrome plate (Shenzhen Jiuwei photoelectricity Limited); the pattern is then photolithographically processed, which may be carried out using conventional methods, as described in the MicroChem operating procedure (website: http:// MicroChem. com/pdf/SU-82000DataSheet2025thru2075Ver4. pdf); the substrate used was a 2 inch area silicon wafer; the photoresist used was SU-8 thick negative photoresist from MicroChem. After spin coating, pre-baking, exposure, post-baking, and development, the pattern is carried by the photoresist on the silicon wafer.
Then, pattern replication was performed on the resulting mold using polydimethylsiloxane: placing the processed silicon wafer with the photoresist in a dryer, adding 20 μ L of tridecafluorosilane (purchased by sigma company, cas number 51851-37-7), and vacuumizing for 1 h; pouring a mixture (mass ratio is 10: 1) of a prepolymer of polydimethylsiloxane and a curing agent, removing bubbles for 1 h in vacuum, and then baking for 1 h in an oven at 80 ℃ to form a solid polydimethylsiloxane; the solid is elastic and can be taken off from the silicon wafer; the polydimethylsiloxane was removed and the pattern transferred to the top of the polydimethylsiloxane. The polydimethylsiloxane is cut into a proper size according to the requirement, and then the polydimethylsiloxane can be used as a seal for micro-contact printing, as shown in figure 1.
4. Adsorption of oligomeric dopamine on seal surface
The seal is soaked in an oligomeric dopamine aqueous solution for 30 min, wherein the concentration of the seal is 0.02-2 mg/mL, the pH value of the seal is 4.0-7.0. As shown in fig. 2.
5. Printing oligomeric dopamine on the surface of a quartz plate, and inoculating cells
Taking the seal out of the solution, drying, flatly placing the seal on the surface of the quartz plate modified by methoxy polyethylene glycol silane, placing a 50 g weight on the seal, taking down the weight after 1 min, and removing the seal, so that the oligomeric dopamine is printed on the surface of the quartz plate, and a pattern for patterned growth of cells is formed. The quartz plate printed with the oligodopamine was placed in a 24-well plate, and the cell suspension (the cells were murine fibroblasts, NIH 3T3, the concentration of the cell suspension was 10) just digested was added5one/mL) was placed on a substrate and incubated for 1 h (cell incubator, 37 ℃, 5% CO)2DMEM +10% FBS +1% double antibody) as shown in fig. 3, the cells formed a pattern on the substrate, the pattern was formed by selectively adhering the cells only at the position where the oligomeric dopamine was printed, thereby forming a predetermined pattern, and the cells had good spreading morphology in the adhered area.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (5)
1. A microcontact printing system for cell patterning, said system comprising oligomeric dopamine polymerized from dopamine monomer having a molecular weight between 1000 and 10000D and having the following structural formula:
the system is characterized in that a seal with a pattern is soaked in an aqueous solution of oligomeric dopamine, then is transferred to a substrate with biological adhesion-resistant modification, and is subjected to cell culture to obtain an observable pattern; the method of achieving cell patterning is as follows:
s1 preparation of a substrate modified with an anti-bioadhesive substance: cleaning and activating the substrate, and grafting polyethylene glycol or amphoteric molecular substances on the substrate to obtain the substrate modified by the anti-biological adhesion substances;
s2, preparing a seal, namely placing a liquid siloxane prepolymer in a template with a micron-scale structure, and curing to obtain the seal with a pattern;
s3, soaking the seal with the pattern in the S2 in an oligomeric dopamine solution, drying, and then placing the seal on a substrate resistant to biological adhesion in S1 to obtain a surface with the oligomeric dopamine pattern;
the pH value of the oligomeric dopamine solution in the S3 is 4.0-7.0, and the concentration is 0.02-2 mg/mL.
2. The micro-contact printing system for cell patterning according to claim 1, wherein the material of the substrate with the pattern is a glass sheet, a silicon wafer, a titanium sheet, a gold sheet or a polystyrene sheet; chemical modification to combat bioadhesion the main substances used are polyethylene glycol-based substances or amphiphilic molecules.
3. The microcontact printing system for cell patterning of claim 2, wherein the polyethylene glycol species is methoxy polyethylene glycol silane, mercapto polyethylene glycol, or polylysine-polyethylene glycol; the amphoteric molecule is carboxylic acid betaine amphoteric ion polymer or sulfobetaine amphoteric ion polymer.
4. The microcontact printing system for cell patterning as claimed in claim 1, wherein the specific steps of washing and activating the substrate in S1 are: the method comprises the steps of performing ultrasonic treatment on a substrate in acetone and ethanol solutions respectively, boiling the substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide for 0.5-1.5 h, cleaning the substrate with water, drying the substrate, performing low-temperature plasma treatment, soaking the substrate in a solution of polyethylene glycol substances, adding triethylamine to react for a certain time, and cleaning and drying the substrate to obtain the catalyst.
5. The microcontact printing system for cell patterning as claimed in claim 1, wherein a surface with oligomeric dopamine pattern is obtained in S3 for selective adhesion of cells, resulting in patterned cells.
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| CN111424013B (en) * | 2019-01-10 | 2023-08-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | CTC capturing and separating substrate based on polystyrene nanospheres and preparation method thereof |
| CN110628618A (en) * | 2019-09-26 | 2019-12-31 | 大连理工大学 | Method for accurately controlling shape of single cell by using PDMS stamp |
| CN111333759A (en) * | 2020-02-26 | 2020-06-26 | 青岛科技大学 | Preparation method of zwitterionic polymer pattern on surface of solid substrate |
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| CN102465119A (en) * | 2010-11-12 | 2012-05-23 | 国家纳米科学中心 | Substrate for cell micropatterning growth as well as preparation method and application thereof |
| KR20130134080A (en) * | 2012-05-30 | 2013-12-10 | 한국과학기술원 | Method of manufacturing surfaces for controlling cell attachment using plasma-treated biopolymer |
| CN104831261A (en) * | 2015-04-03 | 2015-08-12 | 首都师范大学 | Microring electrode and production method thereof |
| KR20170029237A (en) * | 2015-09-07 | 2017-03-15 | 한양대학교 산학협력단 | Substrate with biopolymer pattern for culturing micro-sized cell sheet and method for delivering the same |
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| CN102465119A (en) * | 2010-11-12 | 2012-05-23 | 国家纳米科学中心 | Substrate for cell micropatterning growth as well as preparation method and application thereof |
| KR20130134080A (en) * | 2012-05-30 | 2013-12-10 | 한국과학기술원 | Method of manufacturing surfaces for controlling cell attachment using plasma-treated biopolymer |
| CN104831261A (en) * | 2015-04-03 | 2015-08-12 | 首都师范大学 | Microring electrode and production method thereof |
| KR20170029237A (en) * | 2015-09-07 | 2017-03-15 | 한양대학교 산학협력단 | Substrate with biopolymer pattern for culturing micro-sized cell sheet and method for delivering the same |
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