JP2008136895A - Method and apparatus for treating aqueous cleaning liquid containing temperature-responsive polymer compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an apparatus for washing a substrate having an immobilized temperature-responsive polymer and a free temperature-responsive polymer present on its surface. <P>SOLUTION: The washing method of the substrate having the immobilized temperature-responsive polymer and the free temperature-responsive polymer present on its surface comprises the washing process of washing the surface of the substrate with an aqueous washing liquid having a temperature less than the lower critical solution temperature of the temperature-responsive polymers to obtain the washing liquid containing the free temperature-responsive polymer, the precipitation process of increasing the temperature of the aqueous washing liquid after the washing to a temperature equal to or more than the lower critical solution temperature of the temperature-responsive polymers to precipitate the temperature-responsive polymer in the aqueous washing liquid, and the separation process of separating the temperature-responsive polymer precipitated in the precipitation process from the aqueous washing liquid. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method and apparatus for treating an aqueous cleaning liquid containing a temperature-responsive polymer compound, and a substrate having a surface on which a fixed temperature-responsive polymer compound and a free temperature-responsive polymer compound are present. The present invention relates to a cleaning method and apparatus.

  The cell sheet is a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding. Cell sheets are used in regenerative medicine. The cell sheet can be obtained by culturing cells on a support such as a petri dish, but the cell sheet formed on the support is firmly bonded to the support surface via an adhesion molecule or the like. It is not easy to quickly peel the cell sheet from the culture support without breaking the bonds between the cells.

  Thus, as a method for efficiently peeling the cell sheet from the cell culture support, methods using a cell culture support in which a temperature-responsive polymer compound is immobilized on a cell adhesion surface have been proposed (Patent Documents 1 and 2). ). The temperature-responsive polymer compound is hydrophilic at a temperature lower than the lower critical solution temperature and hydrophobic at a temperature equal to or higher than the lower critical solution temperature in an aqueous solution, so that the temperature-responsive polymer compound is immobilized. The surface of the cell culture support is an environment suitable for the formation of a cell sheet under a temperature condition equal to or higher than the lower critical lysis temperature, and an environment suitable for the peeling of the formed cell sheet at a temperature lower than the lower critical lysis temperature.

Japanese Patent Publication No. 6-104061 JP 2005-27532 A

  Immobilization of the temperature-responsive polymer compound on the surface of the cell culture support, for example, the monomer that is a precursor of the temperature-responsive polymer compound together with the necessary reagents such as a polymerization initiator, By carrying out electron beam irradiation (EB) or the like in the state of being present in the polymer, the polymerization reaction between the monomers and the binding reaction of the polymer to the substrate surface can proceed. When the temperature-responsive polymer compound is immobilized on the substrate surface of the cell culture support by this procedure, a large amount of free temperature-responsive polymer compound that is not immobilized on the substrate surface exists on the substrate surface. It becomes. Therefore, the surface of the substrate is washed with an aqueous cleaning solution to remove the free temperature-responsive polymer compound.

  Since the cleaning liquid generated in this way has a high BOD (biochemical oxygen demand), it is not preferable to directly dispose of it in the environment. Moreover, if the water after washing | cleaning can be reused, the consumption of water can be suppressed.

  Accordingly, an object of the present invention is to provide an effective means for removing the temperature-responsive polymer compound from the aqueous cleaning liquid containing the temperature-responsive polymer compound and reusing the cleaning liquid after the removal.

The present invention includes the following inventions.
(1) A method for treating an aqueous cleaning solution containing a temperature-responsive polymer compound, wherein the temperature of the aqueous cleaning solution containing the temperature-responsive polymer compound is increased to a temperature equal to or higher than the lower critical solution temperature of the temperature-responsive polymer compound. The method comprising: a precipitation step of precipitating a temperature-responsive polymer in an aqueous cleaning solution; and a separation step of separating the temperature-responsive polymer precipitated in the precipitation step from the aqueous cleaning solution.

(2) A method for cleaning a substrate in which an immobilized temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface, wherein the surface of the substrate is coated with the temperature-responsive polymer A washing step of obtaining an aqueous washing solution containing a free temperature-responsive polymer compound by washing with an aqueous washing solution having a temperature lower than the lower critical solution temperature of the compound, and the temperature-responsive polymer compound after the washing The method comprising the precipitation step of precipitating the temperature-responsive polymer in the aqueous cleaning liquid by raising the temperature to the lower critical solution temperature or higher, and the separation step of separating the temperature-responsive polymer precipitated in the precipitation step from the aqueous cleaning liquid .

(3) The method according to (2), wherein the temperature of the aqueous cleaning liquid after the separation step is reduced to less than the lower critical solution temperature and then reused in the cleaning step.
(4) The base material on which the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound exist on the surface has a monomer that is a precursor of the temperature-responsive polymer compound on the surface of the base material. The method according to (2) or (3), which is formed by causing the polymerization reaction and the binding reaction to the substrate surface to proceed on the substrate surface.

(5) The substrate on which the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound are present on the surface has a monomer that is a precursor of the temperature-responsive polymer compound on the substrate surface, and The method according to (2) or (3), which is formed by allowing a prepolymer to exist and causing a polymerization reaction and a binding reaction to the substrate surface to proceed on the substrate surface.
(6) The method according to any one of (2) to (5), wherein the substrate is a cell culture support.

(7) An apparatus for treating an aqueous cleaning liquid containing a temperature-responsive polymer compound, the liquid tank storing the aqueous cleaning liquid, and the temperature of the aqueous cleaning liquid stored in the liquid tank of the temperature-responsive polymer compound The said apparatus provided with the heating means which raises more than a lower critical solution temperature, and the isolation | separation means which isolate | separates the deposit of the temperature-responsive polymer formed by the said heating from an aqueous cleaning liquid.

(8) A cleaning apparatus for cleaning a substrate on which the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound are present, the surface of the substrate being subjected to the temperature Cleaning means for removing free temperature-responsive polymer compound from the surface of the substrate by washing with an aqueous cleaning solution having a temperature lower than the lower critical solution temperature of the responsive polymer compound, and temperature-responsive polymer compound generated by the washing A liquid tank containing a water-based cleaning liquid containing, a heating means for raising the temperature of the water-based cleaning liquid stored in the liquid tank to a temperature equal to or higher than a lower critical solution temperature of the temperature-responsive polymer compound, and the heating. Separating means for separating the temperature-responsive polymer precipitate from the aqueous cleaning liquid.

(9) A cooling means for lowering the temperature of the aqueous cleaning liquid after the separation below the lower critical solution temperature of the temperature-responsive polymer compound, and a supply means for supplying the aqueous cleaning liquid after cooling to the cleaning means A device according to (8).

  According to the present invention, it is possible to reduce the BOD of washing wastewater generated when a substrate having a temperature-responsive polymer compound immobilized on the surface thereof is produced. The cleaning liquid from which the temperature-responsive polymer compound has been removed according to the present invention can be reused for the purpose of washing and removing the temperature-responsive polymer compound.

(Temperature responsive polymer compound and substrate)
The temperature-responsive polymer compound used in the present invention refers to a polymer compound in which a hydrophobic state and a hydrophilic state reversibly change in water according to temperature conditions. The temperature-responsive polymer compound used in the present invention is a polymer compound that becomes hydrophobic and insoluble in water above a predetermined temperature and becomes hydrophilic and soluble in water below a predetermined temperature. It is. The predetermined temperature is referred to as a “lower critical solution temperature”. The behavior of the temperature-responsive polymer compound is schematically shown in FIG.

  As the temperature-responsive polymer compound that can be suitably used in the present invention, a lower critical solution temperature is lower than the cell culture temperature (usually about 37 ° C.) and is hydrophobic under the cell culture temperature conditions. Suitable temperature-responsive polymer compounds include, for example, polymer compounds described in Patent Document 1, and specifically, for example, poly-N-iso, propylacrylamide (lower critical solution temperature (LCST) = 32 ° C. ), Poly-Nn-propyl acrylamide (LCST = 21 ° C.), poly-Nn-propyl methacrylamide (LCST = 32 ° C.), poly-N-ethoxyethyl acrylamide (LCST = about 35 ° C.), poly- N-tetrahydrofurfuryl acrylamide (LCST = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (LCST = about 35 ° C.), poly-N, N-diethylacrylamide (LCST = 32 ° C.) and the like . Examples of other polymers include poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N- Polyalkylene oxide block copolymers represented by alkyl-substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, and polyalkylenes An oxide block copolymer is mentioned. These polymers are prepared, for example, by homopolymerization or copolymerization of monomers such that the monomer homopolymer has LCST = 0-80 ° C. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds may be used. In addition, when it is necessary to regulate LCST depending on the type of proliferating cells, when it is necessary to enhance the interaction between the coating substance and the cell culture support, or to adjust the hydrophilic / hydrophobic balance of the cell support. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above polymer and other polymer used in the present invention, or a mixture of the above polymer and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  In the present invention, the “substrate” is not particularly limited as long as the above-described temperature-responsive polymer compound can be chemically or physically immobilized on the surface thereof. Specifically, polystyrene, polyethylene, polycarbonate, Examples of the base material include urethane acrylate and nylon. The substrate is typically a cell culture support. Examples of the shape of the cell culture support include a sheet shape and a petri dish shape. The petri dish-shaped cell culture support includes, as at least the bottom thereof, a base material on which a temperature-responsive polymer compound is immobilized on the surface facing the petri dish.

  The present invention relates to a method for cleaning a substrate on which a fixed temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface, or a method for treating an aqueous cleaning solution generated as waste water by the cleaning. The “substrate having the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound on the surface” as used herein is typically a temperature-responsive polymer compound on the substrate surface. It is formed by allowing the monomer, which is a precursor, to be present, or the monomer and a prepolymer that has been polymerized to some extent, to advance the polymerization reaction and the binding reaction to the substrate surface on the substrate surface. Various methods such as electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, etc., preferably electrons are used as a method for proceeding the polymerization reaction and the binding reaction to the substrate surface on the substrate surface. The method of advancing a polymerization reaction and the covalent bond formation reaction between the produced | generated polymerization compound and a base-material constituent material by an irradiation process is mentioned. In this case, a polymerization initiator or the like can coexist as necessary. On the surface of the base material treated in this manner, there are immobilized temperature-responsive polymer compounds and free temperature-responsive polymer compounds that have not been immobilized.

(Aqueous cleaning solution)
In the present invention, the “aqueous cleaning liquid” is typically water, but is not limited thereto, and may be an aqueous solution in which various water-soluble salts and hydrophilic organic solvents (for example, alcohols) are dissolved.

(Method and apparatus for treating aqueous cleaning liquid containing temperature-responsive polymer compound)
The present invention is a method for treating an aqueous cleaning solution containing a temperature-responsive polymer compound, wherein the temperature of the aqueous cleaning solution containing the temperature-responsive polymer compound is increased to a temperature equal to or higher than the lower critical solution temperature of the temperature-responsive polymer compound. The method includes a precipitation step of precipitating the temperature-responsive polymer in the aqueous cleaning solution by the step, and a separation step of separating the temperature-responsive polymer deposited in the precipitation step from the aqueous cleaning solution. The invention also relates to an apparatus for carrying out the method. The “aqueous cleaning solution containing a temperature-responsive polymer compound” to be treated is typically a group in which an immobilized temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface. It is generated as a discharged liquid when the material is washed with an aqueous washing liquid having a temperature lower than the lower critical solution temperature of the temperature-responsive polymer compound. Hereinafter, an embodiment of the present invention will be described with reference to FIG.

  The aqueous cleaning liquid containing the temperature-responsive polymer compound is stored in the liquid tank 21. The liquid tank 21 is provided with a heating device (heating means) 22, and the temperature of the aqueous cleaning liquid in the liquid tank 21 is higher than the lower critical solution temperature of the temperature-responsive polymer compound by the heater (heating means) 22. Enhanced. In order to perform heating efficiently, the liquid tank 21 may further be provided with stirring means (typically, a magnetic stirrer, a stirring blade, etc.) for stirring the aqueous cleaning liquid to be processed. The temperature-responsive polymer compound in the aqueous cleaning liquid is precipitated as a solid by heating in the liquid tank 21. In the precipitation step, the temperature of the aqueous cleaning liquid is preferably increased to T + 0 to 20 ° C. when the lower critical solution temperature of the temperature-responsive polymer compound is T ° C. The aqueous cleaning liquid containing the precipitate generated by heating is transferred to a filter (separation means) 23 and filtered, and the cleaning water from which the temperature-responsive polymer compound has been removed is recovered as a filtrate, and the temperature-responsive polymer is recovered. Is recovered as a residual solid. The separation means is not limited to a filter, and a precipitation separation tank that collects the precipitated temperature-responsive polymer as a precipitate and collects washing water from which the temperature-responsive polymer compound has been removed as a supernatant is used. Can do. Moreover, the combination of a centrifuge tube and a centrifuge can also be used as a separating means. It is preferable that the separation means be capable of maintaining the temperature of the separation object above the lower critical solution temperature during the separation operation.

  Moreover, it is easy to integrate the liquid tank provided with the heating means and the separation means. For example, an aqueous cleaning solution containing a temperature-responsive polymer compound is directly put into a precipitation separation tank equipped with a heating means, and the temperature-responsive polymer compound is precipitated as a precipitate in the precipitation separation tank, and the deposited temperature responsiveness In the embodiment in which the polymer compound is recovered as a precipitate and the washing water from which the temperature-responsive polymer compound has been removed is recovered as a supernatant, the precipitation separation tank is regarded as an integrated liquid tank and separation means. be able to.

(Cleaning method and apparatus for base material having immobilized temperature-responsive polymer compound and free temperature-responsive polymer compound on the surface)
The present invention also provides a cleaning method for a substrate in which an immobilized temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface, the surface of the substrate being subjected to the temperature-responsive property. A washing step of washing with an aqueous washing liquid having a temperature lower than the lower critical solution temperature of the polymer compound to obtain an aqueous washing liquid containing a free temperature-responsive polymer compound, and the temperature of the aqueous washing liquid after washing is set to a high temperature responsiveness. A precipitation step of precipitating the temperature-responsive polymer in the aqueous cleaning liquid by raising the molecular compound to a temperature lower than the lower critical solution temperature of the molecular compound; and a separation step of separating the temperature-responsive polymer precipitated in the precipitation step from the aqueous cleaning liquid. It relates to said method. The invention also relates to an apparatus for carrying out the method. Hereinafter, an embodiment of the present invention will be described with reference to FIG. In FIG. 3, the names and functions of the components having the same reference numerals as those in FIG. 2 are the same as those described with reference to FIG.

  A cell having a temperature-responsive polymer compound and a free temperature-responsive polymer compound immobilized on a surface of a water-based cleaning solution having a temperature lower than the lower critical solution temperature using a cleaning water jet device (cleaning means) 31 It sprays on the culture support body (base material) 32. However, the cleaning method is not limited to the injection of cleaning water as shown in FIG. For example, it is also possible to prepare a liquid tank in which cleaning water is stored and perform the cleaning by immersing the substrate to be cleaned in the liquid tank. The temperature of the aqueous cleaning solution used for cleaning is preferably T-1 to 32 ° C when the lower critical solution temperature of the temperature-responsive polymer compound is T ° C. In FIG. 3, the substrate to be cleaned is a petri dish-shaped cell culture support 32, but is not limited thereto. In FIG. 3, a plurality of petri dish-shaped cell culture supports 32 are depicted as being simultaneously washed, but the present invention is not limited to this, and it is possible to wash one by one. The discharged liquid generated by the cleaning contains a temperature-responsive polymer compound derived from the surface of the substrate. The discharged liquid is collected by the collector 33 and stored in the liquid tank 21. The procedure for treating the discharged liquid stored in the liquid tank 21 is as described in the section “Method and apparatus for treating aqueous cleaning liquid containing temperature-responsive polymer compound”. Finally, the aqueous cleaning liquid from which the temperature-responsive polymer compound has been removed is separated from the temperature-responsive polymer compound.

  The aqueous cleaning liquid from which the temperature-responsive polymer compound has been removed by the above procedure can be reused in the cleaning process. FIG. 4 shows a typical apparatus configuration in the case of reuse. In FIG. 4, the names and functions of the constituent elements to which the same reference numerals as those in FIG. 2 or FIG. 3 are added are the same as the constituent elements described with reference to FIG. The aqueous cleaning liquid from which the temperature-responsive polymer compound has been removed is a temperature below the lower critical solution temperature of the free temperature-responsive polymer compound present on the substrate to be cleaned by the cooler (cooling means) 41, preferably the lower limit. When the critical melting temperature is T ° C., it is cooled to T-1 to 32 ° C. The cooled cleaning liquid is supplied to the cleaning water injection device (cleaning means) 31 by the pump (supply means) 42 and reused in the cleaning process.

A substrate cleaning system as shown in FIG. 3 was constructed and the following tests were performed.
Water having a water temperature of 25 ° C. was used as washing water.

The substrate to be cleaned was prepared by the following procedure. An isopropyl alcohol solution of N-isopropylacrylamide (40 wt%) and polyisopropylacrylamide (2 wt%) is applied to a 130 μm polystyrene sheet (1 m ² ) with a wire bar # 4 so that the coating thickness is about 5 g / m ^2. It was coated and dried with a dryer at 60 ° C. for 1 minute. Next, the coated surface was irradiated with an electron beam in two portions. The irradiation amount was 5 Mrad for the first time and 25 Mrad for the second time. After electron beam irradiation, it was immersed in water at 5 ° C. for 12 hours.

  The substrate prepared in the above procedure was washed by spraying water with a water temperature of 25 ° C., and 1000 ml of waste water was collected.

  When the temperature of the collected waste water was increased to 45 ° C. higher than the lower critical solution temperature (32 ° C.) of the polymer, the temperature-responsive polymer was precipitated and precipitated as a solid content.

  The precipitated temperature-responsive polymer was separated by suction filtration of the drained water after precipitation (filter paper No. 3 150 mm).

  The amount of the temperature-responsive polymer compound in the wastewater was quantified by measuring the solid content weight of the wastewater before evaporation after evaporation of water. Further, the amount of the temperature-responsive polymer in the filtrate after filtration was quantified by the same procedure. The removal amount (reduction rate) was evaluated by comparing the amount of the temperature-responsive polymer in the washing liquid before and after the filtration determined in this way.

  As a comparative example, the same operation as in the above experiment (Example) was performed except that the washing waste water was not heated to 45 ° C., and the removal amount of the temperature-responsive polymer compound was evaluated.

  The results are shown in Table 1. It became clear that the temperature-responsive polymer compound in the waste water can be removed by cooling the waste water after washing.

It is a figure which shows typically the behavior under the temperature conditions of the temperature-responsive high molecular compound which has a lower limit critical solution temperature below the said temperature or above. It is a figure which shows the structure of the apparatus for performing the processing method of the aqueous cleaning liquid containing a temperature-responsive high molecular compound based on this invention. It is a figure which shows the structure of the apparatus for performing the washing | cleaning method of the base material which the fixed temperature responsive high molecular compound and free temperature responsive high molecular compound which exist on the surface based on this invention exist. In accordance with the present invention, there is provided a method for cleaning a substrate on which a fixed temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface, the method for reusing cleaning water It is a figure which shows the structure of an apparatus.

Explanation of symbols

21: liquid tank, 22: heater, 23: filter, 31: washing water injection device, 32: cell culture support, 33: collector, 41: cooler, 42: pump

Claims (9)

  A method for treating an aqueous cleaning solution containing a temperature-responsive polymer compound, wherein the temperature of the aqueous cleaning solution containing a temperature-responsive polymer compound is increased to a temperature equal to or higher than the lower critical solution temperature of the temperature-responsive polymer compound. The method comprising: a precipitation step of precipitating the temperature-responsive polymer; and a separation step of separating the temperature-responsive polymer precipitated in the precipitation step from the aqueous cleaning liquid.   A method for cleaning a substrate in which a fixed temperature-responsive polymer compound and a free temperature-responsive polymer compound are present on the surface, the surface of the substrate being a lower limit of the temperature-responsive polymer compound A washing step of washing with an aqueous washing liquid having a temperature lower than the critical dissolution temperature to obtain an aqueous washing liquid containing a free temperature-responsive polymer compound, and the temperature of the aqueous washing liquid after washing is a lower criticality of the temperature-responsive polymer compound The method comprising: a precipitation step of precipitating a temperature-responsive polymer in the aqueous cleaning liquid by raising the temperature to a melting temperature or higher; and a separation step of separating the temperature-responsive polymer precipitated in the precipitation step from the aqueous cleaning liquid.   The method according to claim 2, wherein the temperature of the aqueous cleaning liquid after the separation step is reduced to less than the lower critical solution temperature and then reused in the cleaning step.   The substrate on which the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound exist on the surface causes the monomer that is a precursor of the temperature-responsive polymer compound to exist on the substrate surface, The method according to claim 2 or 3, wherein the method is formed by advancing a polymerization reaction and a binding reaction to the substrate surface on the substrate surface.   The substrate on which the immobilized temperature-responsive polymer compound and the free temperature-responsive polymer compound are present on the surface is coated with a monomer and a prepolymer that are precursors of the temperature-responsive polymer compound on the substrate surface. The method according to claim 2 or 3, wherein the method is formed by causing the polymerization reaction and the binding reaction to the substrate surface to proceed on the surface of the substrate.   The method according to any one of claims 2 to 5, wherein the substrate is a cell culture support.   An apparatus for treating a water-based cleaning liquid containing a temperature-responsive polymer compound, the liquid tank storing the water-based cleaning liquid, and the temperature of the water-based cleaning liquid stored in the liquid tank is the lower critical solution of the temperature-responsive polymer compound The said apparatus provided with the heating means which raises more than temperature, and the isolation | separation means which isolate | separates the deposit of the temperature-responsive polymer formed by the said heating from an aqueous cleaning liquid.   A cleaning apparatus for cleaning a substrate on which a fixed temperature-responsive polymer compound and a free temperature-responsive polymer compound are present, wherein the surface of the substrate has a high temperature-responsive property. Washing means for removing free temperature-responsive polymer compound from the surface of the substrate by washing with an aqueous washing solution having a temperature lower than the lower critical solution temperature of the molecular compound, and an aqueous system containing the temperature-responsive polymer compound generated by the washing A liquid tank for storing the cleaning liquid, a heating means for increasing the temperature of the aqueous cleaning liquid stored in the liquid tank to be equal to or higher than a lower critical solution temperature of the temperature-responsive polymer compound, and a temperature responsiveness formed by the heating Separating means for separating the polymer precipitate from the aqueous cleaning liquid.   The cooling means for lowering the temperature of the water-based cleaning liquid after the separation below the lower critical solution temperature of the temperature-responsive polymer compound, and the supply means for supplying the water-based cleaning liquid after cooling to the cleaning means. 8. The apparatus according to 8.

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