CN102465119B - Substrate for cell micropatterning growth as well as preparation method and application thereof - Google Patents
Substrate for cell micropatterning growth as well as preparation method and application thereof Download PDFInfo
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- CN102465119B CN102465119B CN201010544606.3A CN201010544606A CN102465119B CN 102465119 B CN102465119 B CN 102465119B CN 201010544606 A CN201010544606 A CN 201010544606A CN 102465119 B CN102465119 B CN 102465119B
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Abstract
The invention provides a substrate for cell micropatterning growth. The substrate comprises an inert surface on which cells cannot be adhered and patterns on the inert surface, wherein the patterns are formed from substances which are allowable for cell adhesion, and the inert surface is not completely covered by the patterns. The invention further provides a preparation method of the substrate, wherein a one-step printing step is adopted in the method for preparing the substrate, so that the traditional method for preparing the substrate for the cell micropatterning growth is greatly simplified, and the range for using the substrate is broadened. The invention further provides application of the substrate to the cell micropatterning growth.
Description
Technical field
The present invention relates to cell culture technology, particularly, the present invention relates to a kind of substrate that can make cell patterned growth and its preparation method and application.
Background technology
The patterning of cell, is a kind of technology of vitro culture, cell is confined to some region growing of base plane, and cell can not move to the place outside localized area.The method of cell patterning is different from and conventional directly cell is seeded in to the mode of cultivating in culture dish, and the latter makes cell in culture dish middle berth spread, become the cell monolayer film of homogeneous.
The technology of cell patterning has very important application for the impact of cell migration and cell for the response of medicine irritation on the interaction between research cell and interaction, cell and the cell of substrate, shape.In addition, the technology of cell patterning can be construct external model, is organizing the behavior of level research cell that platform is provided.Relevant bibliographical information has, at article Jiang, X.Y., Bruzewicz, D.A., Wong, A.P., Piel, M., Whitesides, G.M., Proc.Natl.Acad.Sci.U.S.A., 102 (4), in 975-978 (2005), author utilizes the method for self-assembled monolayer and micro-contact printing, cell is fixed into water droplet shape, then cell is discharged.Cell after release moves towards more blunt one end in its form.And for example, by cell being fixed in the patterns of different sizes, find cell easy Growth and Differentiation on compared with the pattern of big scale (10 microns of >), and in the pattern of less (10 microns of <) easily apoptosis (Chen C.S., Mirksich, M., Huang, S., Whitesides G.M., and Ingber, D.E., Science, 276,1425-1428 (1997)).In these articles, the substrate of patterning has very large using value in the laboratory of some underlying issues that is used to study cytobiology.
At present more existing technology can be fixed on substrate surface by cell patterning, for example self-assembled monolayer.Self-assembled monolayer is to utilize one end of molecule and substrate to have very strong reactive force, causes the molecule unitary film that chemisorption and regular arrangement form on substrate.For example thiol molecule just can be assembled formation unitary film at gold surface, silane molecule at glass or silicon base surface.Thiol molecule can be imprinted on gold surface by the method for printing.Different zones in gold surface just can obtain the thiol molecule of different functional groups like this.The thiol molecule of different functional groups is different for the interaction of cell, and have plenty of the cell of permission and attach in the above, the attaching of resisting cell having, the latter is called inactive surfaces.According to Search Results, the typical example of correlation technique has: one), by the method for printing, the thiol molecule that cell can be adhered to is printed onto in metal base, the another kind of thiol molecule that the upper cell of other region bubbles of substrate can not adhere to; Or the silane molecule that cell can be adhered to is printed onto in glass or silicon base, the another kind of silane molecule that the upper cell of other region bubbles of substrate can not adhere to; Two) with miniflow pipeline and substrate, form some closed systems, pass into cell suspending liquid in closed system, after after a while, cell is grown in pipeline, removes pipeline, just can leave banded cell pattern; Three) by micro-machined method, first prepare a film that contains pattern hole, film is attached in substrate, then cell suspending liquid is layered on above substrate, cell will sink to film surface gradually.Open film, cell just forms pattern in the region of hole; Four) mode such as laser, anodizing reduction for surface that can not adhere to existing cell is carried out micro-pattern etch, thereby at the pattern of the supported cell adhesion in region of processing.
Although above method can well be controlled cell at the patterning of substrate, but need two kinds of molecules to assemble on surface, and there is certain restriction (as the first) for the substrate of assembling, improved like this cost prepared by substrate, or maintenance patterning (as second and third kind) that can not be long-term, or depend on the equipment (as the 4th kind) of write-through.
Therefore, exist making the cell method of patterned growth and demand of this substrate in substrate at present, exploitation can keep the patterning of cell for a long time, can reduce costs again, the method for the scope of application of expanded substrate, must promote greatly the research of stechiology, and the structure of external model.
Summary of the invention
The substrate that the object of the invention is to overcome prior art is not generally used in laboratory, needs specific installation, the defect that price is more expensive.The inventor, through conscientiously research, has developed a series of substrates based on preparing at the general material of biology laboratory, and these substrates can be used in the patterning of cell after treatment, can address the deficiencies of the prior art like this, thereby complete the present invention.
Therefore, an object of the present invention is to provide a kind of substrate for cell patterned growth.Another object of the present invention is to provide the preparation method of described substrate.In addition, a further object of the present invention is to provide the application of described substrate.
The object of the invention is to be achieved through the following technical solutions:
On the one hand, the invention provides a kind of substrate for cell patterned growth, described substrate comprises:
A) inactive surfaces that cell can not adhere to; With
B) pattern in described inactive surfaces, this pattern is by allowing the material of cell adhesion to form, and described inactive surfaces is not all by described pattern covers.
Preferably, the material of described permission cell adhesion is poly-Dopamine HCL.
Wherein, described substrate can be polystyrene plastic sheet, comprises the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer (PLL-g-PEG) molecule on described polystyrene sheet;
Or described substrate is sheet glass or silicon chip, on described sheet glass or silicon chip, comprise the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is metal titanium sheet or niobium sheet, on described titanium sheet or niobium sheet by interior be titanium or niobium outward successively, titanium oxide or niobium oxides and the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is indium tin oxide conductive glass (ITO), on described indium tin oxide conductive glass by interior be glass outward successively, indium tin oxide layer and the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Preferably, described poly-lysine-polyethyleneglycol-graft copolymer molecule is the PLL-g-PEG of different copolymer degree, the further preferred 20～375k of PLL wherein, and PEG is preferred 2～5k further, and in PLL, the ratio of Methionin and PEG chain further preferably 3～5;
Or described substrate is by polyethylene glycol dimethacrylate illumination polymer formation;
Or described substrate is sheet glass or silicon chip, on described sheet glass or silicon chip by the interior inactive surfaces that there is successively metal level and formed by the thiol molecule ending up with polyoxyethylene glycol outward;
Preferably, described metal level comprises gold layer; Further preferably, described golden bed thickness 20～50nm;
Preferably, described metal level from sheet glass or silicon chip by interior titanium adhesion layer and the gold layer of comprising successively outward; Further preferably, described titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm;
Preferably, described thiol molecule is HS (CH
2) n (OCH
2cH
2) mOH (n > 3, m > 2), preferably HS (CH
2)
11(OCH
2cH
2)
6oH.
In addition, the pattern being formed by the material that allows cell adhesion in substrate inactive surfaces is arbitrary graphic pattern, includes but not limited to the irregular pattern or the array that point, line, bar, band, piece, irregularly shaped etc., consist of;
Any cell of the cell that can be adhered to by substrate for surviving after can adhering to and adhere to, includes but not limited to eukaryotic cell and bacterium.
On the other hand, the invention provides the preparation method of above-mentioned substrate, the method comprises, the substrate with the inactive surfaces that cell can not adhere to is provided, by the pattern that allows cell adhesion by micro-contact printing on the inactive surfaces of this substrate.
Preferably, the preparation method of described substrate comprises the following steps:
1) prepare the inactive surfaces of substrate:
Selection has polystyrene sheet, sheet glass, silicon chip, titanium sheet, niobium sheet or the indium tin oxide conductive glass of suitable size, is placed in air plasma oxidizer and carries out oxide treatment, and oxidization time is more than 1 minute.Hydroxyethyl piperazine second thiosulfonic acid (HEPES) solution (pH 7.4) of PLL-g-PEG that substrate after oxidation is placed in to 0.5～10g/L is more than 0.5 hour, thereby obtains having the substrate of inactive surfaces;
Alternatively, the benzoin dimethylether light trigger of 1% (mass ratio) will be added in polyethylene glycol dimethacrylate liquid, dissolve, under the UV-light of 250-400nm, (> 60mW) irradiates more than 2 minutes, obtains polyethylene glycol dimethacrylate illumination polymkeric substance;
Alternatively, selection has sheet glass or the silicon chip of suitable size, clean clean after surperficial evaporated metal layer (with reference to Pale-Grosdemange C., Simon E.S., Prime K.L., and Whitesides G.M., J.Am., Chem.Soc., 113,12-20 (1991)), then by evaporation the substrate of metal level be placed in the water of the thiol molecule with polyoxyethylene glycol ending of 2～10mM concentration or the spirituous solution soaking at room temperature of spending the night, thereby obtain having the substrate of inactive surfaces;
2) prepare seal:
Prepare seal, make to contain projection on a surface of this seal, described protruding composition will be formed on suprabasil pattern, and each protruding height is 1～40 micron, and the area of projection is 10000 square nanometers～1 square millimeter; The material of preparing seal preferably adopts polydimethylsiloxane;
3) micro-contact printing:
It is 8-10 that seal is put into pH, in the dopamine solution that concentration is 0.1-100g/L, spends the night, and makes poly-being incorporated on seal of Dopamine HCL spontaneous oxidation form the i.e. poly-Dopamine HCL of one layer of polymeric film, then takes out seal, dries up; Wherein, the preferred pH of described dopamine solution is 8-9, concentration 0.5～10g/L;
The seal drying up is lain in to step 1) in the inactive surfaces of preparation, make to contact with inactive surfaces containing figuratum one side, after exerting pressure and making poly-Dopamine HCL transfer in substrate, take seal away, just obtained the inactive surfaces with poly-Dopamine HCL pattern.
In the above-mentioned method of preparing substrate, step 1) the described metal level in comprises the gold layer of thick 20～50nm; Further preferably, described metal level comprises that direct evaporation is at the titanium adhesion layer of suprabasil 2～10nm and the gold layer of the 20～50nm of evaporation on titanium adhesion layer;
Preferably, in step 1) in, described thiol molecule of take polyoxyethylene glycol ending is HS (CH
2) n (OCH
2cH
2) mOH (n > 3, m > 2), the preferably HS (CH of 1～5mM
2)
11(OCH
2cH
2)
6oH solution.
In the above-mentioned method of preparing substrate, described step 2) by following steps, undertaken:
First, with the method processing mold of photoetching: design the pattern needing with mapping software, and print by film printer; Then adopt ordinary method to carry out lithography process to described pattern; Select conventional silicon chip and photoresist material, through spin coating, front baking, exposure, after dry, develop after, described pattern is just carried by the photoresist material on silicon chip;
Then, to the mould obtaining, using polydimethylsiloxane to carry out pattern copies: the silicon chip with photoresist material processing is gone up to polydimethylsiloxane and carry out duplicating molded; Then more than the baking oven of 80 degree toasts half an hour, make polydimethylsiloxane form solid; Above silicon chip, take polydimethylsiloxane off, pattern has just been transferred to above polydimethylsiloxane; Cut as required the polydimethylsiloxane that polymerization is good, can be used as the seal of micro-contact printing.
In the above-mentioned method of preparing substrate, step 3) dopamine solution in is the Tris-HCl buffer salt solution of the Dopamine HCL of pH8.5,2g/L; Preferably, after seal is contacted with inactive surfaces containing figuratum one side, on seal, exert pressure 10～60 seconds, take seal away, just obtained the inactive surfaces with poly-Dopamine HCL pattern.
On the one hand, the invention provides above-mentioned substrate at culturing cell again, make the application in cell patterned growth.In addition, the present invention also provides the application of described substrate in making cell controllable release, and described substrate is preferably gold substrate.
Detailed description of the present invention below: the invention provides the device that cell is adhered to suprabasil method and realizes the method in oldered array mode.
Described method is to use elastomerics seal that permission cell adhesion material is printed onto to inactive surfaces and forms specific pattern.
Described device comprises:
(1) substrate; Described substrate comprises the inactive surfaces that cell can not adhere to.
Described substrate can be to be coated with the glass of metal level or silicon chip, metal level is up followed successively by titanium adhesion layer and gold layer from sheet glass or silicon chip, then at gold layer, is assembled with the thiol molecule ending up with polyoxyethylene glycol above or contains the thiol molecule of phosphate group and/or tertiary nitrogen groups simultaneously.The structure of described metal level is that titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm.
(2) elastomerics seal.On described seal, there is one side containing irregular micro-structure.
Described elastomeric material is polydimethylsiloxane.
Described micro-structure is concaveconvex structure arbitrarily, can be array, can be also banded pattern.
Described micro-structure is the pattern that protrudes certain plane of seal, and its protrusion height is at 1～40 micron.
The area of described micro-structure is 10000 square nanometers to 1 square millimeter.
Cell for patterning can be eukaryotic cell, can be also bacterium, microorganism.
According to an embodiment of the invention, the invention provides cell is adhered to same suprabasil method in oldered array mode, comprise following step:
1) preparation of surface metal-layer: evaporation titanium layer and gold layer successively in clean substrate;
2) preparation of the inadhesive inactive surfaces of cell: the HS (CH that the substrate for preparing metal level is placed in to 1～5mM
2)
11(OCH
2cH
2)
6the immersion of spending the night in OH solution, can obtain inactive surfaces.
3) use the method processing mold of photoetching:
First, with the pattern described in L-Edit software design, and print by film printer; Then described pattern is carried out to lithography process; Because described pattern area is little, can be made on the silicon chip of 3 cun of areas simultaneously; What photoresist material was used is the negative glue of the thick type of SU-8 of MicroChem company.Through spin coating, front baking, exposure, after dry, develop after, described pattern is just carried by the photoresist material on silicon chip;
4) to the mould obtaining, using polydimethylsiloxane to carry out pattern copies: the silicon chip with photoresist material processing is gone up to polydimethylsiloxane and carry out duplicating molded; Then the baking oven at 80 degree toasts 1 hour, and polydimethylsiloxane has just formed solid; This solid is elastic, can above silicon chip, take off; Take polydimethylsiloxane off, pattern has just been transferred to above polydimethylsiloxane.Cut as required the polydimethylsiloxane that polymerization is good, can be used as the seal of micro-contact printing.
5) the Tris-HCl buffer salt solution of seal being put into the Dopamine HCL of 2g/L spends the night, pH furnishing 8.5.Dopamine HCL is spontaneous oxidation polymerization in above-mentioned condition, and on seal, forms polymeric film.After spending the night, take out seal, dry up.
6) micro-contact printing: the seal drying up is lain in inactive surfaces, contact with gold surface containing figuratum one side, then place a counterweight above seal.After 10～60s, take seal away, just obtained the inactive surfaces with poly-Dopamine HCL pattern.
7) patterning of cell: cell suspending liquid is directly added in to the inactive surfaces with poly-Dopamine HCL pattern, through obtaining afterwards the cell of patterning for 2 hours.
The present invention just can obtain by a step printing can be by the method for the substrate of cell patterning.Compared with prior art, the present invention at least has following advantage:
One, a kind of substrate for patterning culturing cell provided by the invention, does not need to carry out special processing and gets final product inoculating cell, and nontoxic to cell; The cell pattern that adopts poly-Dopamine HCL to form pattern and further obtain can maintain at about 3 days to 3 weeks (seeing embodiment);
Two, method steps of the present invention is simple, the surface that only need to directly provide cell not adhere to, then with the material that the method for micro-contact printing directly can adhere to cell, be printed onto surface, for example by poly-Dopamine HCL, with physical contact form, directly stick in inactive surfaces, method is simple, be adapted at operating in common laboratory, and relatively can save reagent;
Three, in the prior art, the inadhesive inactive surfaces of cell that conventionally adopts gold-plated sheet glass or silicon chip to prepare, or the inactive surfaces obtaining with the silane molecule assembling that contains polyoxyethylene glycol functional group on silicon chip.Method of the present invention is also applicable to the polystyrene culture dish that biology laboratory is conventional, and can extend to titanium, niobium, conductive glass and polyoxyethylene glycol illumination polymkeric substance.Therefore, the method has broken through for realizing the restriction of the gold-plated substrate of use of cell patterning, the selection of having widened substrate kind;
Four, the mode that substrate of the present invention and method are also applicable to analyse by electrolysis discharges the cell fixing, thereby dynamically controls cell at the patterning of substrate.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the schematic diagram of preparing polydimethylsiloxane seal;
Fig. 2 is that Dopamine HCL forms the schematic diagram of poly-Dopamine HCL at seal surface aggregate;
The glass surface that Fig. 3 modifies at PLL-g-PEG for poly-Dopamine HCL forms the schematic diagram of pattern by micro-contact printing;
Fig. 4 is mouse fibroblast cell (NIH 3T3) after the polystyrene surface that contains poly-Dopamine HCL pattern is cultivated 23 days, the pattern of formation;
Fig. 5 is mouse fibroblast cell after the polyethylene glycol dimethacrylate illumination polymer surfaces that contains poly-Dopamine HCL pattern is cultivated 2 days, the pattern of formation;
Fig. 6 is intestinal bacteria, cultivates the pattern forming for 4 hours in gold surface;
Fig. 7 is mouse fibroblast cell after gold surface is cultivated 6 hours, after gold surface is carried out electrolysis and analysed, and the result of the shape of every 2 hours observation of cell.
Embodiment
Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit by any way scope of invention.
In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all indicate when occurring first, identical reagent used is if no special instructions, all identical with the content of indicating first thereafter.
the poly-Dopamine HCL pattern of glass sheet surface printing that 1 couple of PLL-g-PEG of embodiment modifies
1, sheet glass is carried out to oxide treatment.
Sheet glass (25.4*76.2mm, sailing boat board slide glass, article No. 7101) is cut into 5 * 5mm size, be positioned over successively in acetone and water ultrasonic cleaning 10 minutes, the vitriol oil and hydrogen peroxide are mixed with the ratio of 3: 1, and the cleaned slide of mixed liquid dipping 30 minutes, dries up.Sheet glass is placed in air plasma oxidizer and is oxidized 3min.
2, sheet glass is carried out to inertia modification.
Sheet glass after oxidation is put into hydroxyethyl piperazine second thiosulfonic acid (HEPES) solution of PLL-g-PEG solution of 0.1g/L, and (pH 7.4,10mM), soak 2 hours, and the PLL-g-PEG adopting is PLL (20)-g[3.5]-PEG (5), be that PLL is 20k, PEG is 5k, and in PLL, the ratio of Methionin and PEG chain is 3.5.
3, prepare polydimethylsiloxane seal.
First, with L-Edit software (The layout Editor Win 32 8.30, Tanner EDA, A division of Tanner Research, Inc.) the nail-like pattern that formed by trilateral and rectangle of design, and be printed on (purchased from the Suzhou precise and tiny Photoelectric Co., Ltd. of U.S.) above chromium plate; Then described pattern is carried out to lithography process, lithography process can adopt usual method to carry out, the operating process that can provide referring to MicroChem company (network address: http://www.microchem.com/products/pdf/SU-82000DataSheet2025thru 2075Ver4.pdf); What substrate was used is the silicon chip of 3 cun of areas; What photoresist material was used is the negative glue of the thick type of SU-8 of MicroChem company.Through spin coating, front baking, exposure, after dry, develop after, described pattern is just carried by the photoresist material on silicon chip;
Then, to the mould obtaining, using polydimethylsiloxane to carry out pattern copies: the silicon chip with photoresist material processing is gone up to polydimethylsiloxane and carry out duplicating molded; Then the baking oven at 80 degree toasts 1 hour, and polydimethylsiloxane has just formed solid; This solid is elastic, can above silicon chip, take off; Take polydimethylsiloxane off, pattern has just been transferred to above polydimethylsiloxane.Cut as required the polydimethylsiloxane that polymerization is good, can be used as the seal of micro-contact printing, as shown in Figure 1.
4, on seal surface, form poly-Dopamine HCL.
The polymerization of spending the night in the Tris-HCl solution of the Dopamine HCL that is 2g/L in concentration by seal bubble, polymerizing condition is pH 8.0～8.5, as shown in Figure 2.
5, will gather Dopamine HCL and be printed on glass surface
Seal is taken out from solution, dry up, lie in the glass surface of PLL-g-PEG modification through 30 seconds, take off seal, thereby will gather Dopamine HCL, be printed on glass surface, formed the pattern for cell patterned growth, as shown in Figure 3.
patterned growth in the polystyrene substrate that embodiment 2 cells are modified at PLL-g-PEG
1, p-poly-phenyl ethene thin slice carries out oxide treatment.
Polystyrene (disposable use plastic culture dish likes to pursue progress medical plastic company limited) culture dish is cut into the slice, thin piece of 5 * 5mm size, slice, thin piece is put into air plasma oxidizer and be oxidized 3min.
2, p-poly-phenyl ethene thin slice carries out inertia modification, (pH 7.4 wherein to adopt hydroxyethyl piperazine second thiosulfonic acid (HEPES) solution of PLL-g-PEG solution of 10g/L, 10mM), and the PLL-g-PEG adopting is PLL (375)-g[5]-PEG (5), be that PLL is 375k, PEG is 5k, and in PLL, the ratio of Methionin and PEG chain is 5.
3, prepare polydimethylsiloxane seal.
4, on seal surface, form poly-Dopamine HCL.
5, will gather Dopamine HCL and be printed on thin polystyrene sheet surface.
Wherein the step of 2-5 is consistent with the corresponding steps in embodiment 1.
6, at the polystyrene surface inoculating cell that is printed on poly-Dopamine HCL.
The thin polystyrene sheet that is printed on poly-Dopamine HCL is placed in culture dish, by the cell suspending liquid under just digesting (cell is mouse fibroblast cell, NIH 3T3, the concentration of cell suspending liquid is 10
5individual/mL) be placed on substrate, through the cultivations of 23 days (cell culture incubator, 37 ℃, 5%CO2, cell culture medium is that DMEM+10%FBS+1% is dual anti-, during change substratum), obtained cell as shown in Figure 4 and in substrate, formed pattern.
the patterning of embodiment 3 cells on polyethylene glycol dimethacrylate illumination polymkeric substance is raw
long
1, the preparation of polyethylene glycol dimethacrylate illumination polymkeric substance
By polyethylene glycol dimethacrylate liquid, (sigma company buys, No. CAS is 25852-47-5) in add the benzoin dimethylether light trigger of 1% (mass ratio) (sigma company buy, No. CAS is 24650-42-8), vibration is dissolved, dropping is in polystyrene culture dish, under the UV-light of 250-400nm, (> 60mW) irradiates more than 2 minutes, obtains curing polyethylene glycol dimethacrylate illumination polymkeric substance.In culture dish, add water, the immersion of spending the night.Polyethylene glycol dimethacrylate illumination polymkeric substance meeting automatic trip, from polystyrene surface, forms film.Film is placed in baking oven and dries (40 degree, 30 minutes).
2, prepare polydimethylsiloxane seal.
3, on seal surface, form poly-Dopamine HCL.
4, will gather Dopamine HCL and be printed on polyethylene glycol dimethacrylate illumination polymer surfaces.
Wherein the step of 2-4 is consistent with the corresponding steps in embodiment 1.
5,, at the polyethylene glycol dimethacrylate illumination polymer surfaces inoculating cell (identical with step 6 in embodiment 2) that is being printed on poly-Dopamine HCL, cultivate the cell that obtains after 2 days as shown in Figure 5 at the pattern structure of polyethylene glycol dimethacrylate illumination polymkeric substance.
patterned growth on the gold plaque that embodiment 4 bacteriums are modified at thiol molecule
1, the preparation of surface metal-layer
By this area routine techniques, evaporation titanium layer and gold layer successively at the bottom of clean silicon wafer-based.Wherein, first at the titanium adhesion layer of substrate surface evaporation 10nm, then on titanium layer the gold layer of evaporation 40nm (with reference to Pale-Grosdemange C., Simon E.S., Prime K.L., and Whitesides G.M., J.Am., Chem.Soc., 113,12-20 (1991)).
2, the preparation of the polydimethylsiloxane seal of nail-like array pattern.
3, the preparation of the inadhesive inactive surfaces of bacterium, (pH 7.4 wherein to adopt hydroxyethyl piperazine second thiosulfonic acid (HEPES) solution of PLL-g-PEG solution of 5g/L, 10mM), and the PLL-g-PEG adopting is PLL (200)-g[2]-PEG (3), be that PLL is 200k, PEG is 3k, and in PLL, the ratio of Methionin and PEG chain is 2.
Gold-plated sheet glass is placed on to concentration soaked overnight in the HS of 2mM (CH2) 11 (OCH2CH2) 6OH solution, obtains the inactive surfaces of self-assembled monolayer.
4, on seal surface, form poly-Dopamine HCL.
5, will gather Dopamine HCL and be printed on gold plaque surface.
Wherein, 2,4 is identical with embodiment 1 corresponding steps with 5 operation steps.
6, the inoculation of bacterium.
The gold plaque that is printed on poly-Dopamine HCL is placed in culture dish, and by bacterium liquid, (intestinal bacteria, DSMZ of Institute of Micro-biology buys, ATCC11775, the OD of bacterium liquid
600value is 0.4, is equivalent to 10
8cFU/mL, the culture solution of bacterium is broth culture) being seeded in substrate surface, culture condition is 37 degree shaking table shaking culture.Through 4 hours, just can obtain bacterium and in substrate, form pattern, as shown in Figure 6, wherein Fig. 6, for cultivating the bacterial growth situation after 4 hours, can see that bacterium carries out patterned growth on the nail-like pattern of substrate surface.
the cell of 5 pairs of patternings of embodiment carries out controlled release
1, the substrate with poly-Dopamine HCL pattern obtaining with embodiment 4 step 1-5 is taken out, and use step 6 inoculating cell of embodiment 2, then cell is cultivated in incubator 6 hours.
2, gold plaque is carried out to electrolysis and analyse: by voltage stabilized source, to being placed on the voltage 30s of the apply-1.5V of substrate in substratum, with substratum, wash 3 times.
Substrate after electrolysis is analysed is reentered among substratum, with microscope, carries out continuous shooting observation, can see that cell moves out gradually from pattern, as shown in Figure 7.
Claims (26)
1. for a substrate for cell patterned growth, described substrate comprises:
A) inactive surfaces that cell can not adhere to; With
B) pattern in described inactive surfaces, this pattern is by allowing the material of cell adhesion to form, and described inactive surfaces is not all by described pattern covers;
Wherein, the material of described permission cell adhesion is poly-Dopamine HCL;
And described substrate is sheet glass or silicon chip, on described sheet glass or silicon chip, by the interior inactive surfaces that has successively metal level and formed by the thiol molecule ending up with polyoxyethylene glycol outward, described thiol molecule is HS (CH
2)
11(OCH
2cH
2)
6oH;
Or described substrate is polystyrene plastic sheet, on described polystyrene sheet, comprise the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is sheet glass or silicon chip, on described sheet glass or silicon chip, comprise the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is metal titanium sheet or niobium sheet, on described titanium sheet or niobium sheet by interior be titanium or niobium outward successively, titanium oxide or niobium oxides and the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is indium tin oxide conductive glass, on described indium tin oxide conductive glass by interior be glass outward successively, indium tin oxide and the inactive surfaces being formed by poly-lysine-polyethyleneglycol-graft copolymer molecule;
Or described substrate is by polyethylene glycol dimethacrylate illumination polymer formation.
2. substrate as claimed in claim 1, wherein, described metal level comprises gold layer.
3. substrate as claimed in claim 2, wherein, described golden bed thickness 20～50nm.
4. substrate as claimed in claim 1, wherein, described metal level from sheet glass or silicon chip by interior titanium adhesion layer and the gold layer of comprising successively outward.
5. substrate as claimed in claim 4, wherein, described titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm.
6. substrate as claimed in claim 1, wherein, described poly-lysine-polyethyleneglycol-graft copolymer molecule is the PLL-g-PEG of different copolymer degree.
7. substrate as claimed in claim 6, wherein, PLL is the PLL of 20～375k.
8. substrate as claimed in claim 6, wherein, PEG is the PEG of 2～5k.
9. substrate as claimed in claim 6, wherein, in PLL, the ratio of Methionin and PEG chain is 3～5.
10. substrate as claimed in any one of claims 1-9 wherein, wherein, the pattern being formed by the material that allows cell adhesion in substrate inactive surfaces comprises by point, line, bar, band, piece or the irregularly shaped irregular pattern forming or array.
11. substrates as claimed in claim 10, wherein, the cell of described cell for surviving after can being adhered to and adhere to by substrate, described cell is eukaryotic cell or bacterium.
The preparation method of 12. 1 kinds of substrates as described in any one in claim 1-11, wherein, described method comprises: the substrate with the inactive surfaces that cell can not adhere to is provided, by the pattern that allows cell adhesion by micro-contact printing on the inactive surfaces of this substrate;
Wherein, said method comprising the steps of:
1) prepare the inactive surfaces of substrate:
Selection has polystyrene sheet, sheet glass, silicon chip, titanium sheet or the niobium sheet of suitable size, is placed in air plasma oxidizer and carries out oxide treatment, and oxidization time is more than 1 minute; The hydroxyethyl piperazine second thiosulfonic acid solution that substrate after oxidation is placed in to the PLL-g-PEG that pH7.4, concentration are 0.5～10g/L is more than 0.5 hour, thereby obtains having the substrate of inactive surfaces;
Or, the benzoin dimethylether light trigger of 1% mass ratio will be added in polyethylene glycol dimethacrylate liquid, dissolve, under the UV-light of 250-400nm, >60mW, irradiate more than 2 minutes, obtain polyethylene glycol dimethacrylate illumination polymkeric substance, as the inactive surfaces of substrate;
Or, selection has sheet glass or the silicon chip of suitable size, after clean clean in surperficial evaporated metal layer, then by evaporation the substrate of metal level be placed in the water of the thiol molecule with polyoxyethylene glycol ending of 2～10mM concentration or the spirituous solution soaking at room temperature of spending the night, thereby obtain having the substrate of inactive surfaces;
2) prepare seal:
Prepare seal, make to contain projection on a surface of this seal, described protruding composition will be formed on suprabasil pattern, and each protruding height is 1～40 micron, and the area of projection is 10000 square nanometers～1 square millimeter;
3) micro-contact printing:
It is 8～10 that seal is put into pH, in the dopamine solution that concentration is 0.1-100g/L, spends the night, and makes poly-being incorporated on seal of Dopamine HCL spontaneous oxidation form the i.e. poly-Dopamine HCL of one layer of polymeric film, then takes out seal, dries up;
The seal drying up is lain in the inactive surfaces of preparing in step 1), make to contact with inactive surfaces containing figuratum one side, after exerting pressure and making poly-Dopamine HCL transfer in substrate, take seal away, just obtained the inactive surfaces with poly-Dopamine HCL pattern.
13. methods as claimed in claim 12, wherein, dopamine solution pH is 8-9, concentration is 0.5～10g/L.
14. methods as claimed in claim 12, wherein, step 2) material of preparing seal in adopts polydimethylsiloxane.
15. methods as claimed in claim 12, wherein, in step 1), the metal level of described evaporation comprises the gold layer of thick 20～50nm.
16. methods as claimed in claim 15, wherein, described metal level comprises that direct evaporation is at the titanium adhesion layer of suprabasil 2～10nm and the gold layer of the 20～50nm of evaporation on titanium adhesion layer.
17. methods as claimed in claim 15, wherein, described thiol molecule solution of take polyoxyethylene glycol ending is HS (CH
2) n (OCH
2cH
2) mOH solution, n>3 wherein, m>2.
18. methods as claimed in claim 17, wherein, the HS (CH that described thiol molecule solution of take polyoxyethylene glycol ending is 1～5mM
2)
11(OCH
2cH
2)
6oH solution.
19. methods as described in any one in claim 12-18, wherein, described step 2) by following steps, undertaken:
First, with the method processing mold of photoetching: design the pattern needing with known mapping software, and print by film printer; Then adopt ordinary method to carry out lithography process to described pattern; Select conventional silicon chip and photoresist material, through spin coating, front baking, exposure, after dry, develop after, described pattern is just carried by the photoresist material on silicon chip;
Then, to the mould obtaining, using polydimethylsiloxane to carry out pattern copies: the silicon chip with photoresist material processing is gone up to polydimethylsiloxane and carry out duplicating molded; Then more than the baking oven of 80 degree toasts half an hour, make polydimethylsiloxane form solid; Above silicon chip, take polydimethylsiloxane off, pattern has just been transferred to above polydimethylsiloxane; Cut as required the polydimethylsiloxane that polymerization is good, can be used as the seal of micro-contact printing.
20. methods as described in any one in claim 12-18, wherein, the dopamine solution in described step 3) is the Tris-HCl buffer salt solution of the Dopamine HCL of pH8.5,2g/L.
21. methods as claimed in claim 19, wherein, the dopamine solution in described step 3) is the Tris-HCl buffer salt solution of the Dopamine HCL of pH8.5,2g/L.
22. methods as claimed in claim 20 wherein, after seal is contacted with inactive surfaces containing figuratum one side, are exerted pressure 10～60 seconds on seal, take seal away, have just obtained the inactive surfaces with poly-Dopamine HCL pattern.
23. methods as claimed in claim 21 wherein, after seal is contacted with inactive surfaces containing figuratum one side, are exerted pressure 10～60 seconds on seal, take seal away, have just obtained the inactive surfaces with poly-Dopamine HCL pattern.
24. as described in any one in claim 1-11 substrate at culturing cell, make the application in cell patterned growth.
25. application of substrate in making cell controllable release as described in any one in claim 1-11.
26. application as claimed in claim 25, wherein, described substrate is gold substrate.
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| CN105016294B (en) * | 2015-06-04 | 2017-03-08 | 天津大学 | A kind of method preparing advanced microstructure poly-dopamine thin film |
| CN105886472A (en) * | 2016-05-13 | 2016-08-24 | 中山大学 | Patterned substrate for constructing in-vitro tumour model |
| CN107267387B (en) * | 2017-08-03 | 2020-02-07 | 长春理工大学 | Cell culture substrate capable of positioning, controlling and measuring potential, preparation method and application |
| CN109082155A (en) * | 2017-08-24 | 2018-12-25 | 中山大学 | A kind of application of tannic acid as micro-contact printing ink in cell patterning |
| CN108641892B (en) * | 2018-04-17 | 2021-11-16 | 广州波奇亚标准及检测技术有限公司 | Micro-contact printing system for cell patterning |
| CN109776842B (en) * | 2019-01-11 | 2020-06-09 | 西南交通大学 | Regioselectivity controllable fixed bimolecular film 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 |
| CN111621468A (en) * | 2020-05-21 | 2020-09-04 | 上海慧灯医疗科技有限公司 | Method for preparing induced pluripotent stem cell-derived cardiomyocytes and fixing method and application thereof |
| CN112724737B (en) * | 2021-01-14 | 2022-02-22 | 中山大学 | Dopamine ink and method and application thereof for preparing micro-nano pattern |
| CN114668893B (en) * | 2022-03-11 | 2023-05-23 | 华南理工大学 | Method for preparing nerve repair material in high flux based on surface patterning experiment platform |
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