CN110205243A - The manufacturing method of cell attachment device and the cell attachment device - Google Patents
The manufacturing method of cell attachment device and the cell attachment device Download PDFInfo
- Publication number
- CN110205243A CN110205243A CN201810167026.3A CN201810167026A CN110205243A CN 110205243 A CN110205243 A CN 110205243A CN 201810167026 A CN201810167026 A CN 201810167026A CN 110205243 A CN110205243 A CN 110205243A
- Authority
- CN
- China
- Prior art keywords
- cell
- warming
- magnetic
- layer
- macromolecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
-
- C—CHEMISTRY; METALLURGY
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
-
- 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
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
- C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating
Abstract
The present invention provides a kind of cell attachment device, lies in a surface of a substrate, forms a cell attaching layer, and being formed then at the surface of the cell attaching layer has the magnetic warming layer of a plurality of warmings high molecular one, and each warming macromolecule has a plurality of magnetic particles.When variation effect of the magnetism warming layer in a physical features, so that a plurality of cells are able to be attached on the cell attaching layer or go to attach by the cell attaching layer.Cell attachment device through the invention can formerly control temperature to one first temperature and lure that the warming macromolecule on substrate generates structure change into, make cell attachment on the cell attaching layer.The separately magnetic particle temperature rise with alternating magnetic field (HAC) to making in warming macromolecule enables cell to maintain the effect of cell attachment in the second temperature lower than first temperature.
Description
Technical field
The present invention relates to a kind of technologies of cell attachment, particularly relate to a kind of utilization physical features variation alternating magnetic field change
Warming macromolecule with magnetic particle generate cell attachment device that structure change is attached at cell on substrate with
Method and its manufacturing method.
Background technique
In the normal tissue, blastema (homotypic cell) and heteroplasia cell (heterotypic cell) be must
The functionality that there could be normal tissue need be grown on corresponding position.Therefore the graphical permutation technology (cell of cell
Partterning technology) it on raw doctor's engineering field is critically important research topic.The concept that cell graphically arranges
It is to be suggested in generation nineteen sixty；The substantially patterning arrangement of cell is divided into passive type and active two kinds.
Passive type mainly relies on arranging cells on substrate to attach ligand (cell-adhesion ligand) and cell table
Face recipient (receptor), which combines, reaches cell arrangement, and the reaction time of the method long and can not relatively be reused.And it leads
Dynamic formula mainly relies on that apply an external force seem sound wave, light pincers, dielectrophoresis etc., carries out figure to guide cell to the position defined
Change arrangement.These active cell arrangement methods are being answered although technology is reversible, reaction efficiency is good and the operating time is shorter
With or some limitation, for example, microstructure be easy to cause cell to cause changing on cell inner frame during attaching
Become, and target cell can not be screened and make graphical arrangement.Although dielectrophoresis technology can be used to manipulate group's cell, necessary
It to be operated under the culture environment of low electric conductivity, and need high-frequency electric current to carry out manipulation cell and avoid causing cell membrane
Injury, in addition to this, a large amount of microelectrode preparations, which also increase, uses upper limitation；Though light pincers can be used to manipulate single cell,
It is constrained to that instrument is relatively expensive and the limitation of operation power is to prevent from causing heat injury to cell.
In the prior art, such as Tang et al., " Temperature-Responsive Polymer has been delivered in 2012
Modified Surface for Cell Sheet Engineering”,Polymer,15Aug,2012,4,1478-1498,
A kind of technology of warming type macromolecule progress cell attachment is proposed in one text.In cell attaching layer (tissue culture
Polystyrene, TCPS) the warming macromolecule layer that the variation that one layer can use temperature changes hydrophilic and hydrophobic is formed on surface,
It is made of poly-N-isopropyl acrylamide (poly (N-isopropylacrylamide), poly (NIPAAm)).But,
Although warming macromolecule layer can be higher than lower critical solution temperature (lower critical solution temperature,
When LCST), hydrophobic state is formed, so that cell is able to be attached on TCPS, conversely, when temperature is lower than LCST, warming
Macromolecule layer is then transformed into hydrophilic state, so that the cell on TCPS goes to attach.
Although in the prior art, can produce the cell attachment device for attaching cell effect, it must be maintained at and be higher than
The phenomenon that cell attachment is just had when LCST operating temperature, is unable to maintain that cell attachment effect under normal temperature state, furthermore also difficult
To allow these cells to line up specific pattern.Although the aforementioned technology for also having cell arrangement in the prior art, but there is its deficiency
Place, synthesis be aforementioned, it is therefore desirable to which a kind of cell attachment apparatus and method solve problem of the prior art.
Summary of the invention
It is an object of the invention to: a kind of cell attachment device and the manufacturing method of the cell attachment device are provided, solved
Certainly the above-mentioned technical problems in the prior art.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of cell attachment device, it is characterized in that including:
One substrate；
One cell attaching layer is formed in the surface of the substrate；And
One magnetic warming layer, is formed in the surface of the cell attaching layer, which has a plurality of magnetic warmings
Macromolecule, each magnetism warming macromolecule has a warming macromolecule and a plurality of magnetic particles, when acting on the magnetism temperature
When feeling the physical features variation of layer, a plurality of cells are able to be attached on the cell attaching layer or go to paste by the cell adsorption layer
It is attached.
The cell attachment device, in which: the cell attaching layer is polyethyleneimine (polyethylenimine), should
Warming macromolecule is poly-N-isopropyl acrylamide (poly (N-Isopropylacrylamide)).
The cell attachment device, in which: the object features are the combination of temperature, magnetic field or both.
The cell attachment device, in which: also there is a magnetic texure, which has a patterning, should
Magnetic texure is set between the cell attaching layer and the substrate or in the side being set to outside the substrate.
The cell attachment device, in which: the patterning is with a plurality of concentric arrangement of geometrical pattern structures.
A kind of manufacturing method of cell attachment device, it is characterized in that including the following steps:
A plurality of magnetic warming macromolecules are manufactured, each magnetic warming macromolecule has one first warming macromolecule, often
One first warming macromolecule has a plurality of magnetic particles；
One substrate is provided, and forms a cell adsorption layer on the substrate；And
Using a plurality of magnetic warming macromolecules in forming a magnetic warming layer on the cell adsorption layer, when acting on this
When the physical features variation of magnetic warming layer, a plurality of cells are able to be attached on the cell attaching layer or are adsorbed by the cell
Layer goes to attach.
The manufacturing method of the cell attachment device, in which: formed before the cell adsorption layer, further include having in the base
A laminated magnetic film is formed on plate, there is a patterning, and when forming the magnetism warming layer, this is a plurality of with magnetism
Warming macromolecule is corresponding to the specific position arrangement in the patterning.
The manufacturing method of the cell attachment device, in which: further include having that there is a specific pattern in the side of the substrate
The permanent magnet of case has a patterning, and when forming the magnetism warming layer, a plurality of temperature with magnetic particle
Feel macromolecule to arrange along the profile of the patterning.
The manufacturing method of the cell attachment device, in which: the object features are the combination of temperature and magnetic field, the cell
Adhering device a plurality of magnetic warming macromolecules in first temperature are contraction state, so that cell is adsorbed in cell suction
On attached layer, in a second temperature, by the effect of the changes of magnetic field, so that the warming only in the corresponding patterning is high
Molecule maintains contraction state, and cell should mutually adsorbed on the specific region of patterning by the cell adsorption layer.
The manufacturing method of the cell attachment device, in which: it further include the tangible second warming macromolecule that pluralizes, and
When forming the magnetism warming layer, the magnetism warming macromolecule and the second warming macromolecule mixed solution, coating will be contained
In on the cell adsorption layer, so that corresponding on the region of the patterning, there is the magnetism warming macromolecule to be formed in the cell
On adsorption layer, and on other regions, there is the second warming macromolecule to be formed on the cell adsorption layer.
Compared with prior art, the invention has the advantages that:
The present invention provides a kind of cell attachment apparatus and method, utilizes and warming macromolecule is allowed to have magnetic particle to be formed
Magnetic warming macromolecule, the magnetism warming macromolecule attach cell except through the variation of temperature, can further pass through friendship
The control of varying magnetic field, so that cell still can maintain the state attached under conditions of being lower than LCST temperature, to avoid existing
Have in technology, cell is attached by external force, destruction is led to the problem of to cell, and generate the condition not having to being lower than LCST
Under, allow cell attachment or go attach effect.
The present invention provides a kind of cell attachment apparatus and method, can under conditions of using LCST temperature change is controlled
It cell attachment or is gone except attaching with allowing, more can use the arrangement pattern of externally-applied magnetic field control cell, and then can simulated each
The different histocyte of kind, to increase the field of its application.In particular, passing through the knot of the laminated magnetic film with specific pattern
Structure is attached to the warming macromolecule with magnetic particle on the specific position of patterning, and then can pass through
Alternating magnetic field, control attach the position and region of cell attachment, form specific cell pattern.
The present invention provides a kind of cell attachment apparatus and method, by having the either magnetic warming layer of magnetic warming layer
With the structure of laminated magnetic film, attaching cell can be allowed to generate cell attachment under conditions of not needing injection magnetic particle and go
The effect of attaching, and then the integrality of cell can be maintained, expand the application range of research and development.
Detailed description of the invention
Fig. 1 is cell attachment device first embodiment diagrammatic cross-section of the invention.
Fig. 2A and 2B is warming macromolecule in the variation of hydrophilic-hydrophobic property schematic diagram for being higher than LCST temperature with being lower than LCST temperature.
Fig. 2 C and Fig. 2 D is the warming macromolecule with magnetic particle when being lower than LCST temperature, by action of alternating magnetic field
And the variation of hydrophilic-hydrophobic property schematic diagram generated.
Fig. 3 A~Fig. 3 D is a flow diagram of production cell attachment device of the invention.
Fig. 4 A is cell attachment device second embodiment diagrammatic cross-section of the invention.
Fig. 4 B is the patterning schematic diagram of thin magnetic film.
Fig. 5 A and Fig. 5 B is that cell attachment device of the invention attaches cell and forms specific cells pattern schematic diagram.
Fig. 6 is another embodiment schematic diagram of magnetic adhering device of the invention.
Shown in Fig. 7 A~Fig. 7 F, which is to form the cell attachment device second embodiment Structure and Process of Fig. 4 A and Fig. 4 B to show
It is intended to.
Description of symbols: 2,2a- cell attachment device；20- substrate；21- cell attaching layer；22- magnetism warming layer；
23- magnetism warming macromolecule；230- warming macromolecule；231- magnetic particle；25- laminated magnetic film；250- patterning；26-
Warming layer；27- permanent magnet；3,3a- cell；90- alternating magnetic field.
Specific embodiment
Alterations will be referred to below, and various exemplary embodiments can be more fully described, show in alterations
Some exemplary embodiments.However, concept of the present invention may embody in many different forms, and should not be construed as limited by herein
Middle illustrated exemplary embodiments.Specifically, provide these exemplary embodiments make the present invention will to be detailed and complete,
And the scope for concept of the present invention sufficiently being conveyed to those who familiarize themselves with the technology.Similar number indicates similar component always.Below will
Illustrate the manufacturing method of the cell attachment apparatus and method and the cell attachment device with various embodiments cooperation schema,
However, following embodiments are not intended to limit the invention.
Refering to Figure 1, the figure is cell attachment device first embodiment diagrammatic cross-section of the invention.In this implementation
In example, which includes a substrate 20, a cell attaching layer 21 and a magnetic warming layer 22.The substrate 20
Material have no certain restrictions, the either flexible substrate of glass substrate, silicon substrate can be used, such as: plastic substrate etc.,
System that but not limited to this.It is formed with a cell attaching layer 21 on the overhead surface of the substrate 20, cell can be provided and be attached to it
On.In one embodiment, which can be polyethyleneimine (polyethylenimine, PEI), but not with this
For limitation, the people of art technology can select cell attachment layer material appropriate according to demand.
The upper surface of the cell attaching layer 21 is then formed with the magnetism warming layer 22, can be by acting on cell patch
The variation of the exterior object feature of adsorption device 2, such as: change environment temperature or be to provide an alternating magnetic field (HAC), and changes
Its hydrophily and hydrophobicity.The magnetism warming layer 22 has a plurality of magnetic warming macromolecules 23, each magnetism warming macromolecule
23 there is a magnetic warming macromolecule 230 and a plurality of magnetic particles 231 to be attached on its surface or inside it.One
In embodiment, it is poly-N-isopropyl acrylamide (poly (N- which, which has,
Isopropylacrylamide), poly (NIPAAm)), system that but not limited to this.The magnetic particle 231, in one embodiment
For nanometer magnetic particle.It please refers to shown in Fig. 2A and 2B, in the present embodiment, which has a low Critical Solution
Temperature (lower critical solution temperature, LCST), in one embodiment, be 32~34.4 DEG C it
Between, the present embodiment is 32 DEG C, it is to be noted that the LCST temperature system is not limited to this embodiment depending on the property of material
System.
In fig. 2, when environment temperature is higher than LCST, such as: each magnetic temperature at 37 DEG C, in magnetic warming layer 22
Sense macromolecule 23 will become hydrophobic state and shrink, so that the cell that originally inflated magnetic warming macromolecule 23 covers
Associated layer 21 can reveal, region A as shown in Figure 2 A.At this point, if environment it is interior have cell 3 if, one implement
In example, which is liquid, which can be attached on cell attaching layer 21.Conversely, as shown in Figure 2 B, when environment temperature is low
When LCST, such as: 28 DEG C, then the magnetic warming macromolecule 23 of magnetic warming layer 22 can be transformed into hydrophily from hydrophobicity,
Structure expand and be blinded by original region A so that cell 3 go attach and departing from cell attaching layer 21.
It is to change magnetic warming layer hydrophilic and hydrophobic schematic diagram by alternating magnetic field 90 for another example shown in Fig. 2 C and 2D.Scheming
In 2C, when environment temperature is lower than LCST temperature, such as at 28 DEG C of room temperature, although magnetic 23 meeting of warming macromolecule normally
Become hydrophily and expand, and then be blinded by cell attaching layer 21, but passes through 90 pairs of alternating magnetic field magnetic warming macromolecules 23
231 generation of magnetic particle effect, as shown in Figure 2 D, so that generating temperature with the magnetic warming macromolecule 23 of magnetic particle 231
Degree variation namely the effect of alternating magnetic field 90 make magnetic warming macromolecule 23 heat up, and make the magnetism with magnetic particle 231 warm
Sense macromolecule 23 generates hydrophobic state namely warming macromolecular structure is shunk, and then cell 3 is allowed to be attached at cell attaching layer
21 either maintain cell 3 to be persistently attached on cell attaching layer 21.It, can be in room temperature by the control of alternating magnetic field 90
Under, allowing should be originally that cell 3 goes the case where attaching, and maintain or become cell 3 is attached at 21 state of cell attaching layer, greatly
Increase the application environment of refinement born of the same parents adhering device 2.For example, as soon as user does not have to maintenance environment deliberately using in situation
Temperature, to maintain cell to be attached on cell attaching layer 21, though under room temperature, by the effect of alternating magnetic field, i.e.,
It can achieve the effect for maintaining cell to be attached, can so reduce cost needed for maintaining environment temperature, include device and energy
Source also can increase the convenience of transport.
The mode that manufacture Fig. 1 structure will be illustrated next please refers to shown in Fig. 3 A and Fig. 3 D, which is that production of the invention is thin
One flow diagram of born of the same parents' adhering device.Firstly, carrying out the warming high score that warming high molecular material is converted into amination by step 30
Sub- material, in this step, by warming high molecular material, such as: poly (NIPAAm) is dissolved in methanol with hydrazine N2H2, heating
It flows back to 90 DEG C of generations, is down to room temperature after reaction, the poly (NIPAAm) of amination is freeze-dried to obtain after dialysis.
The person of connecing carries out step 31 and generates magnetic warming type glue material.It is by phosphinylidyne diimine (1-Ethyl-3- (3- in this step
Dimethylaminopropyl)-carbodiimide, EDC) and n-hydroxysuccinimide (N-
Hydroxysuccinimide, NHS) it is molten with the magnetic nanoparticle CM-dextran/Fe3O4 of 40wt% with 5,10,20 respectively
In deionized water, using the COOH functional group of EDC/NHS activation CM-dextran, then connects and the obtained amination of step 31 is added
Poly (NIPAAm), both make to be grafted, obtain magnetic warming type glue material, the magnetism temperature with magnetic particle formed
Feel 23 structural schematic diagram of macromolecule, as shown in Figure 3A, which is close spherical, and magnetic nanoparticle 231 is grafted
In magnetic 23 surface of warming macromolecule.
As shown in Figure 3B, step 32 is then carried out, provides a substrate 20, feature is as previously mentioned, this will not be repeated here.It connects
The step of getting off, carrying out step 33, form cell attaching layer.As shown in Figure 3 C, the substrate is made to be soaked in 1.0wt% in step 33
PEI (polyethylenimine) solution, and be dried, such as: spontaneously dry at room temperature, to form film thickness about
For 1 μm of cell attaching layer.Then, step 34 is carried out in forming magnetic warming macromolecule 23 and shape on the cell attaching layer
At magnetic warming layer 22.This step system drips the deionized water containing Fig. 3 A structure in the substrate of Fig. 3 B, by electronegative
Poly (NIPAAm) and electropositive PEI is closely attracting by positive negative electric force, continues and carries out rotary coating, makes poly
(NIPAAm) it is evenly applied to substrate 20, completes poly (NIPAAm) magnetic warming layer 22, as shown in Figure 3D.
It please refers to shown in Fig. 4 A, which is cell attachment device second embodiment diagrammatic cross-section of the invention.In this reality
Apply in example, cell attachment device 2a is substantially similar to Fig. 1, institute's difference, the cell attachment device 2a of the present embodiment in
A laminated magnetic film 25 is had more between the cell attaching layer 21 and the substrate 20, there is a patterning 250, so that this is multiple
Several magnetic warming macromolecules 23 with magnetic particle 231 are arranged along the profile of the patterning 250.In an embodiment
In, as shown in Figure 4 B, which there are a plurality of concentric geometries to be constituted, which implements one
It is rectangular configuration in example.The structure system of geometrical pattern is noted that depending on the demand of user, not with the present embodiment
Concentric geometrical pattern be limitation.In addition, in one embodiment, the cell attaching layer 21 in non-corresponding geometrical pattern region 251
On, then form the warming layer 26 constituted without the magnetic warming macromolecule 23 of magnetic particle 231.
By structure shown in Fig. 4 A, it can control after cell attachment device 2a attaches cell and be formed by pattern, so that
Cell attachment device 2a can simulate the cell of specific function, such as: liver, heart etc., and then application field can be increased, have
Help the exploitation of drug either therapeutic modality.For the structure shown in Fig. 4 A and Fig. 4 B, in the condition for being higher than LCST temperature
Under, such as: 37 DEG C, the state as shown in earlier figures 2A, since magnetic warming macromolecule 23 is hydrophobicity, the at this time magnetic of substrate
Warm-natured 23 structure both shrinks of sense macromolecule form the state such as Fig. 5 A so that cell 3 is able to be attached on cell attaching layer 21.When
Environment temperature is lower than the LCST of substrate, such as: at 27 DEG C, state as shown in Figure 2 B, magnetic 23 structure of warming macromolecule expansion
Lure that cell 3 is detached from cell attaching layer 21 into.At this point, again by applying alternating magnetic field (HAC), so that with magnetic particle 231
Magnetic warming macromolecule 23 because magnetic particle 231 by alternating magnetic field (HAC) effect and temperature rises, and then can make to have
Be magnetic its hydrophobic state of magnetic warming macromolecule 23 maintenance of particle 231, so that the cell 3 attached thereon continues to tie up
Hold attaching state.The magnetic warming macromolecule 23 that the patterning of laminated magnetic film 25 is not corresponded to as other, due to temperature
The event already below LCST is spent, magnetic 23 structure of warming macromolecule changes and becomes hydrophily, so that being originally attached at cell
The cell 3a of associated layer 21 is detached from cell attaching layer 21.The ambient temperature conditions higher than LCST temperature are first imposed by above-mentioned, are made
Cell is all attached on cell attaching layer 21, it is rear be lower than LCST by control ambient temperature conditions, then apply alternating magnetic field,
So that the cell 3 of specific region maintains on cell attaching layer 21, and then generating, there is the specific cell for attaching cell pattern to paste
Adsorption device 2a.
Patterned cell arrangement principle will be illustrated next, since in the present embodiment, patterning is a plurality of concentric
Rectangular configuration be therefore formed with the magnetic warming macromolecule with a plurality of magnetic particles 231 along the profile of rectangular configuration
23.When temperature is lower than LCST and applies alternating magnetic field HAC, cell is dissipated by caused by concentric rectangles structure turning point
Ease field attracts, so that cell 3 can concentrate the warming for being attached at and having magnetic particle accordingly in the turnover place of patterning 250
On macromolecule, and form graphical cell culture substrate as shown in Figure 5 B.In addition, when cell attachment device is placed in room temperature and nothing
When alternating magnetic field HAC exposes photograph, total cell can be completed and go to attach, reach another application.
It please refers to shown in Fig. 6, which is another embodiment schematic diagram of magnetic adhering device of the invention.In the present embodiment
In, substantially similar to Fig. 4 A, difference is that the present embodiment system is taken using the permanent magnet 27 with specific shape or pattern
For the laminated magnetic film 25 of Fig. 4 A.Same mode is formed in the position of corresponding permanent magnet 27 with magnetic particle 231
Magnetic warming macromolecule 23, and remaining region is then formed with the magnetic warming macromolecule 23 without magnetic particle.As for utilization
Alternating magnetic field controls the mode of cell attachment, then as previously mentioned, this will not be repeated here.
The method that attaching cell of the invention will be illustrated next, provides the cell as shown in Fig. 4 A or Fig. 6 first with step 40
Adhering device 2a or 2b.Then into type step 41, when the first operating temperature of Yu Yi, a plurality of cells is made to be attached at cell patch
Adsorption device 2a.In one embodiment, which is higher than the high molecular LCST temperature of warming, and the present embodiment is 37 DEG C, but
It is not limited system.Under first operating temperature, the cell results that cell attachment device 2a is attached are as shown in Figure 5A
State namely cell 3 be covered with cell attachment device on the high molecular surface of warming.Then into type step 42, one is provided
Alternating magnetic field HAC is acted on cell attachment device 2a.Then, at step 43, one is reduced to from first operating temperature
Second operating temperature, by the effect of the alternating magnetic field, so that at least part in a plurality of cells is attached at this carefully
On born of the same parents' adhering device 2a, as shown in Figure 5 B there is patterned cell attachment structure to be formed.In one embodiment, this second
Operating temperature is LCST temperature high molecular lower than warming, 28 DEG C in the present embodiment.
It please refers to shown in Fig. 7 A~7F, which is the cell attachment device second embodiment structure stream to form Fig. 4 A Yu Fig. 4 B
Journey schematic diagram.In the present embodiment, step 50 is carried out first, is made warming profile material poly (NIPAAm).In one embodiment,
The reaction flask containing 40mL deionized water is added in NIPAAm and chain-transferring agent (methyl3-mercaptopropionate) by system
In, it is passed through nitrogen, is heated.Initiator potassium peroxydisulfate (potassium peroxydisulfate, KPS), which is added, keeps it anti-
It should polymerize, obtain warming profile material poly (NIPAAm).Then step 51 is carried out, the warming macromolecule containing magnetic particle is made
The magnetic warming type glue material constituted.It in one embodiment, is that step 50 warming high molecular material and hydrazine N2H2 is molten
Solution is heated to 90 DEG C of generation reflux, is down to room temperature after reaction, is freeze-dried to obtain amination after dialysis in methanol
poly(NIPAAm).The person of connecing, by phosphinylidyne diimine (1-Ethyl-3- (3-dimethylaminopropyl)-
Carbodiimide, EDC) with n-hydroxysuccinimide (N-hydroxysuccinimide, NHS) respectively with 5,10,20 with
The magnetic nanoparticle CM-dextran/Fe3O4 of 40wt% is dissolved in deionized water, using EDC/NHS activation CM-dextran's
COOH functional group connects the poly (NIPAAm) that amination is added, and is grafted the two, obtains magnetic warming type glue material, the height
23 structure of molecule is close spherical, and magnetic nanoparticle 231 grafts on magnetic 23 surface of warming macromolecule.
Then step 52 is carried out, coats AZ photoresist 200 on the base plate 20, the structure such as Fig. 7 A is formed, then such as Fig. 7 B institute
Show, then cover light shield 201 and be etched by light with UV, connects with electron beam evaporation plating ferromagnetic thin film 202, form the structure such as Fig. 7 C.Such as
Shown in Fig. 7 D, photoresist 200 is removed, patterned thin magnetic film is completed and is fixed on glass substrate, pattern is concentric square structure, line
Wide and spacing is all 12 μm, and substrate can magnetize using externally-applied magnetic field, generates magnetic and dissipates field.It connects and carries out step 53, by fixed magnetic
The glass substrate of property film, is soaked in PEI (polyethylenimine) solution of 1.0wt%, and spontaneously dry at room temperature,
To form cell attaching layer 21, thickness is about 1 μm, as seen in figure 7e.It connects and carries out step 54, as shown in Figure 7 F, then with temperature
The mixed solution drop of sense type aqueous colloidal solution and magnetic warming type aqueous colloidal solution invests on the cell attaching layer 21 of substrate 20, is revolved
Turn to coat, in aqueous colloidal solution drying process, the magnetic warming macromolecule 23 containing magnetic particle 231 can be attracted to thin magnetic film
The graphic structure of layer 21, and the magnetic warming macromolecule 23 without containing magnetic particle 231 can then be attached to not patterning
On cell attaching layer 21, wait it is to be dried after, that is, complete the preparation of cell attachment device as shown in Figure 4 A.
Described above to be merely exemplary for the purpose of the present invention, and not restrictive, those of ordinary skill in the art understand,
Without departing from the spirit and scope defined by the claims, can many modifications may be made, variation or equivalent, but will all fall
Enter within protection scope of the present invention.
Claims (10)
1. a kind of cell attachment device, it is characterized in that including:
One substrate；
One cell attaching layer is formed in the surface of the substrate；And
One magnetic warming layer, is formed in the surface of the cell attaching layer, which has a plurality of magnetic warming high scores
Son, each magnetism warming macromolecule has a warming macromolecule and a plurality of magnetic particles, when acting on the magnetism warming layer
A physical features variation when, a plurality of cells are able to be attached on the cell attaching layer or go to attach by the cell adsorption layer.
2. cell attachment device according to claim 1, it is characterised in that: the cell attaching layer is polyethyleneimine
(polyethylenimine), which is poly-N-isopropyl acrylamide (poly (N-
Isopropylacrylamide))。
3. cell attachment device according to claim 1, it is characterised in that: the object features are temperature, magnetic field or both
Combination.
4. cell attachment device according to claim 1, it is characterised in that: also have a magnetic texure, the magnetic texure
With a patterning, which is set between the cell attaching layer and the substrate or in being set to outside the substrate
Side.
5. cell attachment device according to claim 4, it is characterised in that: the patterning is with a plurality of concentric rows
The geometrical pattern structure of column.
6. a kind of manufacturing method of cell attachment device, it is characterized in that including the following steps:
A plurality of magnetic warming macromolecules are manufactured, each magnetic warming macromolecule has an one first warming macromolecule, and every 1 the
One warming macromolecule has a plurality of magnetic particles；
One substrate is provided, and forms a cell adsorption layer on the substrate；And
Using a plurality of magnetic warming macromolecules in forming a magnetic warming layer on the cell adsorption layer, when acting on the magnetism
When the physical features variation of warming layer, a plurality of cells are able to be attached on the cell attaching layer or are gone by the cell adsorption layer
It attaches.
7. the manufacturing method of cell attachment device according to claim 6, it is characterised in that: formed the cell adsorption layer it
Before, further include having to form a laminated magnetic film on the substrate, there is a patterning, and forming the magnetism warming layer
When, a plurality of specific position arrangements that there is magnetic warming macromolecule to correspond in the patterning.
8. the manufacturing method of cell attachment device according to claim 6, it is characterised in that: further include having in the substrate
Side has the permanent magnet of a specific pattern, has a patterning, and when forming the magnetism warming layer, this is a plurality of
Warming macromolecule with magnetic particle is arranged along the profile of the patterning.
9. the manufacturing method of cell attachment device according to claim 7 or 8, it is characterised in that: the object features are temperature
The combination of degree and magnetic field, the cell attachment device a plurality of magnetic warming macromolecules in first temperature are contraction state,
So that cell is adsorbed on the cell adsorption layer, in a second temperature, by the effect of the changes of magnetic field, so that only right
Should the warming macromolecule in patterning maintain contraction state, allow cell mutually should patterning specific region
On by the cell adsorption layer adsorb.
10. the manufacturing method of cell attachment device according to claim 7 or 8, it is characterised in that: further include being formed again
Several second warming macromolecules, and when forming the magnetism warming layer, the magnetism warming macromolecule and second temperature will be contained
Feel macromolecule mixed solution, be coated on the cell adsorption layer, so that there is the magnetism temperature on the region of the corresponding patterning
Sense macromolecule is formed on the cell adsorption layer, and on other regions, there is the second warming macromolecule to be formed in the cell
On adsorption layer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810167026.3A CN110205243A (en) | 2018-02-28 | 2018-02-28 | The manufacturing method of cell attachment device and the cell attachment device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810167026.3A CN110205243A (en) | 2018-02-28 | 2018-02-28 | The manufacturing method of cell attachment device and the cell attachment device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110205243A true CN110205243A (en) | 2019-09-06 |
Family
ID=67778620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810167026.3A Pending CN110205243A (en) | 2018-02-28 | 2018-02-28 | The manufacturing method of cell attachment device and the cell attachment device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110205243A (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0387975A1 (en) * | 1989-03-16 | 1990-09-19 | W.R. Grace & Co.-Conn. | Cell culture substrate cell sheet, cell cluster and preparations thereof |
| TW591106B (en) * | 2000-10-06 | 2004-06-11 | Ind Tech Res Inst | Carrier for cell attachment or fixation and its process for preparation |
| TW200724623A (en) * | 2005-12-30 | 2007-07-01 | Ind Tech Res Inst | Thermosensitive nanostructure for hyperthermia treatment |
| JP2008232716A (en) * | 2007-03-19 | 2008-10-02 | Canon Inc | Magnetic particle and manufacturing method therefor |
| CN101701207A (en) * | 2009-11-06 | 2010-05-05 | 华南师范大学 | Preparation method for temperature sensing cornea epithelial cell culture material and application |
| JP2011223944A (en) * | 2010-04-21 | 2011-11-10 | Dainippon Printing Co Ltd | Transfer substrate for micro-patterned vascular endothelial cell |
| WO2014013238A1 (en) * | 2012-07-16 | 2014-01-23 | The University Of Nottingham | Scaffold |
| US20140127774A1 (en) * | 2008-04-09 | 2014-05-08 | Massachusetts Institute Of Technology | Synthetically Functionalized Living Cells |
| WO2014143871A2 (en) * | 2013-03-15 | 2014-09-18 | Garnet Biotherapeutics, Inc. | Thermoresponsive polymer applications for adherent cell culture and recovery |
| US20150093823A1 (en) * | 2013-10-02 | 2015-04-02 | President And Fellows Of Harvard College | Environmentally Responsive Microstructured Hybrid Actuator Assemblies For Use in Mechanical Stimulation of Cells |
| WO2016187614A1 (en) * | 2015-05-21 | 2016-11-24 | Univerity of South Florida | Cell culture substrate for rapid release and re-plating |
| WO2017046629A1 (en) * | 2015-09-15 | 2017-03-23 | Stellenbosch University | A method of culturing cells |
| WO2017062600A1 (en) * | 2015-10-06 | 2017-04-13 | University Of South Florida | High-throughput platform for bioprinting tissue modules |
| TW201726909A (en) * | 2016-01-29 | 2017-08-01 | 國立中央大學 | Cell culture device, method of forming the same, and method of partially detaching stem cells from the same |
-
2018
- 2018-02-28 CN CN201810167026.3A patent/CN110205243A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0387975A1 (en) * | 1989-03-16 | 1990-09-19 | W.R. Grace & Co.-Conn. | Cell culture substrate cell sheet, cell cluster and preparations thereof |
| TW591106B (en) * | 2000-10-06 | 2004-06-11 | Ind Tech Res Inst | Carrier for cell attachment or fixation and its process for preparation |
| TW200724623A (en) * | 2005-12-30 | 2007-07-01 | Ind Tech Res Inst | Thermosensitive nanostructure for hyperthermia treatment |
| JP2008232716A (en) * | 2007-03-19 | 2008-10-02 | Canon Inc | Magnetic particle and manufacturing method therefor |
| US20140127774A1 (en) * | 2008-04-09 | 2014-05-08 | Massachusetts Institute Of Technology | Synthetically Functionalized Living Cells |
| CN101701207A (en) * | 2009-11-06 | 2010-05-05 | 华南师范大学 | Preparation method for temperature sensing cornea epithelial cell culture material and application |
| JP2011223944A (en) * | 2010-04-21 | 2011-11-10 | Dainippon Printing Co Ltd | Transfer substrate for micro-patterned vascular endothelial cell |
| WO2014013238A1 (en) * | 2012-07-16 | 2014-01-23 | The University Of Nottingham | Scaffold |
| WO2014143871A2 (en) * | 2013-03-15 | 2014-09-18 | Garnet Biotherapeutics, Inc. | Thermoresponsive polymer applications for adherent cell culture and recovery |
| US20150093823A1 (en) * | 2013-10-02 | 2015-04-02 | President And Fellows Of Harvard College | Environmentally Responsive Microstructured Hybrid Actuator Assemblies For Use in Mechanical Stimulation of Cells |
| WO2016187614A1 (en) * | 2015-05-21 | 2016-11-24 | Univerity of South Florida | Cell culture substrate for rapid release and re-plating |
| WO2017046629A1 (en) * | 2015-09-15 | 2017-03-23 | Stellenbosch University | A method of culturing cells |
| WO2017062600A1 (en) * | 2015-10-06 | 2017-04-13 | University Of South Florida | High-throughput platform for bioprinting tissue modules |
| TW201726909A (en) * | 2016-01-29 | 2017-08-01 | 國立中央大學 | Cell culture device, method of forming the same, and method of partially detaching stem cells from the same |
Non-Patent Citations (2)
| Title |
|---|
| KENICHI NAGASE等: "Poly(N-isopropylacrylamide)-based thermoresponsive surfaces provide new types of biomedical applications", 《BIOMATERIALS》 * |
| ZHONGLAN TANG等: "Temperature-Responsive Polymer Modified Surface for Cell Sheet Engineering", 《POLYMERS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Medina‐Sánchez et al. | Swimming microrobots: Soft, reconfigurable, and smart | |
| Ganguly et al. | 3D printed magnetic polymer composite hydrogels for hyperthermia and magnetic field driven structural manipulation | |
| Tu et al. | Motion manipulation of micro‐and nanomotors | |
| Gong et al. | Multiscale soft–hard interface design for flexible hybrid electronics | |
| Yan et al. | Controllable location of inorganic nanoparticles on block copolymer self-assembled scaffolds by tailoring the entropy and enthalpy contributions | |
| Nunes et al. | Multifunctional shape and size specific magneto-polymer composite particles | |
| Shin et al. | Design of an electro-stimulated hydrogel actuator system with fast flexible folding deformation under a low electric field | |
| Kavaldzhiev et al. | Biocompatible 3D printed magnetic micro needles | |
| Cui et al. | Intelligent polymer‐based bioinspired actuators: from monofunction to multifunction | |
| CN104039951B (en) | For guiding the device of cell migration and implementing the bootstrap technique of this device | |
| Skorb et al. | “Smart” surface capsules for delivery devices | |
| Hu et al. | Scaffolds with anisotropic structure for neural tissue engineering | |
| Taylor et al. | Biomimetic and biologically compliant soft architectures via 3D and 4D assembly methods: a perspective | |
| Tan et al. | Micro-and nano-assembly of composite particles by electrostatic adsorption | |
| Zhang et al. | Femtosecond laser nanofabrication of hydrogel biomaterial | |
| De France et al. | 2.5 D hierarchical structuring of nanocomposite hydrogel films containing cellulose nanocrystals | |
| Lee et al. | Electrolyte solution-assisted electrospray deposition for direct coating and patterning of polymeric nanoparticles on non-conductive surfaces | |
| Lee | 3D nanoprinting technologies for tissue engineering applications | |
| Zhu et al. | Recent advances in patterning natural polymers: from nanofabrication techniques to applications | |
| Lin et al. | Spontaneously micropatterned silk/gelatin scaffolds with topographical, biological, and electrical stimuli for neuronal regulation | |
| Park et al. | Fabrication and applications of stimuli‐responsive micro/nanopillar arrays | |
| CN105019043A (en) | Electrostatic spinning patterning collection method and electrostatic spinning apparatus | |
| SwaminathanáIyer | Systematic assessment of surface functionality on nanoscale patterns for topographic contact guidance of cells | |
| Yang et al. | Recent advance in cell patterning techniques: Approaches, applications and future prospects | |
| Sanjuan-Alberte et al. | Remotely controlled in situ growth of silver microwires forming bioelectronic interfaces |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190906 |