CN111635496B - Temperature-sensitive cell culture plate and preparation method thereof - Google Patents
Temperature-sensitive cell culture plate and preparation method thereof Download PDFInfo
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- 238000004113 cell culture Methods 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 229920000642 polymer Polymers 0.000 claims abstract description 41
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 25
- 239000010703 silicon Substances 0.000 claims abstract description 25
- YHHSONZFOIEMCP-UHFFFAOYSA-N 2-(trimethylazaniumyl)ethyl hydrogen phosphate Chemical compound C[N+](C)(C)CCOP(O)([O-])=O YHHSONZFOIEMCP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229950004354 phosphorylcholine Drugs 0.000 claims abstract description 23
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 238000012650 click reaction Methods 0.000 claims abstract description 4
- 239000000178 monomer Substances 0.000 claims abstract description 4
- 150000003376 silicon Chemical class 0.000 claims abstract description 4
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 90
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 75
- 238000006243 chemical reaction Methods 0.000 claims description 56
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 54
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 40
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 36
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 24
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- VHRYZQNGTZXDNX-UHFFFAOYSA-N methacryloyl chloride Chemical compound CC(=C)C(Cl)=O VHRYZQNGTZXDNX-UHFFFAOYSA-N 0.000 claims description 18
- 239000002244 precipitate Substances 0.000 claims description 17
- BCUQTZFLMHEKRN-UHFFFAOYSA-N CC(CCCCCCCCCC[Si](Cl)(Cl)Cl)OC(C(C)(C)Br)=O Chemical compound CC(CCCCCCCCCC[Si](Cl)(Cl)Cl)OC(C(C)(C)Br)=O BCUQTZFLMHEKRN-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 11
- PMNIHDBMMDOUPD-UHFFFAOYSA-N 2-[2-(2-azidoethoxy)ethoxy]ethanol Chemical compound OCCOCCOCCN=[N+]=[N-] PMNIHDBMMDOUPD-UHFFFAOYSA-N 0.000 claims description 9
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 9
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 9
- UWHCKJMYHZGTIT-UHFFFAOYSA-N Tetraethylene glycol, Natural products OCCOCCOCCOCCO UWHCKJMYHZGTIT-UHFFFAOYSA-N 0.000 claims description 8
- 238000002390 rotary evaporation Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 7
- 229960005055 sodium ascorbate Drugs 0.000 claims description 7
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- DWFKOMDBEKIATP-UHFFFAOYSA-N n'-[2-[2-(dimethylamino)ethyl-methylamino]ethyl]-n,n,n'-trimethylethane-1,2-diamine Chemical compound CN(C)CCN(C)CCN(C)CCN(C)C DWFKOMDBEKIATP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 230000001680 brushing effect Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 4
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 claims description 3
- 239000003999 initiator Substances 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 101710141544 Allatotropin-related peptide Proteins 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 31
- 230000021164 cell adhesion Effects 0.000 abstract description 16
- 238000003795 desorption Methods 0.000 abstract description 9
- 210000000170 cell membrane Anatomy 0.000 abstract description 6
- 230000010261 cell growth Effects 0.000 abstract description 4
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- 125000001165 hydrophobic group Chemical group 0.000 abstract description 2
- 230000000379 polymerizing effect Effects 0.000 abstract description 2
- 239000013256 coordination polymer Substances 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 description 12
- 238000001179 sorption measurement Methods 0.000 description 11
- 235000019441 ethanol Nutrition 0.000 description 8
- -1 propionyloxy Chemical group 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
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- 102000004169 proteins and genes Human genes 0.000 description 4
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- 238000002834 transmittance Methods 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 238000010907 mechanical stirring Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 238000012323 Endoscopic submucosal dissection Methods 0.000 description 1
- 206010030201 Oesophageal ulcer Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
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- 125000003636 chemical group Chemical group 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
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- 239000011557 critical solution Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 208000028299 esophageal disease Diseases 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
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Abstract
The invention discloses a temperature-sensitive cell culture plate, which takes a modified silicon wafer as a substrate, and the surface of the silicon wafer is connected with a temperature-sensitive polymer brush p (EO) containing an azide end group side chain 4 MA, phosphocholine modified by click reaction on p (EO) 4 The MA side chain terminus; wherein, p (EO) 4 MA with (EO) 4 MA is obtained by polymerizing monomers. The invention also discloses a preparation method of the temperature-sensitive cell culture plate, which comprises the following steps: preparation (EO) 4 MA preparation of temperature sensitive Polymer Brush p (EO) 4 MA surface, preparing temperature sensitive cell culture plate modified by choline phosphate, the technical scheme disclosed by the invention does not utilize the change of hydrophilic and hydrophobic groups of temperature sensitive polymer to control desorption, but utilizes p (EO) 4 The heat shrinkage of the MA temperature-sensitive polymer brush actively reduces the accessibility of CP in the polymer structure to cell membranes, so that cell sheets attached to the surface of the adhesive plate are automatically separated, a relatively complete cell sheet layer is obtained, the cell adhesion can be remarkably accelerated, the cell growth is accelerated, and the cell adhesion strength of a cell culture plate can be flexibly regulated and controlled.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a temperature-sensitive cell culture plate and a preparation method thereof.
Background
Among the numerous tissue engineering studies, cell sheet engineering is considered to be one of the most effective tissue reconstruction techniques. The cell sheet is a monolayer similar to a tissue, becomes a new tool of regenerative medicine, is applied to human clinical research, such as cell transplantation, tissue repair and the like, and particularly can be used for esophageal ulcer treatment after corneal reconstruction and endoscopic submucosal dissection.
In the traditional technology, after cells are cultured on a silicon plate, the cells are separated by adopting trypsin or a mechanical stirring mode, so that the cells and extracellular matrix are damaged, and the trypsin digests intercellular conjugates to separate the cells from each other, so that the integrity of cell sheets is damaged, and the biological skills are influenced.
At present, in order to avoid damage to cells and extracellular matrix, obtaining a relatively intact cell sheet is generally performed by culturing the cell sheet on a temperature-sensitive cell culture plate. The main method for preparing temperature sensitive cell culture plates is to graft or coat a temperature sensitive polymer onto the culture plate. When the cell sheet needs to be separated, the temperature is reduced to be below the critical solution temperature (LCST), at the moment, temperature-sensitive polymer molecules are in an extension state, some structures in the temperature-sensitive polymer molecules can form hydrogen bonds and the like with water molecules, the hydrophilicity is shown, the adhesion strength to cells is reduced, and therefore the cell sheet is released, and when the temperature is higher than the LCST, the molecular chains of the temperature-sensitive polymer molecules are in a curling state, the hydrogen bonds can be generated among the molecular chains, the hydrophobicity is shown, and the cell adhesion is facilitated.
At present, in order to improve the performance of cell adhesion and accelerate cell growth, the most important way is to add adhesion proteins from other individuals, and such external addition can cause immunogenic reaction of cell sheets in the using process, and the properties of proteins mediating cell adhesion and protein layers adsorbed on the surface of materials are changed continuously, so that it is difficult to perform system analysis and precise regulation of cell-to-material surface adhesion behaviors through cell adhesion proteins, and the adhesion period is long.
Through retrieval, an invention patent with the application number of CN200610015326.7 discloses an intelligent membrane for cell culture and desorption and a preparation method thereof. According to the technical scheme disclosed by the invention, acrylic Acid (AAC) and Chitosan (CS) are subjected to condensation reaction to obtain a CS-AAc macromonomer containing C = C with reaction activity, and then the CS-AAc macromonomer and N-isopropylacrylamide are copolymerized to prepare the temperature-sensitive intelligent membrane, wherein a branched chain of a copolymerized molecular chain has a-CONH bond, so that cell adhesion is promoted. However, although the invention solves the problem of cell adhesion to a certain extent by introducing the-CONH bond to the branched chain of the copolymer, and has better biocompatibility, the original technical mode is not broken through in the desorption process, and the hydrophilicity of the copolymer is improved by reducing the temperature below LCST, so that the cell sheet is desorbed.
An invention patent with the application number of CN201210228270.9 discloses a cell sheet intelligent desorption hydrogel and application thereof. The technical scheme disclosed by the invention can promote cell adhesion, avoid damage to cell activity, functions and the like caused by a traditional enzymolysis method, and reduce complications related to traditional tissue engineering. In essence, the cell culture substrate is still subjected to desorption and adsorption through the change of the hydrophilic and hydrophobic properties of the cell culture substrate.
Generally, the current technical scheme is that heating or cooling is adopted to enable the temperature to be higher than or lower than LCST, so that the temperature-sensitive polymer is hydrophobic and hydrophilic, and then the cells are adsorbed and desorbed. A new temperature-sensitive cell culture plate is needed, and the adsorption and desorption of cells are realized by a new way.
Disclosure of Invention
In order to solve the problems in the background art and achieve the purpose of providing a novel temperature-sensitive cell culture plate, the invention provides a temperature-sensitive cell culture plate and a preparation method thereof.
A temperature-sensitive cell culture plate is prepared by using modified silicon wafer as substrate, and connecting temperature-sensitive polymer brush p (EO) containing azide end group side chain on surface of silicon wafer 4 MA, phosphocholine modified by click reaction on p (EO) 4 The MA side chain terminus;
wherein, p (EO) 4 MA with (EO) 4 MA is obtained by polymerizing monomers.
Further, the surface of the silicon slice is firstly modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane, and the (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane is an initiator of ATRP reaction to initiate (EO) 4 And (3) polymerization of MA.
Further, said (EO) 4 MA has the structural formula
A preparation method of a temperature-sensitive cell culture plate comprises the following steps:
a. preparation (EO) 4 MA; firstly, carrying out a first reaction, sequentially adding tetraethylene glycol, triethylamine and tetrahydrofuran into a dry single-neck flask, then dissolving p-toluenesulfonyl chloride in tetrahydrofuran and dropwise adding the tetrahydrofuran into a reaction system, removing generated precipitate after room temperature reaction, extracting by using dichloromethane as an extracting agent, adding sodium azide and dimethylformamide into a solution after rotary evaporation to remove dichloromethane, removing the precipitate after reaction, extracting by using dichloromethane, and MgSO (MgSO) 4 Drying to obtain 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol, carrying out a second reaction, sequentially adding 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol, tetrahydrofuran and triethylamine into a flask, dropwise adding methacryloyl chloride into a reaction system, reacting at room temperature, removing precipitate, extracting with dichloromethane, and carrying out MgSO (MgSO) as a solvent, wherein the solvent is a solvent, and the solvent is a solvent 4 Drying to obtain (EO) 4 MA；
b. Preparation of thermo-sensitive Polymer Brush p (EO) 4 A MA surface; placing the silicon slice modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane in absolute ethyl alcohol, and adding (EO) 4 Dissolving MA, 1,4,7, 10-hexamethyl triethylene tetramine and CuBr in ethanol, taking out the silicon wafer after reaction in an oxygen-free environment, washing the surface of the silicon wafer with anhydrous toluene and anhydrous methanol to obtain the thermo-sensitive polymer brush p (EO) 4 A MA surface;
c. preparing a temperature-sensitive cell culture plate modified by the choline phosphate; dissolving phosphocholine, copper sulfate pentahydrate and sodium ascorbate with methanol aqueous solution to obtain mixed solution, and brushing the temperature sensitive polymer obtained in step b with p (EO) 4 And (3) soaking the MA surface into the mixed solution for reaction, removing the reaction solution, and washing the surface with methanol to obtain the phosphocholine-modified temperature-sensitive cell culture plate.
Further, in the first reaction of the step a, 1.0mol of tetraethylene glycol, 0.25mol to 0.35mol of triethylamine and 50ml of tetrahydrofuran are sequentially added into a dry single-neck flask, then 0.2mol to 0.5mol of p-toluenesulfonyl chloride is dissolved in 50ml of tetrahydrofuran and is dropwise added into a reaction system to react for 12 hours at room temperature; after dichloromethane is removed by rotary evaporation, 0.25mol to 0.28mol of sodium azide and 120ml of dimethylformamide are added into the solution, and the mixture reacts for 24 hours at the temperature of between 70 and 80 ℃; in the second reaction in the step a, after dropwise adding methacryloyl chloride into a reaction system, reacting for 12 hours at room temperature;
in step b, the silicon wafer modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane is placed in 10ml to 15ml of absolute ethanol, and 1.74mmol to 3.48mmol (EO) 4 MA, 1,4,7, 10-hexamethyl triethylene tetramine with the concentration of 0.028mmol-0.042mmol and CuBr with the concentration of 0.014mmol-0.029mmol are dissolved in ethanol, the ethanol is used as a solvent, the influence on the reaction is small, and the reaction is kept at the temperature of 45 ℃ to 50 ℃ in an oxygen-free environment for 2h to 22h;
in step c, 50mg to 300mg of phosphocholine, 2mg to 6mg of copper sulfate pentahydrate and 5mg to 15mg of sodium ascorbate are dissolved with 10ml to 15ml of an aqueous solution of methanol to obtain a mixed solution, and then p (EO) 4 The MA surface was immersed in the above mixed solution and reacted at 40 ℃ for 24 hours.
Further, preparation (EO) 4 In the second reaction of MA, 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol, triethylamine, methacryloyl chloride =1 (1.5-3) and 1.2, in molar ratio.
And further, the dripping time of the p-toluenesulfonyl chloride in the step a is 2h-3h, an ice bath is used in the dripping process, after the dripping is finished, the ice bath is removed, and the reaction is carried out at normal temperature.
Further, the dropping time of the methacryloyl chloride in the step a is 2-3 h, an ice bath is used in the dropping process, after the dropping is finished, the ice bath is removed, and the reaction is carried out at normal temperature.
Further, in the methanol aqueous solution in the step c, the volume ratio of methanol to water is (4-5): 1.
In the technical scheme, the knot of (11- (2-bromine-2-methyl) propionyloxy) dodecyl trichlorosilaneStructure is as
After being modified with (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane, (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane reacts with hydroxyl groups on the surface of a silicon plate to form a siloxane bond, and then, (EO) 4 MA is polymerized to p (EO) by surface-initiated atom transfer radical method (SI-ATRP) 4 MA, result form e.g
Wherein (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane is used as a surface initiator, and finally, choline Phosphate (CP) reacts with azido through click reaction to form a compound in a form such as
Wherein a + b = n.
In the technical scheme of the invention, the phosphocholine (CP) is a zwitterionic monomer, and compared with polar head group-Phosphocholine (PC) of phospholipid bilayer on the cell membrane skeleton structure, the CP has the same chemical group with the polar head group-Phosphocholine (PC), but in contrast, each CP molecule can form two groups with the PC
The pairing completes the electrostatic adsorption, and since the PC groups are widely distributed outside the cell membrane, the CP is electrostatically bound to the cell membrane, thereby forming a general biofilm "adhesive". The CP functional molecules are modified at the tail end of the polymer, cells and the polymer are rapidly combined at multiple points through the interaction of CP-PC, the rapid adhesion of the cells is realized, and in addition, the surface material modified by the CP has very good biological pollution resistance while promoting the adhesion of the cells. CP has excellent biocompatibility as a zwitterion, which also makes it an ideal choice for constructing cell-adhesive biomedical materials.
Because of using p (EO) with CP end modified on side chain 4 MA, cellular uptake,The desorption process is different from the adsorption and desorption processes on the traditional thermo-sensitive polymer molecular brush, and the tail end of the side chain is modified with p (EO) of CP 4 When the temperature is lower than LCST, the MA thermo-sensitive polymer brush adheres cells rapidly by means of CP-PC interaction, thereby accelerating cell growth, and has good universality because the outer surface of the cell membrane of the nucleated cell contains PC.
Compared with the prior art, the invention has the beneficial effects that:
1. instead of using traditional mechanical stirring, trypsin or the change of hydrophilic and hydrophobic groups of temperature-sensitive polymer to control desorption, p (EO) is used 4 The heat shrinkage of the MA temperature-sensitive polymer brush actively reduces the accessibility of CP in the polymer structure to cell membranes, so that cell sheets attached to the surface of the sticky plate are automatically separated, and a relatively complete cell sheet layer is obtained;
2. use of phosphocholine, i.e., CP, to modify p (EO) 4 The MA side chain end can obviously accelerate cell adhesion, thereby accelerating cell growth;
3. the adhesion strength of the cell culture plate to the cells can be measured by p (EO) 4 The molecular weight, grafting ratio and CP usage of the MA polymer brush are regulated and controlled.
Drawings
FIG. 1: a schematic representation of the functioning of a cell culture plate that regulates initial cell adhesion and isolation.
FIG. 2: a graph showing the amount of adsorbed red blood cells of the cell culture plate prepared in example 2 and the blank silicon wafer as a function of time.
FIG. 3: example 2 ultraviolet transmittance profile of PBS solution (1 mg/mL) of the isolated polymer segment on the surface of the cell culture plate prepared in example 2.
FIG. 4: when CHO cells were cultured for two days using the cell culture plate prepared in example 3, the temperature was raised from 36 ℃ to 39 ℃ to induce cell detachment.
FIG. 5: graph comparing the number of adhesion of the red blood cells in 10min for the cell culture plates prepared in example 2, example 3 and example 4 and the blank silicon wafer.
Detailed Description
The present invention is illustrated but not limited by the above description, and all technical solutions obtained in the light of the idea of the present invention should be included in the protection scope of the present patent. In the following examples: (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane is available from Shanghai Michell chemical technology, inc., sodium azide and copper sulfate pentahydrate are available from pharmaceutical group, chemicals, inc., ascorbic acid sodium salt is available from Shanghai Mingsheng Biochemical reagents, inc., and other reagents are available from Aladdin reagents (Shanghai) Inc.
Example 1
(1) Sequentially adding 1.0mol of tetraethylene glycol, 0.3mol of Triethylamine (TEA) and 50mL of Tetrahydrofuran (THF) into a dry single-neck flask, dissolving 0.5mol of p-toluenesulfonyl chloride in 50mL of tetrahydrofuran, dropwise adding into a reaction system for 2h, using an ice bath during dropwise adding, removing the ice bath after dropwise adding is finished, and reacting at room temperature for 12h; the salt precipitate formed by the reaction was removed and extracted with Dichloromethane (DCM). After removing DCM by rotary evaporation, 0.28mol of sodium azide and 120mL of Dimethylformamide (DMF) are added into the solution, and the mixture is reacted for 24h at 70-80 ℃; removing salt precipitate, DCM extracting, mgSO 4 Drying, purifying to obtain 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol (A (EO) 4 ). Adding the purified A (EO) into a single-neck flask in sequence 4 And THF and TEA, dropwise adding methacryloyl chloride into the reaction system for 2h, using an ice bath during the dropwise adding process, and removing the ice bath after the dropwise adding process is finished, wherein (EO) 4 N 3 Reacting TEA and methacryloyl chloride at room temperature for 12h, wherein the molar ratio of TEA to methacryloyl chloride is 1; removing salt precipitate, DCM extracting, mgSO 4 Drying and purifying to obtain (EO) 4 MA。
(2) The (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane-modified silicon wafer was placed in 10mL of a chamber without any solventIn aqueous ethanol, and adding the (EO) 4 MA1.74mmol, 1,4,7, 10-hexamethyl triethylene tetramine (HMTETA) 0.028mmol and CuBr0.014mmol are dissolved in the ethanol and reacted for 2h at 45-50 ℃ in an oxygen-free environment. After the reaction, the silicon wafer was taken out and the surface was rinsed with anhydrous toluene and anhydrous methanol to obtain p (EO) 4 And (3) brushing the surface with MA temperature-sensitive polymer.
(3) Dissolving 50mg of phosphocholine, 2mg of copper sulfate pentahydrate and 5mg of sodium ascorbate in 10mL of aqueous methanol solution at a volume ratio of methanol to water of 4 4 Soaking the surface of the MA polymer brush into the mixed solution, and reacting for 24 hours at 40 ℃; removing the reaction solution, and washing the surface with methanol to obtain the temperature-sensitive cell culture plate modified by the choline phosphate.
The principle of cell adhesion and detachment is shown in FIG. 1.
Example 2
(1) Sequentially adding 1.0mol of tetraethylene glycol, 0.35mol of Triethylamine (TEA) and 50mL of Tetrahydrofuran (THF) into a dry single-neck flask, then dissolving 0.3mol of p-toluenesulfonyl chloride in 50mL of tetrahydrofuran and dropwise adding into a reaction system for 3h, using an ice bath during dropwise adding, removing the ice bath after dropwise adding is finished, and reacting at room temperature for 12h; the salt precipitate formed by the reaction was removed and extracted with Dichloromethane (DCM). After removing DCM by rotary evaporation, 0.28mol of sodium azide and 120mL of Dimethylformamide (DMF) are added into the solution, and the mixture is reacted for 24 hours at 70-80 ℃; removing salt precipitate, DCM extracting, mgSO 4 Drying, purifying to obtain 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol (A (EO) 4 ). Adding the purified A (EO) into a single-neck flask in sequence 4 Adding methacryloyl chloride into the reaction system dropwise for 2 hours, using an ice bath during the dropwise addition, and removing the ice bath after the dropwise addition is finished, wherein (EO) 4 N 3 Reacting TEA and methacryloyl chloride at room temperature for 12h, wherein the molar ratio of TEA to methacryloyl chloride is 1; removing salt precipitate, DCM extracting, mgSO 4 Drying and purifying to obtain (EO) 4 MA。
(2) Modifying (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilaneThe treated silicon wafer was placed in 10mL of absolute ethanol, and the (EO) solution was added 4 MA2.9mmol, 1,4,7, 10-Hexamethyltriethylenetetramine (HMTETA) 0.035mmol and CuBr0.022mmol are dissolved in the ethanol and reacted for 14h at 45-50 ℃ in an oxygen-free environment. After the reaction, the silicon wafer was taken out and the surface was rinsed with anhydrous toluene and anhydrous methanol to obtain p (EO) 4 And (3) brushing the surface with MA temperature-sensitive polymer.
(3) Dissolve 100mg of phosphocholine, 3mg of copper sulfate pentahydrate and 7.5mg of sodium ascorbate salt with 10mL of aqueous methanol solution at a methanol to water volume ratio of 5 4 Immersing the surface of the MA polymer brush into the mixed solution, and reacting for 24 hours at 40 ℃; removing the reaction solution, and washing the surface with methanol to obtain the temperature-sensitive cell culture plate modified by the choline phosphate.
The principle of cell adhesion and detachment is shown in FIG. 1. As shown in FIG. 2, the temperature-sensitive cell culture plate obtained in this example had higher cell adhesion efficiency than the blank silicon plate. FIG. 3 shows a graph of UV transmittance of PBS solution (1 mg/mL) of polymer segments separated from the surface of the temperature-sensitive cell culture plate prepared in this example, wherein the two curves show the heating from low temperature to high temperature and the cooling from high temperature to low temperature, respectively, and the graph shows that the polymer brush is p (EO) modified with CP when the polymer brush is in an extended state as a whole below LCST 4 The MA polymer brush exhibits good ultraviolet transmittance properties, and when the temperature is above the LCST, the polymer brush exhibits an irregular shrinkage state, so that the ultraviolet transmittance is reduced.
Example 3
(1) Sequentially adding 1.0mol of tetraethylene glycol, 0.25mol of Triethylamine (TEA) and 50mL of Tetrahydrofuran (THF) into a dry single-neck flask, then dissolving 0.2mol of p-toluenesulfonyl chloride in 50mL of tetrahydrofuran and dropwise adding into a reaction system for 2h, using an ice bath during dropwise adding, removing the ice bath after dropwise adding is finished, and reacting at room temperature for 12h; the salt precipitate formed by the reaction was removed and extracted with Dichloromethane (DCM). After removing DCM by rotary evaporation, 0.25mol of sodium azide and 120mL of Dimethylformamide (DMF) are added into the solution, and the mixture is reacted for 24 hours at 70-80 ℃; removing salt depositsExtracting with DCM, mgSO 4 Drying, purifying to obtain 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol (A (EO) 4 ). Adding the purified A (EO) into a single-neck flask in sequence 4 THF and TEA, adding methacryloyl chloride dropwise into the reaction system for 2h, using ice bath during the dropwise addition, and removing the ice bath after the dropwise addition is finished, wherein (EO) 4 N 3 The molar ratio of TEA to methacryloyl chloride is 1.5; removing salt precipitate, DCM extracting, mgSO 4 Drying and purifying to obtain (EO) 4 MA。
(2) (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane modified silicon wafer was placed in 10mL of absolute ethanol, and the (EO) 4 MA2.32mmol, 1,4,7, 10-hexamethyl triethylene tetramine (HMTETA) 0.028mmol and CuBr0.014mmol are dissolved in the ethanol and reacted for 8h at 45-50 ℃ in an oxygen-free environment. After the reaction, the silicon wafer was taken out and the surface was rinsed with anhydrous toluene and anhydrous methanol to obtain p (EO) 4 MA temperature sensitive polymer brush surface.
(3) Dissolving 200mg of phosphocholine, 5mg of copper sulfate pentahydrate and 12.5mg of sodium ascorbate in 15mL of aqueous methanol solution, wherein the volume ratio of methanol to water in the aqueous methanol solution is 5 4 Immersing the surface of the MA polymer brush into the mixed solution, and reacting for 24 hours at 40 ℃; removing the reaction solution, and washing the surface with methanol to obtain the temperature-sensitive cell culture plate modified by the choline phosphate.
The principle of cell adhesion and detachment is shown in FIG. 1.
Example 4
(1) Sequentially adding 1.0mol of tetraethylene glycol, 0.25mol of Triethylamine (TEA) and 50mL of Tetrahydrofuran (THF) into a dry single-neck flask, dissolving 0.3mol of p-toluenesulfonyl chloride in 50mL of tetrahydrofuran, dropwise adding into a reaction system for 2h, using an ice bath during dropwise adding, removing the ice bath after dropwise adding is finished, and reacting at room temperature for 12h; the salt precipitate formed by the reaction was removed and extracted with Dichloromethane (DCM). After removal of DCM by rotary evaporation, 0.25mol of sodium azide and 120mL of Dimethylformamide (DMF) are added to the solution and the reaction is carried out at 70-80 deg.CThe reaction time is 24 hours; removing salt precipitate, extracting with DCM, and MgSO 4 Drying, purifying to obtain 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol (A (EO) 4 ). Adding the purified A (EO) into a single-neck flask in sequence 4 THF and TEA, adding methacryloyl chloride dropwise into the reaction system for 2h, using ice bath during the dropwise addition, and removing the ice bath after the dropwise addition is finished, wherein (EO) 4 N 3 The molar ratio of TEA to methacryloyl chloride is 1.5; removing salt precipitate, DCM extracting, mgSO 4 Drying and purifying to obtain (EO) 4 MA。
(2) (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane modified silicon wafer was placed in 15mL of absolute ethanol, and the (EO) 4 MA3.48mmol, 1,4,7, 10-Hexamethyltriethylenetetramine (HMTETA) 0.042mmol and CuBr0.029mmol were dissolved in the above ethanol and reacted at 45-50 deg.C for 22h in an oxygen-free environment. After the reaction, the silicon wafer was taken out and the surface was rinsed with anhydrous toluene and anhydrous methanol to obtain p (EO) 4 And (3) brushing the surface with MA temperature-sensitive polymer.
(3) 300mg of phosphocholine, 6mg of copper sulfate pentahydrate and 15mg of sodium ascorbate were dissolved in 15mL of aqueous methanol solution at a volume ratio of methanol to water of 4 4 Immersing the surface of the MA polymer brush into the mixed solution, and reacting for 24 hours at 40 ℃; and removing the reaction solution, and washing the surface with methanol to obtain the temperature-sensitive cell culture plate modified by the phosphocholine.
The principle of cell adhesion and detachment is shown in FIG. 1.
As shown in FIG. 5, a graph comparing the number of adhesion of the cell culture plates prepared in examples 2, 3 and 4 with the blank silicon wafer to red blood cells within 10min is shown. It can be seen that the total amount of adhesion to erythrocytes of the cell culture plates prepared in examples 2, 3 and 4 is higher than that of the blank silicon plate within 10min, which indicates that the technical scheme disclosed by the invention has the effect of improving the adsorption efficiency compared with the traditional technical scheme, and in addition, the cell culture plates prepared in examples 2, 3 and 4 have the effect of improving erythrocytes within 10minThe difference in adsorption effect was also seen in that the adsorption efficiency of the cell culture plate prepared in example 4 was the best, the adsorption efficiency of the cell culture plate prepared in example 3 was the second best, the adsorption efficiency of the cell culture plate prepared in example 2 was weaker than that of the cell culture plate prepared in example 3, and in summary, the amount of CP was the main factor causing the difference in adsorption efficiency, and those skilled in the art could also change p (EO) 4 The molecular weight and the grafting rate of the MA polymer brush are used for regulating and controlling the adsorption efficiency.
Claims (7)
1. A temperature-sensitive cell culture plate, comprising: the modified silicon chip is used as a substrate, and the surface of the silicon chip is connected with a thermosensitive polymer brush p (EO) containing an azide end group side chain 4 MA, phosphocholine modified by click reaction on p (EO) 4 MA side chain terminal; wherein, p (EO) 4 MA with (EO) 4 MA is obtained by monomer polymerization; the surface of the silicon slice is firstly modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane, (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane is an initiator of ATRP reaction, and (EO) is initiated 4 Polymerization of MA; said (EO) 4 MA has the structural formula
2. A preparation method of a temperature-sensitive cell culture plate is characterized by comprising the following steps: comprises the following steps:
a. preparation (EO) 4 MA; firstly, carrying out a first reaction, sequentially adding tetraethyleneglycol, triethylamine and tetrahydrofuran into a dry single-neck flask, then dissolving p-toluenesulfonyl chloride in tetrahydrofuran and dropwise adding the tetrahydrofuran into a reaction system, removing generated precipitate after room temperature reaction, extracting by using dichloromethane as an extracting agent, adding sodium azide and dimethylformamide into a solution after removing dichloromethane by rotary evaporation, removing the precipitate after reaction, extracting by using dichloromethane, and carrying out MgSO 4 Drying to obtain 2- (2- (2- (2-stack)N-ethoxy) ethanol, performing a second reaction, sequentially adding 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol, tetrahydrofuran and triethylamine into the flask, dropwise adding methacryloyl chloride into the reaction system, reacting at room temperature, removing precipitate, extracting with dichloromethane, and MgSO 4 Drying to obtain (EO) 4 MA；
b. Preparation of thermo-sensitive Polymer Brush p (EO) 4 A MA surface; the silicon wafer modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyltrichlorosilane is placed in absolute ethanol, and (EO) 4 Dissolving MA, 1,4,7,10, 10-hexamethyl triethylene tetramine and CuBr in ethanol, taking out the silicon wafer after reaction in an oxygen-free environment, washing the surface of the silicon wafer with anhydrous toluene and anhydrous methanol to obtain the thermo-sensitive polymer brush p (EO) 4 A MA surface;
c. preparing a temperature-sensitive cell culture plate modified by the choline phosphate; dissolving phosphocholine, copper sulfate pentahydrate and sodium ascorbate with methanol aqueous solution to obtain mixed solution, and brushing the temperature sensitive polymer obtained in step b with p (EO) 4 And (3) immersing the MA surface into the mixed solution for reaction, removing the reaction solution, and washing the surface with methanol to obtain the temperature-sensitive cell culture plate modified by the choline phosphate.
3. The method for preparing a temperature-sensitive cell culture plate according to claim 2, wherein: in the first reaction of the step a, 1.0mol of tetraethylene glycol, 0.25mol to 0.35mol of triethylamine and 50ml of tetrahydrofuran are sequentially added into a dry single-neck flask, then 0.2mol to 0.5mol of p-toluenesulfonyl chloride is dissolved in 50ml of tetrahydrofuran and is dripped into a reaction system to react for 12 hours at room temperature; in the subsequent process, after dichloromethane is removed by rotary evaporation, 0.25mol to 0.28mol of sodium azide and 120ml of dimethylformamide are added into the solution, and the mixture reacts for 24 hours at the temperature of between 70 and 80 ℃; in the second reaction in the step a, after dropwise adding methacryloyl chloride into a reaction system, reacting for 12 hours at room temperature;
in the step b, the silicon slice modified by (11- (2-bromo-2-methyl) propionyloxy) dodecyl trichlorosilane is placed in 10ml to 15ml of absolute ethanol, and 1.74mmol to 3.48mmol(EO) 4 MA, 1,4,7,10, 10-hexamethyl triethylene tetramine with the mmol of 0.028-0.042 mmol and CuBr with the mmol of 0.014-0.029 mmol are dissolved in ethanol and react for 2-22 h at the temperature of 45-50 ℃ in an oxygen-free environment;
in step c, 50mg to 300mg of phosphocholine, 2mg to 6mg of copper sulfate pentahydrate and 5mg to 15mg of sodium ascorbate are dissolved with 10ml to 15ml of an aqueous solution of methanol to obtain a mixed solution, and then p (EO) 4 The MA surface was immersed in the above mixed solution and reacted at 40 ℃ for 24 hours.
4. The method for preparing a temperature-sensitive cell culture plate according to claim 3, wherein: preparation (EO) 4 In the second reaction of MA, the molar ratio of 2- (2- (2- (2-azidoethoxy) ethoxy) ethanol to triethylamine to methacryloyl chloride is =1: 1.5-3: 1.2.
5. The method for preparing a temperature-sensitive cell culture plate according to claim 3, wherein: and (b) dropwise adding the p-toluenesulfonyl chloride in the step a for 2h-3h, using an ice bath in the dropwise adding process, removing the ice bath after the dropwise adding is finished, and reacting at normal temperature.
6. The method for preparing a temperature-sensitive cell culture plate according to claim 3, wherein: and (b) dropwise adding methacryloyl chloride for 2-3 h in the step a, using an ice bath in the dropwise adding process, removing the ice bath after the dropwise adding is finished, and reacting at normal temperature.
7. The method for preparing a temperature-sensitive cell culture plate according to claim 3, wherein: in the methanol water solution in the step c, the volume ratio of methanol to water is (4-5) to 1.
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