CN105566567A - Temperature-sensitive polymer material with adjustable hardness and preparation method and application thereof - Google Patents

Temperature-sensitive polymer material with adjustable hardness and preparation method and application thereof Download PDF

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CN105566567A
CN105566567A CN201610056059.1A CN201610056059A CN105566567A CN 105566567 A CN105566567 A CN 105566567A CN 201610056059 A CN201610056059 A CN 201610056059A CN 105566567 A CN105566567 A CN 105566567A
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temperature sensitive
cell
hardness
cross
linked
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杜亚楠
樊星亮
朱路
王冰洁
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
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    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/10—Esters
    • C08F222/1006—Esters of polyhydric alcohols or polyhydric phenols
    • C08F222/102—Esters of polyhydric alcohols or polyhydric phenols of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
    • 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

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Abstract

The invention provides a temperature-sensitive polymer material with adjustable hardness and a preparation method and application thereof. The temperature-sensitive polymer material with the adjustable hardness is formed by a temperature-sensitive crosslinking material and a non-temperature-sensitive crosslinking material in a three-dimensional netty crosslinking mode. The polymer can be effectively used for cell culture, good hardness controllability, temperature sensibility and cell expansion supportability are achieved, and downstream application can be conducted by collecting cells through a non-invasive mode of regulating the temperature.

Description

Adjustable temperature sensitive polymer material of hardness and its preparation method and application
Technical field
The present invention relates to technical field of biological materials, particularly, relate to adjustable temperature sensitive polymer material of hardness and its preparation method and application.
Background technology
The importance of biomechanics is paid close attention to by increasing investigator.The medicine that the appearance of biomechanics makes tradition only be directed to gene level disease receives strong challenge, cyto-mechanics, extra-cellular matrix structure and or cell mechanical conductive all may many diseases be played a crucial role, comprise atherosclerosis, fibrosis, asthma, osteoporosis, cardiac failure, cancer, the medical treatment even comprising broad sense is disabled as irritable bowel syndrome.Research shows, when grow different hardness equivalent chemistry on the surface time, have the cell of many types can change they self form and gene expression atlas.Pericellular microenvironment is that cell provides complicated chemical-biological activities structure and physical regulating.In this environment, cell carrys out the hardness of its place microenvironment of active probe identification by perception extracellular matrix or flanking cell, and the mechanical disturbance experienced and environmental stress are delivered in cell, adjusts self epigenetics and expresses.Biomechanical environment in fetal development, organize the selective expression affecting gene in homeostasis and pathology, cell epigenetics difference embody in play an important role.Normal liver Young's modulus is less than 1500 handkerchiefs, and liver astroglia cell can keep quiescent condition under these conditions.The Young's modulus of about 1 kPa can keep lung fibroblast to be in good physiological level.But (as 8-22 kPa) will change and be differentiated to form different phenotypes when Growth of Cells is in harder environment.Research proves, the external mechanical force continued to increase can induce more Extracellular Matrix Gene Expression and apposition, thus causes Fibrotic formation, causes intracorporeal organ system dysfunction.
Polymeric biomaterials can be used as Cell culture invitro matrix, and the hydrogel that wherein bio-mechanical property is adjustable is that the conditioned response of research cell for extracellular matrix in different soft and hard stimulates provides strong instrument.The foundation of external model has vital effect for the mechanism understanding wound healing and disease incidence, indispensable especially especially in the phenotype of cell and the research of Function.But, most biomechanics Research mainly concentrates on the formation how mechanical conditions affects adherent cell activity and their extracellular matrixs, the research of biomechanics regulating cell epigenetics is only confined to cells in situ, cell harvesting to the influence of cell often studied persons ignored.How digestion under the prerequisite of not damaging cells characteristic, re-suspended cell still have very large challenge.
Successful regenerative medicine treatment must consider the natural characteristic of cell, makes cell can excite for the favourable signal path of tissue repair thus incorporate natural weave construction.Homeostasis is the dynamic process maintaining cell normal physiological function, comprising all with the inside and outside environment needed for cells survival.Cell culture invitro collection technique comprises enzymic digestion and physics stripping is the method the most often adopted in passage.The effect that tradition regular growth collection method is separated due to enzymic digestion proteolysis or mechanical scraping and the unicellular or cell mass that obtains can cause cell or cell and extracellular matrix to lose with linking.Because enzymatic digestion greatly can change the environment residing for cell self, such as pancreatin can potent cracking almost all in protein peptide chain near the Methionin of carboxyl terminal or arginine, so cause negative impact to the specific expressed of cell possibly, consider that its difference caused on culture condition (as extracellular matrix fragmentation) more likely changes form and the genetic expression of cell.Research shows that trysinization can destroy cell inherent structure and epigenetics feature, and histone methylated in cell, Acetylation Level obviously declines after pancreatin process.Physical separation methods such as cell scraper can show the surviving rate reducing cell, and research finds, the method that physics scrapes stripping may cause the necrocytosis up to 30%.And can't determine now that whether this dead cell can impact the cell of surviving by the material in release cells.Worth the wait, if the cell cultures of routine can not cause interference to cell oneself expression, whether just can distinguish experimental result is thus test the result really wanting to obtain, instead of because due to culture technique condition institute limit and be affected appearance false positive data.
Thus, still need deeply about the research of cell cultures at present.
Summary of the invention
The present invention is intended to solve one of technical problem in correlation technique at least to a certain extent.For this reason, one object of the present invention be to propose one can be effective to cell cultures, can Noninvasive collecting cell, regulating cell genetic expression and cellular form or maintains stability between cell generation, preservation cell surface receptor and extracellular matrix are from the adjustable temperature sensitive polymer material of the hardness destroyed.
In one aspect of the invention, the invention provides the adjustable temperature sensitive polymer material of a kind of hardness (hydrogel can be called for short).According to embodiments of the invention, this hardness adjustable temperature sensitive polymer material is by temperature sensitive cross-linked material and non-temperature sensitive cross-linked material is three-dimensional netted is cross-linked to form, and wherein, temperature sensitive cross-linked material is for being selected from NIPA, N-acryloylpiperidine, N-n-propyl Methacrylamide, N, N-acrylamide, N-sec.-propyl propylene methyl nitrosourea, N-cyclopropyl acrylamide, N-acryl tetramethyleneimine, N, N-ethyl methacrylamide, N-Cvclopropvlmethvl other can, at least one in N-ethyl acrylamide, non-temperature sensitive cross-linked material is be selected from least one in synthetic polymer and natural biologic material, and synthetic polymer is for being selected from acrylamide, polyoxyethylene glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, poly(lactic acid), polyhydroxy acid, poly(lactic acid) alkyd copolymers, polydimethylsiloxane, condensing model, poly-acid esters, polymeric amide, polyamino acid, polyacetal, polybutylcyanoacrylate, polyurethane(s), polypyrrole, polyester, polymethacrylate, polyethylene, at least one in polycarbonate and polyoxyethylene, natural biologic material is for being selected from gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connects at least one in albumen and fibronectin.Contriver finds, according to this hydrogel of the embodiment of the present invention, there is good hardness controllability, temperature sensitivity is supportive with amplification, when being applied to cell cultures, can hardness be changed with institute's culturing cell demand or regulate cytogene and form to express by active control hardness, be hydrophobic in body temperature simultaneously, group's contracting state, become hydrophilic when it is in room temperature, swelling state, and two states can vary with temperature reversible change, and can repeatedly grow in hydrogel surface at the cell that hydrogel surface attaches, its growth conditions can not be interrupted because of cell harvesting, in addition, the cell that hydrogel surface can be made to attach by reducing envrionment temperature initiatively departs from hydrogel, thus complete preservation cell surface receptor and extracellular matrix, maintain the unity of cell characteristics between generation is transmitted, and the stability of epigenetics.
According to embodiments of the invention, temperature sensitive cross-linked material is NIPA, and non-temperature sensitive cross-linked material is acrylamide and polyethyleneglycol diacrylate.
According to embodiments of the invention, the mass ratio of temperature sensitive cross-linked material and non-temperature sensitive cross-linked material is 1:(0.1 ~ 2.0).
According to embodiments of the invention, the hardness Young's modulus of the adjustable temperature sensitive polymer material of hardness is 10 handkerchief ~ 100 kPa, preferably 100 ~ 1500 handkerchiefs.
According to embodiments of the invention, the phase in version critical temperature of the adjustable temperature sensitive polymer material of hardness is 0 ~ 120 degree Celsius, is preferably 4 ~ 33 degrees Celsius.
According to embodiments of the invention, temperature sensitive cross-linked material and three-dimensional netted being cross-linked of non-temperature sensitive cross-linked material are carried out on the surface of base material, wherein, described base material is at least one in slide, plastics, metal and silica gel, is preferably at least one in slide, Tissue Culture Dish, silica gel, PMMA, PCL.
According to embodiments of the invention, the surface of base material utilizes methacrylic acid 3-(Trimethoxy silane) propyl ester to modify in advance.
In another aspect of this invention, the invention provides a kind of method preparing the adjustable temperature sensitive polymer material of foregoing hardness.According to embodiments of the invention, the method comprises: temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent are mixed, and make obtained mixture carry out crosslinking reaction, obtain the adjustable temperature sensitive polymer material of hardness.
Contriver finds, the method technical process is simple, can automatic production, can scale operation be realized, the photopolymer material systems of different shape can be prepared according to the actual requirements simultaneously, as circle, ellipse, Polygons etc., and obtained photopolymer material systems thickness, size are homogeneous, various mid-scale level (>1mm) all can realize, in addition, obtained polymer materials has whole feature and advantage of foregoing polymer materials, and this is no longer going to repeat them.
According to embodiments of the invention, the method preparing foregoing hardness adjustable temperature sensitive polymer material comprises further: temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent are mixed, obtain pre-polymer mix; Pre-polymer mix is placed on the surface of substrate, and covers base material on pre-polymer mix, then make pre-polymer mix carry out crosslinking reaction.Wherein, base material is be selected from least one in slide, Tissue Culture Dish, silica gel, PMMA, PCL and all types of plastic and metal etc.According to embodiments of the invention, the method preparing foregoing hardness adjustable temperature sensitive polymer material comprises further: by temperature sensitive cross-linked material and partial cross-linked dose of mixing, the temperature sensitive mixture obtained is modified in substrate material surface, obtains temperature sensitive substrate material; By non-temperature sensitive cross-linked material and the mixing of surplus linking agent, and the non-temperature sensitive mixture obtained is placed on the hydrophobic surface of substrate, then temperature sensitive substrate material is covered on non-temperature sensitive mixture, makes it carry out described crosslinking reaction.
According to embodiments of the invention, temperature sensitive mixture is modified and comprises further in substrate material surface: the surface temperature sensitive mixture being placed in base material, and react 5 ~ 30 minutes under 20 ~ 60 degrees Celsius or irradiate 10 ~ 1000 seconds under ultraviolet, then gained base material is carried out drying and processing.
According to embodiments of the invention, in the method for the adjustable temperature sensitive polymer material of the foregoing hardness of above-mentioned preparation, crosslinking reaction is undertaken by least one in chemical plastic, optics plastic, physics plastic, hot plastic, ionogen plastic.
According to embodiments of the invention, the condition of chemical plastic is react 5 ~ 30 minutes under room temperature.According to embodiments of the invention, the condition of optics plastic is irradiate 10 ~ 1000 seconds under light, and wherein, the light that optics plastic adopts is at least one in UV-light, visible ray, infrared light.
According to embodiments of the invention, the surface of the substrate adopted in the method for the adjustable temperature sensitive polymer material of the foregoing hardness of above-mentioned preparation utilizes octadecyl trichlorosilane alkane to modify in advance, and the surface of base material utilizes methacrylic acid 3-(Trimethoxy silane) propyl ester to modify in advance.
In another aspect of the invention, the invention provides the purposes of foregoing hardness adjustable temperature sensitive polymer material in one of following: the genetic expression of a. regulating cell and cellular form; B. Noninvasive collecting cell; C. maintain stability between cell generation, preservation cell surface receptor and extracellular matrix are from destruction.Contriver finds, when above-mentioned hydrogel is used for cell cultures, by adjusting the hardness of above-mentioned hydrogel, can Effective Regulation cellular gene expression and cellular form, and cell can be made to be separated with hydrogel by reducing temperature, effectively can avoid the injury that existing trysinization method collecting cell carries out cell, the cell that collection obtains effectively can maintain the stability between cell generation, and preservation cell surface receptor and extracellular matrix are from destruction.
In still another aspect of the invention, the invention provides a kind of method of culturing cell.According to embodiments of the invention, the method comprises: cultivate cell using the adjustable temperature sensitive polymer material of foregoing hardness as carrier; And by reducing temperature, the adjustable temperature sensitive polymer material of cell and hardness is initiatively separated with collecting cell.Utilize the method culturing cell, can Effective Regulation cellular gene expression and cellular form, and cell can be made initiatively to be separated with polymer materials by reducing temperature, effectively can avoid the injury that existing trysinization method collecting cell carries out cell, the cell that collection obtains effectively can maintain the stability between cell generation, and preservation cell surface receptor and extracellular matrix are from destruction.
According to embodiments of the invention, before repopulating cell, being utilized as biological sample in advance provides the matrix of attachment point to carry out surface treatment to the adjustable temperature sensitive polymer material of described hardness.
Accompanying drawing explanation
Fig. 1 shows the photo of the photosensitive polymerization thing material according to the embodiment of the present invention;
Fig. 2 shows and varies with temperature reversible shrink/expanded detected result according to the photosensitive polymerization thing material of the embodiment of the present invention;
Fig. 3 shows the fourier transform infrared spectrometry detected result of the photosensitive polymerization thing material according to the embodiment of the present invention;
Fig. 4 shows the schematic diagram of photosensitive polymerization thing material culturing cell according to the embodiment of the present invention and collecting cell;
Fig. 5 shows the qualitative detection result of the photosensitive polymerization thing material according to the embodiment of the present invention;
Fig. 6 shows the detected result according to the cell surface receptor of the embodiment of the present invention and the signal representation of extracellular matrix;
Fig. 7 shows according to the cell surface receptor of the embodiment of the present invention and the two positive rate detected result of extracellular matrix;
Fig. 8 shows and cultivates and changes in gene expression detected result according to the cell harvesting of the embodiment of the present invention again.
Embodiment
Embodiments of the invention are described below in detail.Embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In one aspect of the invention, the invention provides the adjustable temperature sensitive polymer material of a kind of hardness (comprising hydrogel, silica gel and other high molecular polymers).According to embodiments of the invention, this hardness adjustable temperature sensitive polymer material is by temperature sensitive cross-linked material and non-temperature sensitive cross-linked material is three-dimensional netted is cross-linked to form.
According to embodiments of the invention, the temperature sensitive cross-linked material that can adopt is not particularly limited, as long as can realize the function of hydrophilic and hydrophobic with temperature change.In some embodiments of the invention; temperature sensitive cross-linked material can for being selected from NIPA, N-acryloylpiperidine, N-n-propyl Methacrylamide, N; N-acrylamide, N-sec.-propyl propylene methyl nitrosourea, N-cyclopropyl acrylamide, N-acryl tetramethyleneimine, N, N-ethyl methacrylamide, N-Cvclopropvlmethvl other can, at least one in N-ethyl acrylamide.Thus, polymer materials of the present invention is in hydrophobicity under body temperature environment, and become contraction schedule, when temperature lower (as room temperature condition), hydrogel is wetting ability, becomes swelling state.
According to embodiments of the invention, the non-Thermo-sensitive cross-linked material that can adopt is not particularly limited, and those skilled in the art can select according to actual needs flexibly.In some embodiments of the invention, non-temperature sensitive cross-linked material can for being selected from least one in synthetic polymer and natural biologic material, wherein, synthetic polymer can for being selected from acrylamide, polyoxyethylene glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, poly(lactic acid), polyhydroxy acid, poly(lactic acid) alkyd copolymers, polydimethylsiloxane, condensing model, poly-acid esters, polymeric amide, polyamino acid, polyacetal, polybutylcyanoacrylate, polyurethane(s), polypyrrole, polyester, polymethacrylate, polyethylene, at least one in polycarbonate and polyoxyethylene, natural biologic material can for being selected from gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connects at least one in albumen and fibronectin.Thus, polymer materials is while having good Thermo-sensitive, and have good mechanical property, particularly hardness is adjustable, and then by the hardness of adjustment hydrogel, and can obtain the desirable cell of performance.
According to preferred embodiments more of the present invention, temperature sensitive cross-linked material can be NIPA, and non-temperature sensitive cross-linked material can be acrylamide and polyethyleneglycol diacrylate.The temperature-sensing property of polymer materials, hardness controllability and other performances is improved thereby, it is possible to further.
According to embodiments of the invention, the molecular weight of the polyoxyethylene glycol in polyethyleneglycol diacrylate is not particularly limited.In some embodiments of the invention, the polyoxyethylene glycol in polyethyleneglycol diacrylate includes but not limited to that polyoxyethylene glycol is Macrogol 2000, Macrogol 4000 or polyethylene glycol 6000.Thus, the use properties improving hydrogel is conducive to.
According to embodiments of the invention, in the adjustable temperature sensitive polymer material of hardness, the mass ratio of temperature sensitive cross-linked material and non-temperature sensitive cross-linked material can be 1:(0.1 ~ 2.0).Thus, each component has optimal ratio, and polymer materials has desirable Thermo-sensitive and hardness adjustability, and then during for culturing cell, is conducive to the effect improving culturing cell.
According to embodiments of the invention, the hardness Young's modulus of the adjustable temperature sensitive polymer material of hardness is 10 handkerchief ~ 100 kPa, in preferred embodiments more of the present invention, the hardness Young's modulus of the adjustable temperature sensitive polymer material of hardness can be 100 ~ 1500 handkerchiefs.Thus, polymer materials has suitable hardness, is suitable for cultivating various kinds of cell, and makes cultured cells tool performance in need.
According to embodiments of the invention, the phase in version critical temperature of the adjustable temperature sensitive polymer material of hardness can be 0 ~ 120 degree Celsius, under preferable case, can be 3 ~ 33 degrees Celsius.Thus, polymer materials has desirable phase in version critical temperature, and then during culturing cell, can effectively make cell initiatively depart from polymer materials by reducing temperature, and when complete preservation cell surface receptor and extracellular matrix collecting cell.
According to embodiments of the invention, concrete shape and the size of the adjustable temperature sensitive polymer material of hardness are not particularly limited, and those skilled in the art can select according to actual needs flexibly.Such as, circle, ellipse, Polygons (trilateral, square etc.), heart, Herba Galii Bungei shape etc. is included but not limited to.In some embodiments of the invention, the adjustable temperature sensitive polymer material of hardness can be grade hydrogel, particularly, above-mentioned grade hydrogel refers to that hydrogel is of a size of 0.1 – 900mm, be preferably 0.1 – 100mm, in a concrete example of the present invention, can for the laminar hydrogel that 0.1-0.5mm is thick, radius is 25mm.Thus, application is convenient to.
According to embodiments of the invention, temperature sensitive cross-linked material and three-dimensional netted being cross-linked of non-temperature sensitive cross-linked material are carried out on the surface of base material, wherein, base material can be at least one in slide, plastics, metal and silica gel, in some embodiments of the invention, base material can be at least one in slide, Tissue Culture Dish, silica gel, PMMA (polymethylmethacrylate), PCL (polycaprolactone).Thus, this temperature sensitive polymer material can be made more smooth, be beneficial to application.
In some embodiments of the invention, the surface of base material can utilize methacrylic acid 3-(Trimethoxy silane) propyl ester to modify in advance.Thus, the surface of base material can be made to have good wetting ability, be conducive to being combined with temperature sensitive cross-linked material and non-temperature sensitive cross-linked material.
Contriver finds, according to this polymer materials of the embodiment of the present invention, there is good hardness controllability, temperature sensitivity is supportive with amplification, when being applied to cell cultures, can hardness be changed with institute's culturing cell demand or regulate cytogene and form to express by active control hardness, be hydrophobic in body temperature simultaneously, group's contracting state, become hydrophilic when it is in room temperature, swelling state, and two states can vary with temperature reversible change, and, the cell attached at surface of polymer material can grow at surface of polymer material repeatedly, its growth conditions can not be interrupted because of cell harvesting, in addition, the cell that surface of polymer material can be made to attach by reducing envrionment temperature initiatively departs from polymer materials, thus complete preservation cell surface receptor and extracellular matrix, maintain the unity of cell characteristics between generation is transmitted, and the stability of epigenetics.
In another aspect of this invention, the invention provides a kind of method preparing the adjustable temperature sensitive polymer material of foregoing hardness.According to embodiments of the invention, the method can comprise the following steps: temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent are mixed, and make obtained mixture carry out crosslinking reaction, obtain the adjustable temperature sensitive polymer material of hardness.Thereby, it is possible to fast and effeciently prepare the adjustable temperature sensitive polymer material of foregoing hardness, and step is simple, is easy to control, construct.
According to embodiments of the invention, the concrete kind of above-mentioned temperature sensitive cross-linked material, non-temperature sensitive cross-linked material is consistent with the description in foregoing hardness adjustable temperature sensitive polymer material with ratio, and this is no longer going to repeat them.It should be noted that, the hardness of hydrogel can be controlled by the ratio adjusting temperature sensitive cross-linked material and non-temperature sensitive cross-linked material, specifically, the content of non-temperature sensitive cross-linked material is higher, the hardness of polymer materials is higher, those skilled in the art according to actual needs, can select the polymer materials with suitable stiffness.
According to embodiments of the invention, the concrete kind of the linking agent that can adopt is not particularly limited, as long as temperature sensitive cross-linked material and non-temperature sensitive cross-linked material can be made to carry out cross-linked network.In some embodiments of the invention, linking agent can be ammonium persulphate, N, N, N', N'-tetramethyl-diethylamine and light trigger I2959 etc.Thus, crosslinking reaction speed and efficiency higher, and the performance of the hydrogel prepared is desirable.
According to embodiments of the invention, in this step, can temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent be mixed in a solvent, the concrete kind of the solvent that can adopt is not particularly limited, and includes but not limited to pure water etc.
According to embodiments of the invention, the concrete mode mix temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent and the actual conditions of crosslinking reaction are not particularly limited.
In some embodiments of the invention, directly temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent can be mixed, then the pre-polymer mix obtained is placed on the surface of substrate, then on pre-polymer mix, base material is covered, then pre-polymer mix is made to carry out crosslinking reaction, be specifically as follows and react 5 ~ 30 minutes under room temperature or irradiate 10 ~ 1000 seconds under ultraviolet, obtain the adjustable temperature sensitive polymer material of hardness.In concrete examples more of the present invention, compressing tablet process can be carried out in advance to pre-polymer mix, it can be such as the base material method of forming, particularly, can be die methods, this die methods can comprise the following steps: be placed on the surface of substrate by pre-polymer mix, and covers base material on pre-polymer mix, pre-polymer mix is sandwiched in centre, then reacts 5 ~ 30 minutes under room temperature or irradiate 10 ~ 1000 seconds under ultraviolet.
In other embodiments of the present invention, in advance temperature sensitive cross-linked material can be modified the surface in base material, and then mix with non-temperature sensitive cross-linked material, particularly, can by temperature sensitive cross-linked material and partial cross-linked dose of mixing, then the temperature sensitive mixture obtained is modified in substrate material surface, obtain temperature sensitive substrate material; Then by non-temperature sensitive cross-linked material and the mixing of surplus linking agent, and the non-temperature sensitive mixture obtained is placed on the hydrophobic surface of substrate, then temperature sensitive substrate material is covered on non-temperature sensitive mixture, and makes it carry out crosslinking reaction (such as react 5 ~ 30 minutes under room temperature or irradiate 10 ~ 1000 seconds under ultraviolet).The base material method of forming can be carried out equally.According to embodiments of the invention, above-mentioned modification by temperature sensitive mixture can comprise in substrate material surface: the surface temperature sensitive mixture being placed in base material, and react 5 ~ 30 minutes under 20 ~ 60 degrees Celsius or irradiate 10 ~ 1000 seconds under ultraviolet, then gained base material is carried out drying and processing.Wherein, the actual conditions of drying and processing is not particularly limited, as long as can effectively dry, can be such as conventional furnace drying method, those skilled in the art also can carry out selecting and adjusting according to practical situation.Thereby, it is possible to simple and quick temperature sensitive cross-linked material is modified in substrate material surface.The base material standing storage that temperature sensing material can be modified by this kind of method is in advance for subsequent use, and for the modification of required non-temperature sensing material arbitrarily, simple to operate, convenient.
It should be noted that, the size of the substrate adopted in the present invention and base material and shape and the hydrogel size that will obtain and shape corresponding or identical, those skilled in the art can designed size and shape according to actual needs, thus obtains the hydrogel with suitable size and shape.The concrete material etc. of the substrate that the present invention adopts and base material is not particularly limited, can be known conventional material, such as substrate can be slide, plastics etc., in order to implement aforesaid method of the present invention better, inventors herein propose a kind of new substrate and base material, specifically, the surface of octadecyl trichlorosilane alkane to substrate can be utilized in advance to modify, make the hydrophobic surface that substrate forming property is good, methacrylic acid 3-(Trimethoxy silane) propyl ester is utilized to modify the surface of base material, to form water-wetted surface in advance.In subsequent step, the raw material preparing polymer materials is placed on the hydrophobic surface of the substrate after modification, and contacts with the water-wetted surface of base material.In addition, modifying in the step on substrate material surface by temperature sensitive cross-linked material, temperature sensitive cross-linked material is modified on the water-wetted surface of base material.Thus, substrate and base material functional, be conducive to the preparation of polymer materials, and preparation efficiency is higher.The modification technique method adopted can be surface chemical modification.Thus, simple to operate, convenient and swift.According to embodiments of the invention, the actual conditions of crosslinking reaction is not particularly limited, as long as effectively crosslinking reaction can be carried out, according to some embodiments of the present invention, crosslinking reaction is undertaken by least one in chemical plastic, optics plastic, physics plastic, hot plastic, ionogen plastic.In some embodiments of the invention, the condition of chemical plastic can at room temperature to react 5 ~ 30 minutes.In some embodiments of the invention, the condition of optics plastic can be irradiate 10 ~ 1000 seconds under light, and wherein, the light of employing can be at least one in UV-light, visible ray, infrared light.Thereby, it is possible to ensure that crosslinking reaction is carried out completely, and can not lose time because the reaction times is long.According to embodiments of the invention, the concrete grammar carrying out crosslinking reaction is not particularly limited, and includes but not limited at least one in chemical crosslink technique, physical crosslinking method, radiation cross-linking process, enzyme-catalyzed cross-linking method.
It should be noted that, the describing mode " room temperature " used in the present invention refers to that temperature is 10 ~ 35 degrees Celsius, in addition, the describing mode " chemical plastic " used in the present invention, " optics plastic ", " physics plastic ", " hot plastic ", " ionogen plastic " mainly refers to the initiation conditions of crosslinking reaction, such as " chemical plastic " refers to by under linking agent effect, be tied by chemical bond between macromolecular chain, form netted or body structure polymer crosslinking reaction process, " optics plastic " refers to and cause crosslinking reaction under the condition of illumination, " physics plastic " refers to and passes through electrostatic adhesion, Van der Waals forces etc. carry out crosslinking reaction, " hot plastic " refers to by thermal initiation crosslinking reaction, " ionogen plastic " refers to and forms by electron group and ionization the crosslinking reaction that complex compound carries out.
According to embodiments of the invention, in order to make the cell adhesion performance of the adjustable temperature sensitive polymer material of above-mentioned obtained hardness, the matrix treatments surface of polymer material that biological sample provides attachment point can be adopted as further, improve cell attachment efficiency.Above-mentionedly provide the matrix of attachment point to be not particularly limited for biological sample, include but not limited to that gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin etc.Specifically, adjustable for above-mentioned obtained hardness temperature sensitive polymer material can be soaked in and provide certain hour in the solution of the matrix of attachment point containing for biological sample, make, for biological sample provides the matrix of attachment point to be adsorbed in the adjustable temperature sensitive polymer material surface of hardness, to complete the modification of hydrogel.
According to embodiments of the invention, the above-mentioned processing condition preparing hydrogel also comprise: after completion of the reaction, preserve the step of hydrogel from hydrophobic surface separated and collected.In some embodiments of the invention, the method for collected polymer material for choosing sheet method, can be collected resulting polymers material require and being stored in pure water to keep moistening.Specifically, the method of collected polymer material can be carried out according to following steps: the substrate and the base material that centre are contained polymer materials are placed in liquid (as water), the syringe needle of front end bending is utilized slowly to be separated with the hydrophobic surface of substrate by polymer materials along the slide edge of overing number gel, polymer materials and base material are provoked simultaneously, there is on surface the base material of polymer materials to be placed in culture dish, add water and can preserve for a long time polymer materials.
Contriver finds, the method technical process is simple, can automatic production, can scale operation be realized, the photopolymer material systems of different shape can be prepared according to the actual requirements simultaneously, as circle, ellipse, Polygons etc., and obtained photopolymer material systems thickness, size are homogeneous, various mid-scale level (>1mm) all can realize, in addition, obtained polymer materials has whole feature and advantage of foregoing polymer materials, and this is no longer going to repeat them.
In another aspect of the invention, the invention provides the purposes of foregoing hardness adjustable temperature sensitive polymer material in one of following: the genetic expression of a. regulating cell and cellular form; B. Noninvasive collecting cell; C. maintain stability between cell generation, preservation cell surface receptor and extracellular matrix are from destruction.Contriver finds, when above-mentioned polymer materials is used for cell cultures, by adjusting the hardness of above-mentioned polymer materials, can Effective Regulation cellular gene expression and cellular form, and cell can be made to be separated with hydrogel by reducing temperature, realize Noninvasive cell harvesting, not only effectively can avoid the injury that existing trysinization method collecting cell carries out cell, the cell that collection obtains effectively can also maintain the stability between cell generation, and preservation cell surface receptor and extracellular matrix are from destruction.
In still another aspect of the invention, the invention provides a kind of method of culturing cell.According to embodiments of the invention, the method comprises: cultivate cell using the adjustable temperature sensitive polymer material of foregoing hardness as carrier; And by reducing temperature, the adjustable temperature sensitive polymer material of cell and hardness is initiatively separated with collecting cell.
According to embodiments of the invention, the concrete kind of aforesaid method cultured cells can be adopted to be not particularly limited, include but not limited to following at least one: the mixed culture of any one or several a) in various clone (as 3T3, LX-2, WS-1 etc.) and collection; B) mixed culture of any one or several in various primary cell (as myocardial cell, neuronal cell, epithelial cell etc.) and collection; C) mixed culture of any one or several in stem cell (as mesenchymal stem cells MSCs, induced dry-cell, embryonic stem cell etc.) and collection.
Utilize the method for above-mentioned hardness adjustable temperature sensitive polymer material culturing cell, cell can be comprised the steps: to mix with providing the nutrient solution of nutrition for culturing cell, obtain mixed solution, carry out following steps again: the above-mentioned mixed solution containing cell is added drop-wise to surface of polymer material, slow rotation hydrogel makes mixed solution be evenly distributed on surface of polymer material, until cell attachment after surface of polymer material, enough nutrient solutions are supplemented so that Growth of Cells in 37 DEG C, then, until cell after different hardness surface of polymer material forms specific gene expression and cellular form, add Low-temperature culture liquid (as 4 DEG C or room temperature) and reduce envrionment temperature residing for polymer materials, because of temperature variation cause polymer materials from hydrophobic become hydrophilic, volumetric expansion, thus cause cell cannot continue attachment then initiatively depart from polymer materials, with collecting cell.
According to embodiments of the invention, before cell seeding, the matrix treatments surface of polymer material that biological sample provides attachment point can be utilized as, improve cell attachment efficiency.Above-mentionedly provide the matrix of attachment point to be not particularly limited for biological sample, include but not limited to that gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin etc.Specifically, adjustable for above-mentioned obtained hardness temperature sensitive polymer material can be soaked in and provide certain hour in the solution of the matrix of attachment point containing for biological sample, make, for biological sample provides the matrix of attachment point to be adsorbed in the adjustable temperature sensitive polymer material surface of hardness, to complete the modification of hydrogel.In a preferred embodiment of the invention, for biological sample provides the matrix of attachment point can be gelatin.Thereby, it is possible to effectively improve the adhesive activity of cell.
Contriver finds, utilize the method culturing cell, can Effective Regulation cellular gene expression and cellular form, and cell can be made initiatively to be separated with polymer materials by reducing temperature, effectively can avoid the injury that existing trysinization method collecting cell carries out cell, the cell that collection obtains effectively can maintain the stability between cell generation, and preservation cell surface receptor and extracellular matrix are from destruction.
Embodiments of the invention are described below in detail, below the cell that uses in embodiment be: LX-2 cell: purchased from American Type Culture Collection (ATCC), mouse primary cardiomyocytes: from 1 week rat cardiac tissue separation and Extraction.
The Macrogol 4000 diacrylate (PEGDA4000) used in following embodiment can synthesize according to existing method: under a nitrogen atmosphere, 5g polyoxyethylene glycol (PEG4000) powder is dissolved in 50ml dichloromethane solution while stirring, dropwise slowly add the acrylate chloride solution of 0.47ml and the triethylamine solution of 0.76ml, at room temperature stirring reaction 24h.Then, add the solution of potassium carbonate of 100ml2mol/L with washing, and stratification, collect lower floor's methylene dichloride mixed solution.Methylene dichloride mixed solution is added drop-wise in 500ml anhydrous diethyl ether solvent and precipitates, filter, collect white powder, then drying at room temperature, finally obtain white polyethyleneglycol diacrylate pressed powder.
Embodiment 1
1. octadecyl trichlorosilane alkane (OTS) modifies hydrophobic surface (slide, i.e. substrate) method
1) slide is loaded on staining rack, and is immersed in the liquid detergent aqueous solution, 100W, 40 DEG C ultrasonic 10 minutes;
2) 1) staining rack being loaded with slide is taken out after step, then rinse 3 times, wash clean with distilled water comprehensively;
3) by 2) slide after cleaning boils 20 minutes (optional, can by slide in advance at 300 degrees Celsius of preheating about 1h) in hot NaOH (sodium hydroxide) solution (80 ~ 90 DEG C) of 10%;
4) 3) staining rack being loaded with slide is taken out after step, to be dipped in distilled water 10 minutes, to change the ultrasonic 10min of water, then rinse 3 times with distilled water comprehensively, dry 1h in an oven;
5) 4) after step, under normal temperature, the staining rack being loaded with slide to be immersed in the hexane solution of 1% octadecyl trichlorosilane alkane 10 minutes;
6) by 5) slide after step process soaks 10min in the normal hexane of recovery and rinsing, and soak 10min in ethanol, repeat twice;
7) by 6) step cleaning after slide be put into 4 hours (or spending the night) in 60 DEG C of baking ovens, take out cooling, be in store for.
2. base material (for slide) method modified by methacrylic acid 3-(Trimethoxy silane) propyl ester (TMSPMA)
1) slide is loaded on staining rack, and is immersed in the liquid detergent aqueous solution, 100W, 40 DEG C ultrasonic 10 ~ 20 minutes;
2) 1) staining rack being loaded with slide is taken out after step, to be dipped in distilled water 10 minutes, then to rinse 3 times with distilled water comprehensively;
3) by 2) cleaning after slide in hot NaOH (sodium hydroxide) solution (80 ~ 90 DEG C) of 10%, boil 10 ~ 20 minutes;
4) 3) staining rack being loaded with slide is taken out after step, to be dipped in distilled water 10 minutes, then to rinse 3 times with distilled water comprehensively, dry in an oven or use N 2(nitrogen) dries up;
5) in 600mL dehydrated alcohol, drip 6mLTMSPMA, then in this solution, drip 18mL acetic acid solution (acetic acid is 1:10 with the ratio of water) be made into decorating liquid (each matching while using of this decorating liquid);
6) by 4) slide (on staining rack, ensureing that each of slide fully contacts with solution) after step process puts into 5) decorating liquid for preparing of step fully reacts 10 minutes;
7) soaking and washing 6 in dehydrated alcohol) slide after step process 10 minutes;
8) by 7) slide after step cleaning to be put in 60 DEG C of baking ovens 4 hours, and take out cooling, be in store for.
3. the preparation of different hardness temperature sensitive polymer material
The preparation of reaction soln: NIPA, acrylamide and polyethyleneglycol diacrylate are dissolved in pure water; Again by obtained mixture and ammonium persulphate and N, N, N', N'-tetramethyl-diethylamine mixes, abundant mixing, obtains reaction soln, and the concentration of NIPA in reaction soln is 10g/100ml, the concentration of polyethyleneglycol diacrylate in reaction soln is 1g/100ml or 2.5g/100ml or 10g/100ml, and the concentration of ammonium persulphate in reaction soln is 0.2g/100ml; The concentration of N, N, N', N'-tetramethyl-diethylamine in reaction soln is 0.1g/100ml.
The reaction soln that 147 μ l prepare is dripped the hydrophobic surface of the substrate modified in octadecyl trichlorosilane alkane, it covers the slide that 25mm diameter methacrylic acid 3-(Trimethoxy silane) propyl ester is modified, reaction soln is uniformly distributed.The solution pressed at room temperature is left standstill 10 minutes to carry out crosslinking reaction, after reaction terminates, the pin of front end bending is utilized to be provoked by hydrogel, obtain the temperature sensitive polymer material of different hardness, detect known through hardness Young's modulus, the concentration of polyethyleneglycol diacrylate is that the hardness Young's modulus of the hydrogel that the reaction soln of 1g/100ml or 2.5g/100ml or 10g/100ml prepares is respectively 100 handkerchiefs, 500 handkerchiefs and 1200 handkerchiefs.
Embodiment 2, temperature sensitive polymer material property detect
In this embodiment, temperature sensitive polymer material used all refers to that in embodiment 1, the prepared hardness Young's modulus that obtains is respectively 100 handkerchiefs, the temperature sensitive polymer material of 500 handkerchiefs and 1200 handkerchiefs, hereafter describe or accompanying drawing in, 100 handkerchiefs or 1%PEG all represent that the concentration of polyethyleneglycol diacrylate in embodiment 1 is the hydrogel that the reaction soln of 1g/100ml prepares, 500 handkerchiefs or 2.5%PEG all represent that the concentration of polyethyleneglycol diacrylate in embodiment 1 is the hydrogel that the reaction soln of 2.5g/100ml prepares, 1200 handkerchiefs or 10%PEG all represent that the concentration of polyethyleneglycol diacrylate in embodiment 1 is the hydrogel that the reaction soln of 10g/100ml prepares.
One, temperature sensitive polymer material shape is observed
Fig. 1 is shown in by the photo of temperature sensitive polymer material, and wherein, hydrogel is from left to right followed successively by the temperature sensitive polymer material that hardness Young's modulus is respectively 100 handkerchiefs, 500 handkerchiefs and 1200 handkerchiefs.As seen from Figure 1, the hydrogel of different hardness, all in water white transparency shape, has good light transmission, conveniently observes in cell cultivation process.
Two, temperature sensitive polymer material varies with temperature reversible shrink/expanded
Testing method: with micro-injection pump carry out water droplet injection drip, by observe water droplet at different temperatures at the contact angle of hydrogel surface to study the temperature-sensitive of hydrogel, specific as follows:
The debugging of micro-injection pump: first injection standard is adjusted to the injection standard needed for 1ml asepsis injector: internal diameter 4.53mm, injection flow velocity 100 μ l/min, injection range 500 μ l.
The measurement of contact angle: hydrogel is placed horizontally on clean table top, pure water is dripped lentamente to hydrogel surface respectively under room temperature (16 degrees Celsius) and 37 DEG C of conditions with syringe pump, after drop balance, take pictures and changed by software analysis hydrogel contact angle at different temperatures, detected result is shown in Fig. 2, wherein, Fig. 2 A is temperature sensitive polymer material form photo at different temperatures, and Fig. 2 B is temperature sensitive polymer material contact angle detection result at different temperatures.
Result: as can be seen from Fig. 2 A, temperature sensitive polymer material reversibly shrinks (higher temperatures, as 37 DEG C, is shown in the right figure of Fig. 2 A) according to temperature variation or expands (room temperature is shown in the left figure of Fig. 2 A); As can be seen from Fig. 2 B, temperature sensitive polymer material varies with temperature and reversibly changes hydrophilic (room temperature is shown in the left figure of Fig. 2 B) or hydrophobic (37 DEG C, be shown in the right figure of Fig. 2 B) character.The above results shows that hydrogel has good temperature-sensitive.
Three, temperature sensitive polymer material infrared chromatography test
PerkinElmerSpectrum100FT-IRSpectrometer is used to the test of temperature sensitive polymer material infrared chromatography.Testing method is: peeled off from surface of glass slide by sample (i.e. hydrogel) and drip on optical table, after determining there is no bubble, and collected specimens absorption spectrum.
Fourier transform infrared spectrometry detected result is shown in Fig. 3.As can be seen from the result of Fig. 3, temperature sensitive polymer material is with the difference of hardness, and its chemical group power can change accordingly, but essentially consist is all identical.
Four, temperature sensitive polymer material is for cultivating collecting cell
Temperature sensitive polymer material itself can attach by sustenticular cell, can provide cell attachment sites again crosslinkable biomaterial can improve cell attachment efficiency, the crosslinkable biomaterial that can be used for modifying temperature sensitive polymer material can be following at least one: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connect albumen, fibronectin, sodium alginate.Modifying method is as follows: be modified to example with gelatin, and gelatin preparation is become 0.1% aqueous solution, then temperature sensitive polymer material is immersed in 4 C overnight in aqueous gelatin solution and preserves, namely complete temperature sensitive polymer material and modify.
Then, the performance of temperature sensitive polymer material culturing cell is detected in the following way:
This experiment is for LX-2 cell, and cell adsorption medium takes gelatin.Cell suspension evenly drops to temperature sensitive polymer material surface by dripping method, after cell attachment, supplement enough nutrient solutions for Growth of Cells, cultivate in the incubator of 37 degrees Celsius, Fig. 4 is shown in by the schematic diagram of culturing cell and collecting cell, and the LX-2 cell cultivated with conventional Ti ssueCulturePlastic in contrast.
1. qualitative detection:
Hydrogel with cell is placed in basis of microscopic observation, and Fig. 5 A is shown in by photo, and wherein, scale is 100 microns.The result of Fig. 5 A shows, different hardness hydrogel all can be used for cultivating LX-2 Growth of Cells, LX-2 cell tends to cluster growth in hydrogel surface, cell arrangement is more regular, orderly cell can be formed between cell mass connect, cell on hydrogel increases with hardness and more stretches, and grows and is more prone to unorderedly sprawl arrangement at the cell on control group surface.
Detected the cell and cellular control unit secretion Actin muscle α that are attached to hydrogel surface by immunofluorescence staining, I-type collagen, the situation of fibronectin and IV collagen type, detected result is shown in Fig. 5 B-Fig. 5 E respectively, and wherein, scale is 50 microns.Detected result shows, be attached to the Actin muscle α secreted by cell of hydrogel surface, I-type collagen and fibronectin rise gradually along with the increase of matrix solidity, IV collagen type reduces along with the increase of hardness, illustrates that different hardness hydrogel can active control cellular gene expression and cell morphological characteristic in culturing cell process.
2. the temperature sensitive collecting cell ability of detection by quantitative:
Until cell after different hardness hydrogel surface forms specific gene expression and cellular form, add Low-temperature culture liquid (as 4 DEG C or room temperature) and reduce envrionment temperature residing for hydrogel, because temperature variation causes hydrogel to become hydrophilic, volumetric expansion from hydrophobic, thus cause cell cannot continue attachment then initiatively depart from hydrogel, with collecting cell, in contrast with the cell of traditional trysinization method collection simultaneously.
Detected the signal representation of the cell (namely by cell that alternating temperature is collected) of temperature sensitive collection and compared with control cells (i.e. enzymic digestion collect cell) surface receptor and extracellular matrix respectively by immunofluorescence staining, detected result is shown in Fig. 6.Immunofluorescence dyeing result shows, and collects gained LX-2 cell surface receptor and extracellular matrix still have stronger signal representation through temperature variation, and only has small portion residual through the cell surface receptor of collected by trypsinisation and extracellular matrix.
Detect the two positive rate of the cell of temperature sensitive collection and compared with control cells surface receptor and extracellular matrix respectively by flow cytometer, detected result is shown in Fig. 7.Flow cytometric analysis results confirms to collect gained LX-2 cell surface receptor and extracellular matrix pair positive rate about 20% through temperature variation, but after enzymic digestion, its pair of positive rate is only about 7%.
3. cell harvesting is cultivated and changes in gene expression again
Until cell after different hardness hydrogel surface forms specific gene expression and cellular form, add Low-temperature culture liquid (as 4 DEG C or room temperature) and reduce envrionment temperature residing for hydrogel, cell is departed from from hydrogel surface, collection gained cell is attached at again hydrogel or conventional cell culture dish surface, cultivate after 24 hours, observation of cell form, and detect cellular gene expression by qPCR means, detected result is shown in Fig. 8, wherein, Fig. 8 A is the morphologic observation result of cell before and after collecting cell, Fig. 8 B is cellular gene expression detected result before and after collecting cell, wherein, Relativeexpression represents relative expression quantity, Untreated0h represents original position untreated cell, Thermal0h represents the cell collected through alternating temperature, Trypsin0h represents the cell collected through enzymic digestion, Thermal24hsoft represents the cell of temperature sensitive polymer material after 24 hours of again planting back 100 handkerchiefs through alternating temperature collection, Trypsin24hsoft represents the cell of temperature sensitive polymer material after 24 hours of again planting back 100 handkerchiefs through enzymic digestion collection, Thermal24hrigid represents that collecting plantation again through alternating temperature returns the cell of system after TissueCulturePlastic24 hour, Trypsin24hrigid represents that collecting plantation again through enzymic digestion returns the cell of system after TissueCulturePlastic24 hour.
Result shows: collect gained cell through temperature variation, no matter be again attached at softer or harder substrate surface, agglomerating ordered arrangement still can be kept grow, but cell after trysinization is many grows more irregular in unicellular form; The cellular gene expression persistent levels collected through temperature, stable, without obviously fluctuating, and under the effect of pancreatin, cellular gene expression rises and falls obviously, can follow without evident regularity.The integrity of cell and extracellular matrix preserved by the above results explanation temperature sensitive polymer material non-invasively collecting cell, makes cell can not cause gene and metamorphosis because conventional cell is collected.
Embodiment 3
1. octadecyl trichlorosilane alkane (OTS) modifies hydrophobic surface (slide, i.e. substrate) method, with embodiment 1;
2. slide method modified by methacrylic acid 3-(Trimethoxy silane) propyl ester (TMSPMA), with embodiment 1;
3. the slide that TMSPMA modifies is cross-linked temperature sensing material;
1) NIPA and light trigger I2959 are dissolved in pure water, make final concentration be respectively 10 quality % and 0.5 quality %, form temperature sensitive cross-linked material premixed liquid;
2) the temperature sensitive cross-linked material premixed liquid that 8 μ l prepare is dripped the hydrophobic surface of the substrate modified in octadecyl trichlorosilane alkane, on it, covering diameter is the slide of methacrylic acid 3-(Trimethoxy silane) the propyl ester modification of 25mm, and temperature sensitive cross-linked material premixed liquid is uniformly distributed.Under the solution pressed is placed on ultraviolet pointolite, ultraviolet pointolite energy is 3.5W, height 15cm, reaction times 100s;
3) take out the slide be cross-linked under ultraviolet pointolite, 60 DEG C, within 2 hours, dry, by the temperature sensitive slide 4 DEG C preservation obtained, for subsequent use.
4. the preparation of different hardness temperature sensitive polymer material, with embodiment 1, difference is that the slide by methacrylic acid 3-(Trimethoxy silane) propyl ester is modified changes step 3 into) the temperature sensitive slide that obtains;
5. temperature sensitive polymer material property detects, and with embodiment 2, detected result is similar to Example 2.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not must for be identical embodiment or example.And the specific features of description, structure, material or feature can combine in one or more embodiment in office or example in an appropriate manner.In addition, when not conflicting, the feature of the different embodiment described in this specification sheets or example and different embodiment or example can carry out combining and combining by those skilled in the art.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, and those of ordinary skill in the art can change above-described embodiment within the scope of the invention, revises, replace and modification.

Claims (10)

1. the adjustable temperature sensitive polymer material of hardness, is characterized in that, by temperature sensitive cross-linked material with non-temperature sensitive cross-linked material is three-dimensional netted is cross-linked to form,
Wherein, described temperature sensitive cross-linked material is for being selected from NIPA, N-acryloylpiperidine, N-n-propyl Methacrylamide, N; N-acrylamide, N-sec.-propyl propylene methyl nitrosourea, N-cyclopropyl acrylamide, N-acryl tetramethyleneimine, N; N-ethyl methacrylamide, N-Cvclopropvlmethvl other can, at least one in N-ethyl acrylamide
Described non-temperature sensitive cross-linked material is be selected from least one in synthetic polymer and natural biologic material,
Described synthetic polymer is be selected from least one in acrylamide, polyoxyethylene glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, poly(lactic acid), polyhydroxy acid, poly(lactic acid) alkyd copolymers, polydimethylsiloxane, condensing model, poly-acid esters, polymeric amide, polyamino acid, polyacetal, polybutylcyanoacrylate, polyurethane(s), polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyoxyethylene
Described natural biologic material is be selected from least one in gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, protein-polysaccharide, glycoprotein, hyaluronic acid, layer connection albumen and fibronectin,
Preferably, described temperature sensitive cross-linked material is NIPA, and described non-temperature sensitive cross-linked material is acrylamide and polyethyleneglycol diacrylate.
2. the adjustable temperature sensitive polymer material of hardness according to claim 1, is characterized in that, the mass ratio of described temperature sensitive cross-linked material and non-temperature sensitive cross-linked material is 1:(0.1 ~ 2.0),
Optionally, the hardness Young's modulus of the adjustable temperature sensitive polymer material of described hardness is 10 handkerchief ~ 100 kPa, is preferably 100 ~ 1500 handkerchiefs,
Optionally, the phase in version critical temperature of the adjustable temperature sensitive polymer material of described hardness is 0 ~ 120 degree Celsius, is preferably 3 ~ 33 degrees Celsius.
3. the adjustable temperature sensitive polymer material of hardness according to claim 1, is characterized in that, described temperature sensitive cross-linked material and three-dimensional netted being cross-linked of non-temperature sensitive cross-linked material are carried out on the surface of base material,
Wherein, described base material is at least one in slide, plastics, metal and silica gel, is preferably at least one in slide, Tissue Culture Dish, silica gel, PMMA, PCL,
Optionally, the surface of described base material utilizes methacrylic acid 3-(Trimethoxy silane) propyl ester to modify in advance.
4. prepare a method for the adjustable temperature sensitive polymer material of hardness according to any one of claims 1 to 3, it is characterized in that, comprising:
Temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent are mixed, and makes obtained mixture carry out crosslinking reaction, obtain the adjustable temperature sensitive polymer material of hardness.
5. method according to claim 4, is characterized in that, comprises further:
Described temperature sensitive cross-linked material, non-temperature sensitive cross-linked material and linking agent are mixed, obtains pre-polymer mix;
Described pre-polymer mix is placed on the surface of substrate, and covers base material on described pre-polymer mix, then make described pre-polymer mix carry out described crosslinking reaction.
6. method according to claim 4, is characterized in that, comprises further:
By described temperature sensitive cross-linked material and the described linking agent mixing of part, the temperature sensitive mixture obtained is modified in substrate material surface, obtains temperature sensitive substrate material;
By linking agent mixing described in described non-temperature sensitive cross-linked material and surplus, and the non-temperature sensitive mixture obtained is placed on the surface of substrate, then described temperature sensitive substrate material is covered on described non-temperature sensitive mixture, makes it carry out described crosslinking reaction,
Optionally, described temperature sensitive mixture modified comprise further in described substrate material surface:
Described temperature sensitive mixture is placed in the surface of described base material, and reacts 5 ~ 30 minutes under 20 ~ 60 degrees Celsius or irradiate 10 ~ 1000 seconds under ultraviolet, then gained base material is carried out drying and processing.
7. the method according to claim 5 or 6, it is characterized in that, the surface of described substrate utilizes octadecyl trichlorosilane alkane to carry out hydrophobic modification in advance, and the surface of described base material utilizes methacrylic acid 3-(Trimethoxy silane) propyl ester to modify in advance.
8. the method according to claim 5 or 6, is characterized in that, described crosslinking reaction is undertaken by least one in chemical plastic, optics plastic, physics plastic, hot plastic, ionogen plastic,
Optionally, the condition of described chemical plastic is react 5 ~ 30 minutes under room temperature,
Optionally, the condition of described optics plastic is irradiate 10 ~ 1000 seconds under light, and wherein, described light is at least one in UV-light, visible ray, infrared light.
9. the purposes of the hardness adjustable temperature sensitive polymer material of preparation according to any one of claims 1 to 3 in one of following:
A. regulating cell genetic expression and cellular form;
B. Noninvasive collecting cell;
C. maintain stability between cell generation, preservation cell surface receptor and extracellular matrix are from destruction.
10. a method for culturing cell, is characterized in that, comprising:
Cell is cultivated using the adjustable temperature sensitive polymer material of the hardness according to any one of claims 1 to 3 as carrier;
Described cell is made initiatively to be separated to collect described cell with described hardness adjustable temperature sensitive polymer material by reducing envrionment temperature,
Preferably, before repopulating cell, being utilized as biological sample in advance provides the matrix of attachment point to carry out surface treatment to the adjustable temperature sensitive polymer material of described hardness.
CN201610056059.1A 2016-01-27 2016-01-27 Temperature-sensitive polymer material with adjustable hardness and preparation method and application thereof Pending CN105566567A (en)

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CN106467605A (en) * 2016-09-22 2017-03-01 北京化工大学 One kind is containing temperature sensitive and degradable segment triblock copolymer, preparation method and nano thin-film
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CN110331124B (en) * 2019-06-14 2022-03-22 浙江大学 Conductive polypyrrole/extracellular matrix composite film and preparation method thereof
CN112220967A (en) * 2020-11-26 2021-01-15 山东大学 Targeting antibacterial and in-situ bone-promoting dual-functional material and preparation method and application thereof
CN112220967B (en) * 2020-11-26 2021-08-03 山东大学 Targeting antibacterial and in-situ bone-promoting dual-functional material and preparation method and application thereof

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