WO2017080116A1 - 温度敏感型细胞培养表面及其制备方法 - Google Patents

温度敏感型细胞培养表面及其制备方法 Download PDF

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WO2017080116A1
WO2017080116A1 PCT/CN2016/072754 CN2016072754W WO2017080116A1 WO 2017080116 A1 WO2017080116 A1 WO 2017080116A1 CN 2016072754 W CN2016072754 W CN 2016072754W WO 2017080116 A1 WO2017080116 A1 WO 2017080116A1
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cell culture
temperature
sensitive
culture surface
preparing
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PCT/CN2016/072754
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French (fr)
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袁建华
陈勇
李慧伦
方想元
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广州洁特生物过滤股份有限公司
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Priority to US15/775,695 priority Critical patent/US11286318B2/en
Publication of WO2017080116A1 publication Critical patent/WO2017080116A1/zh

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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
    • C08F112/00—Homopolymers 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 an aromatic carbocyclic ring
    • C08F112/02—Monomers containing only one unsaturated aliphatic radical
    • C08F112/04—Monomers containing only one unsaturated aliphatic radical containing one ring
    • C08F112/06—Hydrocarbons
    • C08F112/08—Styrene
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
    • 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
    • C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
    • C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
    • 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
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L25/00—Compositions of, homopolymers or 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 an aromatic carbocyclic ring; Compositions of derivatives of such polymers
    • C08L25/02—Homopolymers or copolymers of hydrocarbons
    • C08L25/04—Homopolymers or copolymers of styrene
    • C08L25/06—Polystyrene
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2325/00—Characterised by the use of homopolymers or 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 an aromatic carbocyclic ring; Derivatives of such polymers
    • C08J2325/02—Homopolymers or copolymers of hydrocarbons
    • C08J2325/04—Homopolymers or copolymers of styrene
    • C08J2325/06—Polystyrene

Definitions

  • the invention relates to the technical field of cell culture, in particular to a temperature sensitive cell culture surface and a preparation method thereof.
  • tissue regeneration engineering In recent years, the rapid development of tissue regeneration engineering, biomedical engineering, and cell therapy industries has resulted in the acquisition of large numbers of cells, extracellular secretions, and cell therapy products by large-scale culture of animals or human cells in vitro.
  • tissue trauma, skin cell culture and new skin acquisition, tumor cell therapy, cell grooming, cell weight loss, etc. all require in vitro culture of target cells.
  • the cells are mainly divided into suspension cells and adherent cells according to their in vitro culture characteristics.
  • the suspension cells are suspended in the culture medium of the culture device, and the growth of the adherent cells needs to adhere to the surface of the culture device to grow.
  • the adherent cells are cultured, they are mainly processed by trypsin digestion from the adhering surface to complete the collection, and this digestion method will destroy the membrane protein on the cell surface, thereby destroying the integrity of the cells and losing the integrity.
  • the application of cells is greatly limited, especially in the field of cell clinical applications.
  • temperature-sensitive cell culture surfaces are currently available on the market primarily by grafting temperature sensitive materials onto the surface of the culture device.
  • the surface can automatically detach the cells by changing the temperature after the end of the cell culture, thereby reducing the damage to the cells.
  • the temperature sensitive response function is unstable due to the physical coating of the temperature sensitive compound directly on the surface of the cell culture device, and the temperature is moderate during the culture process.
  • the sensitive compound will fall off, so the auto-shedding effect of the cell cannot be guaranteed; most of the other is the grafting of the temperature-sensitive compound on the cell culture surface by high-energy electron beam, and a small part of the grafting by ultraviolet light irradiation or plasma-induced grafting.
  • atom transfer radical-initiated grafting in the way of cultivating surface temperature-sensitive compound grafting, some production equipment is expensive (such as high-energy electron beam, plasma), and some processes are complicated, and the high cost of raw materials is unfavorable to the industry. (eg Atom transfer radical initiation, ultraviolet radiation induced), some have low grafting efficiency and serious environmental pollution (such as plasma).
  • a method for preparing a temperature-sensitive cell culture surface comprises the following steps:
  • the molar ratio of the radical initiator to the temperature sensitive compound is (0.5-50): 100;
  • the cell culture surface is subjected to a reaction at 50-150 ° C for 5-120 min to obtain the temperature-sensitive cell culture surface.
  • the reaction described in the step (2) can be carried out under the protection of nitrogen, as needed.
  • the method of distributing in step (2) is dipping, atomizing, spraying or smearing.
  • the immersing method is: immersing the cell culture surface in the temperature sensitive original solution for 0.1-60 min, and then taking out;
  • the method of atomizing is: first atomizing the temperature sensitive raw liquid into particles having a particle diameter of 2 to 1000 micrometers, and exposing the cell culture surface to the atomized atmosphere for 5 to 100 minutes, and then taking out ;
  • the spraying method is: spraying the temperature sensitive original solution on the cell culture surface by high pressure spraying or air spraying.
  • the temperature sensitive compound is N-isopropyl acrylamide, N-isopropyl methacrylamide, N-isopropyl acrylamide oligomer, N-isopropyl methacryl
  • the material for preparing the cell culture surface is a polyolefin or a polycarboxylate.
  • the polyolefin is polystyrene, polypropylene or polyethylene;
  • the polycarboxylate is a polycarbonate, a poly-fatty acid ester, a polyaryl acid ester or a polyacrylate.
  • the free radical initiator is a peroxide-based initiator (such as benzoyl peroxide, cumene peroxide, potassium persulfate, t-butyl peroxide, etc.), azo Initiator One or more of nitrogen diisobutyronitrile, azobisisoheptanenitrile, azobisisobutylphosphonium hydrochloride (AIBA), azobisisobutyrazoline hydrochloride (AIBI), and the like.
  • a peroxide-based initiator such as benzoyl peroxide, cumene peroxide, potassium persulfate, t-butyl peroxide, etc.
  • azo Initiator One or more of nitrogen diisobutyronitrile, azobisisoheptanenitrile, azobisisobutylphosphonium hydrochloride (AIBA), azobisisobutyrazoline hydrochloride (AIBI), and the like.
  • the solvent is an alcohol (such as methanol, ethanol, propanol, isopropanol), an ester (such as a formate such as methyl formate, ethyl formate, propyl formate, butyl formate) ; acetates such as methyl acetate, ethyl acetate, propyl acetate, butyl acetate, ketones (acetone, methyl ethyl ketone, etc.), chlorinated hydrocarbons (chloroform, dichloromethane, ethyl chloride, etc.) One or more.
  • an alcohol such as methanol, ethanol, propanol, isopropanol
  • an ester such as a formate such as methyl formate, ethyl formate, propyl formate, butyl formate
  • acetates such as methyl acetate, ethyl acetate, propyl acetate, butyl acetate, ketones
  • the present invention also provides a temperature-sensitive cell culture surface prepared by the method for preparing a temperature-sensitive cell culture surface.
  • the present invention also provides a cell culture device having the temperature-sensitive cell culture surface.
  • the cell culture device is a cell culture plate, a cell culture flask, a cell culture dish, a cell culture device provided with a three-dimensional network structure composed of a plurality of fibers, or a cell culture device provided with a particle microcarrier. .
  • the present invention has the following beneficial effects:
  • the method for preparing a temperature-sensitive cell culture surface according to the present invention, rationally arranging the concentration of the temperature-sensitive compound and the radical initiator in the temperature-sensitive stock solution, and distributing the temperature-sensitive stock solution on the cell culture surface, and utilizing common chemistry
  • the free radical initiator directly initiates the graft polymerization of the temperature sensitive compound on the cell culture surface, and obtains the cell culture surface with temperature sensitive response function.
  • the temperature sensitive raw material dosage is used.
  • Low, high grafting efficiency; relative to ultraviolet light irradiation and atom transfer radical initiation, the initiator raw materials are widely available, the cost is low, and the process principle is convenient for large-scale preparation.
  • the temperature-sensitive cell culture surface prepared by the invention enables the temperature-sensitive detachment by the temperature decrease after the culture of the adherent cells is finished, and reduces the damage caused by the chemical digestion and the physical method, and ensures the integrity of the cell membrane protein, and Compared with the prior art, the production cost of the temperature-sensitive cell culture surface is greatly reduced, the grafting rate is high, and the environment is friendly.
  • the distribution manner of the temperature sensitive raw liquid has a key influence on the final grafting rate
  • the present invention preferably realizes the temperature sensitive original liquid by using the impregnation, atomization and spraying methods of specific conditions. Grafting on the cell culture surface can effectively increase the graft ratio.
  • FIG. 1 is a micrograph showing a cell attached to a surface of a device after incubation of a T3T cell at 37 ° C for 48 hours using a temperature-sensitive cell culture surface according to an embodiment of the present invention
  • Figure 2 is a photomicrograph of the auto-desorption of cells in a sheet after the temperature-sensitive cell culture surface of Figure 1 was treated at 20 °C for 10-40 minutes.
  • An azo initiator having a molar ratio of 5:100 to the monomer of 0.5 g of N-isopropylacrylamide was weighed and dissolved in a mixed solvent of 7.5 g of isopropyl alcohol and ethyl acetate; The mixture solution is atomized into particles having a particle diameter of 2 to 1000 ⁇ m, and then the surface of the cell culture plate of the polystyrene material to be treated is exposed to the atomizing vapor atmosphere for 50 minutes to cause the mist to condense on the surface to be treated, and finally This was placed in a nitrogen-filled oven and taken out after being kept at 50-120 ° C for 50 minutes, thereby obtaining a cell culture plate having a temperature-sensitive cell culture surface, and a graft ratio of 2.2 ⁇ g/cm 2 .
  • An azo initiator having a molar ratio of 30:100 to the monomer of 0.6 g of N-isopropylacrylamide was weighed and dissolved in a mixed solvent of 6 g of isopropyl alcohol, butyl acetate and dichloromethane, and then air was used. Spraying the mixture solution on the surface of the cell culture bottle of polystyrene material, and then placing it in an air-filled oven, and taking it out after being kept at 50-120 ° C for 30 minutes, thereby obtaining a cell having a temperature-sensitive cell culture surface.
  • the flask was cultured at a graft ratio of 1.5 ⁇ g/cm 2 .
  • An azo initiator having a molar ratio of 40:100 to 3.5 g of N-isopropylacrylamide monomer was dissolved in 7 g of a mixed solvent of ethyl chloride, ethanol and ethyl formate, and then a high-pressure air mist was used.
  • the mixture solution is atomized into particles having a particle diameter of 2 to 1000 ⁇ m, and then the microsphere carrier prepared by polyethylene having a particle diameter of 40 to 500 ⁇ m to be treated is exposed to the atomizing vapor atmosphere for 10 minutes.
  • the mist vapor is condensed on the surface of the microsphere carrier to be treated, and finally placed in an oven filled with nitrogen, and taken out after being kept at 50-120 ° C for 60 minutes, thereby obtaining a microsphere carrier having a temperature-sensitive cell culture surface, and the graft ratio 1.6 ⁇ g/cm 2 .
  • the spherical particle microcarrier can be placed in a conventional cell culture plate, a cell culture bottle or a cell culture dish, and the culture solution is added, and fine particles are used as a carrier for cell adhesion growth, and suspended in the culture solution by stirring to make the cells in The surface of the carrier is propagated into a monolayer of cells. Since the temperature-sensitive grafting is realized on the granular microcarrier, it is used in combination with a cell culture device such as a conventional cell culture plate, a bottle or a dish, and becomes a temperature-sensitive cell culture device.
  • N-isopropylacrylamide monomer a peroxide initiator with a monomer molar ratio of 1:100
  • the multi-layer fiber three-dimensional network porous support prepared by carbonate is immersed in the temperature-sensitive mixed solution for 0.1-60 minutes, then taken out and air-dried, and finally placed in an oven filled with nitrogen, and taken out after being kept at 80-150 ° C for 30 minutes. That is, a multilayer fiber three-dimensional network porous scaffold having a temperature-sensitive cell culture surface having a graft ratio of 1.5 ⁇ g/cm 2 was obtained .
  • the multi-layer fiber three-dimensional network porous support is a multi-layer network structure, and can be used as a multi-layer temperature-sensitive stent in a conventional cell culture plate, a cell culture bottle or a cell culture dish without changing the conventional routine. Under the premise of cell culture plates, cell culture flasks or cell culture dishes, the specific surface area of cell adherent growth is multiplied, and more cells are harvested under a limited volume.
  • the temperature-sensitive cell culture apparatus prepared in Example 1 was used for T3T cell culture, and the cell attachment micrograph of the T3T cells cultured at 37 ° C for 48 hours was as shown in FIG. 1; then the cells were treated at 20 ° C for 10-40 minutes. A microscope photograph of the automatic desorption of the sheet is shown in Fig. 2.

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Abstract

本发明提供一种温度敏感型细胞培养表面及其制备方法，其包括如下步骤：（1）配制温敏原液：将温敏化合物和自由基引发剂加入至溶剂中，搅拌溶解，得温敏原液；（2）将所述温敏原液分布于细胞培养表面后，将该细胞培养表面置于50-150℃条件下反应5-120min。

Description

温度敏感型细胞培养表面及其制备方法 技术领域
本发明涉及细胞培养技术领域，特别是涉及一种温度敏感型细胞培养表面及其制备方法。
背景技术
近年来，组织再生工程、生物医学工程、细胞治疗行业技术飞速发展，通过将动物或人体细胞在体外进行大规模培养的方式来获得大量细胞、细胞胞外分泌物、细胞治疗产品已成为目前细胞临床应用的重要方向。例如组织创伤、烧伤治疗中皮肤细胞的培养和新皮肤的获得、肿瘤的细胞治疗、细胞美容、细胞减肥等，都需要对目标细胞进行体外培养。
细胞按照其体外培养的特性主要分为悬浮细胞和贴壁细胞，悬浮细胞是悬浮于培养装置中培养液里面的，贴壁细胞的生长需要粘附在培养装置的表面才能生长。当贴壁细胞培养结束后，主要采用胰蛋白酶消化的方式从粘附表面将其处理下来完成收集，而这种消化方法会破坏细胞表面的膜蛋白，进而破坏细胞的完整性，失去完整性的细胞其应用受到极大的限制，尤其是在细胞临床应用领域。
目前市场上已有商品化的温度敏感型细胞培养表面，主要通过将温敏材料接枝到培养装置的表面来实现。该种表面能够在细胞培养结束后，通过改变温度使细胞自动脱落下来，减小对细胞的损伤。
现有的温度敏感性型细胞培养表面的制备方法中，部分通过直接在细胞培养装置表面物理涂布温敏化合物的方式，这种方式实现的温度敏感响应功能不稳定，在培养过程中，温敏化合物会脱落下来，因此细胞自动脱落效果无法保证；其它大部分是通过高能电子束引发温敏化合物在细胞培养表面的接枝，少部分采用紫外光辐照引发接枝、或者等离子引发接枝、或者原子转移自由基引发接枝，这些实现培养表面温敏化合物接枝的方式中，有的生产装备昂贵(例如高能电子束、等离子)，有的工艺复杂、所需原料成本高不利于产业化(例如 原子转移自由基引发、紫外光辐照引发)，有的接枝效率低、环境污染严重(如等离子)。
发明内容
基于此，有必要提供一种接枝率高，生产成本低且环境友好的温度敏感型细胞培养表面的制备方法。
一种温度敏感型细胞培养表面的制备方法，包括如下步骤：
(1)配制温敏原液：将温敏化合物和自由基引发剂加入至溶剂中，搅拌溶解，得温敏原液，所述温敏化合物与所述溶剂的质量比为(2-50)：100，所述自由基引发剂与所述温敏化合物的摩尔比为(0.5-50)：100；
(2)将所述温敏原液分布于细胞培养表面后，将该细胞培养表面置于50-150℃条件下反应5-120min，即得所述温度敏感型细胞培养表面。
根据需要，步骤(2)所述反应可在氮气保护下进行。
在其中一个实施例中，步骤(2)所述分布的方法为浸渍、雾化、喷涂或涂抹。
在其中一个实施例中，所述浸渍的方法为：将所述细胞培养表面浸渍在所述温敏原液中0.1-60min，然后取出；
所述雾化的方法为：先将所述温敏原液雾化为粒径在2-1000微米的颗粒，再将所述细胞培养表面曝露在所述雾化的氛围中5-100min，然后取出；
所述喷涂的方法为：利用高压喷涂或空气喷涂将所述温敏原液喷涂在所述细胞培养表面。
在其中一个实施例中，所述温敏化合物为N-异丙基丙烯酰胺、N-异丙基甲基丙烯酰胺、N-异丙基丙烯酰胺低聚物、N-异丙基甲基丙烯酰胺低聚物中的一种或多种；制备所述细胞培养表面的材料为聚烯烃或聚羧酸酯。
在其中一个实施例中，所述聚烯烃为聚苯乙烯、聚丙烯或聚乙烯；所述聚羧酸酯为聚碳酸酯、聚脂肪酸酯、聚芳香酸酯或聚丙烯酸酯。
在其中一个实施例中，所述自由基引发剂为过氧化物类引发剂(如过氧化苯甲酰、过氧化异丙苯、过硫酸钾、叔丁基过氧化物等)、偶氮类引发剂(如偶 氮二异丁腈、偶氮二异庚腈、偶氮二异丁脒盐酸盐(AIBA)，偶氮二异丁咪唑啉盐酸盐(AIBI)等)中的一种或多种。
在其中一个实施例中，所述溶剂为醇类(如甲醇、乙醇、丙醇、异丙醇)、酯类(如甲酸酯类如甲酸甲酯、甲酸乙酯、甲酸丙酯、甲酸丁酯；乙酸酯类如乙酸甲酯、乙酸乙酯、乙酸丙酯、乙酸丁酯)、酮类(丙酮、丁酮等)、氯代烃类(氯仿、二氯甲烷、氯乙烷等)中的一种或多种。
本发明还提供所述的温度敏感型细胞培养表面的制备方法制备得到的温度敏感型细胞培养表面。
本发明还提供具有所述的温度敏感型细胞培养表面的细胞培养装置。
在其中一个实施例中，所述细胞培养装置为细胞培养板、细胞培养瓶、细胞培养皿、设置有多层纤维组成的三维网状结构的细胞培养装置或设置有颗粒微载体的细胞培养装置。
与现有技术相比，本发明具有以下有益效果：
本发明所述的温度敏感型细胞培养表面的制备方法，合理配置所述温敏原液中温敏化合物和自由基引发剂的浓度，并将该温敏原液分布于细胞培养表面，可利用普通的化学自由基引发剂直接引发温敏化合物在细胞培养表面的接枝聚合，获得具有温度敏感性应答功能的细胞培养表面，相对于高能电子束和等离子接枝，无需购买大型昂贵设备，温敏原料用量低，接枝效率高；相对于紫外光辐照和原子转移自由基引发，引发剂原料来源广泛、成本低、工艺原理便于大规模制备。本发明制备的温敏细胞培养表面使贴壁型细胞培养结束后能够通过温度降低而实现温敏性脱落，减少化学消化脱落或者物理方法脱落对细胞造成的伤害，保证细胞膜蛋白的完整性，且较现有技术极大的降低了温度敏感型细胞培养表面的生产成本，接枝率高，环境友好。
进一步地，在对该制作方法的研究中发现，温敏原液的分布方式对最终的接枝率具有较为关键的影响，本发明优选为采用特定条件的浸渍、雾化、喷涂方式实现温敏原液在细胞培养表面的接枝，可有效提高接枝率。
附图说明
图1为利用本发明一实施例所述温度敏感型细胞培养表面将T3T细胞在37℃培养48小时后细胞贴附于装置表面的显微镜照片；
图2为将图1所述温度敏感型细胞培养表面在20℃处理10-40分钟后细胞成片自动脱附的显微镜照片。
具体实施方式
以下结合具体实施例对本发明的温度敏感型细胞培养表面及其制备方法作进一步详细的说明。
实施例1:
称取0.5gN-异丙基丙烯酰胺单体、与单体摩尔比为5：100的偶氮类引发剂溶解在7.5g异丙醇和乙酸乙酯的混合溶剂中；然后采用雾化器将该混合物溶液雾化为粒径在2-1000微米的颗粒，然后将欲处理的聚苯乙烯材料的细胞培养板表面暴露在该雾化汽氛围中50分钟使雾汽在欲处理的表面凝结，最后将其放置在充满氮气的烘箱中，在50-120℃保温50分钟后取出，即得具有温度敏感型细胞培养表面的细胞培养板，接枝率2.2μg/cm2。
实施例2：
称取0.7g N-异丙基甲基丙烯酰胺单体、与单体摩尔比为20：100的过氧化物类引发剂溶解在10g乙醇和甲酸丙酯、二氯甲烷混合溶剂中，然后采用高压喷涂将温敏混合物溶液均匀地喷涂在聚丙烯材料的细胞培养皿表面，最后将其放置在充满氦气的烘箱中，在90-120℃保温20分钟后取出，即得具有温度敏感型细胞培养表面的细胞培养皿，接枝率1.7μg/cm2。
实施例3：
称取0.6g N-异丙基丙烯酰胺单体、与单体摩尔比为30：100的偶氮类引发剂溶解在6g异丙醇、乙酸丁酯和二氯甲烷混合溶剂中，然后采用空气喷涂将该混合物溶液喷涂在聚苯乙烯材料的细胞培养瓶表面，然后将其放置在充满空气的烘箱中，在50-120℃保温30分钟后取出，即得具有温度敏感型细胞培养表面的细胞培养瓶，接枝率1.5μg/cm2。
实施例4：
称取3.5g N-异丙基丙烯酰胺单体、与单体摩尔比为40：100的偶氮类引发剂溶解在7g氯乙烷、乙醇和甲酸乙酯混合溶剂中，然后采用高压空气雾化法将该混合物溶液雾化为粒径在2-1000微米的颗粒，然后将欲处理的粒径为40-500微米的聚乙烯制备的微球载体暴露在该雾化汽氛围中10分钟使雾汽在欲处理微球载体表面凝结，最后将其放置在充满氮气的烘箱中，在50-120℃保温60分钟后取出，即得具有温度敏感型细胞培养表面的微球载体，接枝率1.6μg/cm2。
该球形颗粒微载体可放置于常规的细胞培养板、细胞培养瓶或细胞培养皿中，加入培养液，以细小的颗粒作为细胞粘附生长的载体，通过搅拌悬浮在培养液内，使细胞在载体表面繁殖成单层细胞。由于在该颗粒微载体上实现了温敏接枝，与常规的细胞培养板、瓶、皿等细胞培养器配套使用，成为温度敏感型的细胞培养装置。
实施例5：
称取0.3g N-异丙基丙烯酰胺单体、与单体摩尔比为1：100的过氧化物类引发剂溶解6g乙醇和乙酸乙酯、丙酮的混合溶剂中，然后将欲处理的聚碳酸酯制备的多层纤维三维网状多孔支架浸渍在温敏混合溶液中0.1-60分钟，然后取出风干，最后将其放置在充满氮气的烘箱中，在80-150℃保温30分钟后取出， 即得具有温度敏感型细胞培养表面的多层纤维三维网状多孔支架，接枝率1.5μg/cm2。
该多层纤维三维网状多孔支架为多层网状结构，可以作为多层的温敏支架置于常规的细胞培养板、细胞培养瓶或细胞培养皿内，在不改变与之配套的常规的细胞培养板、细胞培养瓶或细胞培养皿的规格的前题下，成倍增加细胞贴壁生长的比表面积，在有限的体积下收获更多的细胞。
将实施例1制作得到的温度敏感型细胞培养装置用于T3T细胞培养，T3T细胞在37℃培养48小时后细胞贴附显微镜照片如图1所示；然后在20℃处理10-40分钟后细胞成片自动脱附的显微镜照片如图2所示。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种温度敏感型细胞培养表面的制备方法，其特征在于，包括如下步骤：
    (1)配制温敏原液：将温敏化合物和自由基引发剂加入至溶剂中，搅拌溶解，得温敏原液，所述温敏化合物与所述溶剂的质量比为(2-50)：100，所述自由基引发剂与所述温敏化合物的摩尔比为(0.5-50)：100；
    (2)将所述温敏原液分布于细胞培养表面后，将该细胞培养表面置于50-150℃条件下反应5-120min，即得所述温度敏感型细胞培养表面。
  2. 根据权利要求1所述的温度敏感型细胞培养表面的制备方法，其特征在于，步骤(2)所述分布的方法为浸渍、雾化、喷涂或涂抹。
  3. 根据权利要求2所述的温度敏感型细胞培养表面的制备方法，其特征在于，所述浸渍的方法为：将所述细胞培养表面浸渍在所述温敏原液中0.1-60min，然后取出；
    所述雾化的方法为：先将所述温敏原液雾化为粒径在2-1000微米的颗粒，再将所述细胞培养表面曝露在所述雾化的氛围中5-100min，然后取出；
    所述喷涂的方法为：利用高压喷涂或空气喷涂将所述温敏原液喷涂在所述细胞培养表面。
  4. 根据权利要求1所述的温度敏感型细胞培养表面的制备方法，其特征在于，所述温敏化合物为N-异丙基丙烯酰胺、N-异丙基甲基丙烯酰胺、N-异丙基丙烯酰胺低聚物、N-异丙基甲基丙烯酰胺低聚物中的一种或多种；制备所述细胞培养表面的材料为聚烯烃或聚羧酸酯。
  5. 根据权利要求4所述的温度敏感型细胞培养表面的制备方法，其特征在于，所述聚烯烃为聚苯乙烯、聚丙烯或聚乙烯；所述聚羧酸酯为聚碳酸酯、聚脂肪酸酯、聚芳香酸酯或聚丙烯酸酯。
  6. 根据权利要求1-5任一项所述的温度敏感型细胞培养表面的制备方法，其特征在于，所述自由基引发剂为过氧化物类引发剂和/或偶氮类引发剂。
  7. 根据权利要求1-5任一项所述的温度敏感型细胞培养表面的制备方法，其特征在于，所述溶剂为醇类、酯类、酮类、氯代烃类中的一种或多种。
  8. 权利要求1-7任一项所述的温度敏感型细胞培养表面的制备方法制备得 到的温度敏感型细胞培养表面。
  9. 具有权利要求8所述的温度敏感型细胞培养表面的细胞培养装置。
  10. 根据权利要求9所述的细胞培养装置，其特征在于，该细胞培养装置为细胞培养板、细胞培养瓶、细胞培养皿、设置有多层纤维组成的三维网状结构的细胞培养装置或设置有颗粒微载体的细胞培养装置。
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