JP5862211B2 - Method for determining washing conditions for cell culture supports - Google Patents

Description

  The present invention relates to a method for determining washing conditions for a cell culture support, and more particularly to a method for determining washing conditions for a cell culture support using a temperature-responsive polymer.

  In recent years, cells collected from patients in the field of regenerative medicine are cultured in vitro to produce sheet-like cell aggregates (hereinafter referred to as cell sheets) and transplanted into the living body to achieve medical effects. There are attempts to increase it.

  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 bound to the support surface via adhesion molecules, etc. -It is not easy to peel the cell sheet from the cell culture substrate without breaking the bond between cells. Therefore, methods for efficiently peeling the cell sheet from the cell culture substrate have been studied.

  As a technique for non-invasively peeling the cell sheet from the cell culture substrate without breaking the cell-cell bond, a temperature-responsive polymer such as poly-N-isopropylacrylamide whose cell adhesion level changes at ambient temperature is used. The cell culture support used has been reported. For example, in Patent Document 1, a solution containing an acrylamide monomer is developed on a base material, and the monomer is graft polymerized by irradiating it with an electron beam to immobilize poly-N-isopropylacrylamide, which is a temperature-responsive polymer. Cell culture supports that have been allowed to report have been reported.

JP-A-5-192130

  By the way, unreacted monomers, oligomers, prepolymers and the like remain in the film of the temperature-responsive polymer formed by the polymerization reaction by electron beam irradiation or the like. Therefore, a cell culture support on which a temperature-responsive polymer film is formed usually undergoes washing steps under various conditions. However, a method for determining the washing conditions has not been established so far. In order to accurately assure the quality of cell culture supports, early establishment of a method for determining suitable washing conditions is required.

  The present invention has been made in view of the above circumstances, the purpose of which is, the washing conditions in the washing step necessary to guarantee the quality of the cell culture support using a temperature-responsive polymer, It is to provide a method for simple determination.

  Specifically, the present invention provides the following.

  (1) A method for determining washing conditions for a cell culture support in which a temperature-responsive polymer formed by a polymerization reaction of a polymer-forming material is immobilized on a substrate surface by a covalent bond, the cell culture A washing step for washing the support, a dipping step for immersing the cell culture support after washing in a liquid under conditions capable of extracting a non-immobilized substance not immobilized on the substrate surface, and An absorbance measurement step for measuring the absorbance of the liquid after the dipping step, and calculating the amount of the non-immobilized substance from the measured value of the absorbance obtained in the absorbance measurement step, and the calculated amount of the non-immobilized substance, A comparison step for comparing a predetermined threshold value for the amount of the non-immobilized substance, and selecting appropriate washing conditions for the cell culture support based on the comparison result in the comparison step, The above method.

  (2) The method according to (1), wherein the polymer-forming material contains at least N-isopropylacrylamide monomer.

  According to the method of the present invention, the washing conditions in the washing step necessary for assuring the quality of the cell culture support using the temperature-responsive polymer can be easily determined.

FIG. 1 is a graph showing an absorption spectrum of a solution in which an unwashed cell culture support is immersed. FIG. 2 is a graph showing a change in absorbance at a wavelength of 190 nm of a liquid in which an unwashed cell culture support is immersed. 3A is a graph showing an absorption spectrum for each concentration of an N-isopropylacrylamide solution, and FIG. 3B is a poly-N-isopropylacrylamide solution. FIG. 4 is a graph showing a fitting result of an absorption spectrum of a solution in which an unwashed cell culture support is immersed and an absorption spectrum of a solution containing N-isopropylacrylamide and poly-N-isopropylacrylamide. FIG. 5 is a graph showing an absorption spectrum of a liquid in which a cell culture support is washed or not washed.

  Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and may be implemented with appropriate modifications within the scope of the object of the present invention. can do.

  The method of the present invention is a method for determining washing conditions for a cell culture support in which a temperature-responsive polymer formed by a polymerization reaction of a polymer-forming material is immobilized on a substrate surface by a covalent bond. Since the unreacted monomer, oligomer, prepolymer, etc. remain in the temperature-responsive polymer film formed by the polymerization reaction by electron beam irradiation, etc., the cell culture support on which the temperature-responsive polymer film is formed Usually undergoes a washing step to remove the residue. The present invention is the first to find a method that can easily determine the washing conditions in the washing step necessary for guaranteeing the quality of the cell culture support as described above, which has not been established so far. it makes sense.

[Cell culture support]
First, the cell culture support in the present invention will be described. In the present invention, in the cell culture support, a temperature-responsive polymer formed by a polymerization reaction of a polymer-forming material is immobilized on a substrate surface by a covalent bond. That is, in the cell culture support in the present invention, a temperature-responsive polymer layer is formed on the surface of the substrate.

<Base material>
The type of material is not particularly limited as long as the base material can form a temperature-responsive polymer layer, which will be described later, on one surface, and has water resistance that can withstand cell culture. Typically, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE) ), Vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic and the like.

  The surface of the substrate on which the temperature-responsive polymer layer is formed may be subjected to an easy adhesion treatment. Examples of the easy adhesion treatment include treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, and perfluorooctane sulfonic acid (PFOS).

  Although there is no restriction | limiting in particular in the thickness of a base material layer, It is preferable that it is the thickness which provides flexibility, for example, 10-300 micrometers, Preferably it is 30-250 micrometers.

(Temperature responsive polymer)
The temperature-responsive polymer is a polymer that has a surface that can change from cell adhesiveness to cell non-adhesiveness at a predetermined temperature, and the degree of cell adhesion on the surface changes at a predetermined temperature. As the temperature-responsive polymer, it is preferable to use a polymer that exhibits cell adhesiveness at a temperature at which cells are cultured and exhibits cell non-adhesiveness at a temperature at which the produced cell sheet is peeled. For example, a temperature-responsive polymer has improved affinity to surrounding water at temperatures below the critical dissolution temperature, and the polymer takes up water and swells to make it difficult for cells to adhere to the surface (cell non-adhesiveness). It is preferable that the temperature is equal to or higher than the same temperature so that water is desorbed from the polymer and the polymer contracts to easily adhere cells to the surface (cell adhesion). Such a critical solution temperature is called a lower critical solution temperature. It is preferable to use a temperature-responsive polymer having a lower critical solution temperature T of 0 ° C to 80 ° C, more preferably 0 ° C to 50 ° C. This is because cells can be stably cultured when T is 0 ° C to 80 ° C.

  Suitable temperature-responsive polymers include acrylic polymers or methacrylic polymers, and specifically include poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propylacrylamide (T = 21). ° C), poly-Nn-propylmethacrylamide (T = 32 ° C), poly-N-ethoxyethylacrylamide (T = about 35 ° C), poly-N-tetrahydrofurfurylacrylamide (T = about 28 ° C), Poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), poly-N, N-diethylacrylamide (T = 32 ° C.) and the like can be mentioned.

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. 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, vinyl ether derivatives, and the like. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when a plurality of monomers are used in combination, the polymer formed on the substrate is a heteropolymer.

  The thickness of the temperature-responsive polymer layer is, for example, preferably in the range of 0.5 nm to 300 nm, and more preferably in the range of 1 nm to 100 nm. By making the film thickness in the range of 0.5 nm to 300 nm, it is easy to achieve both cell adhesion and peeling.

  The temperature-responsive polymer layer is prepared by preparing a temperature-responsive polymer layer-forming coating solution containing the above-mentioned monomer that can be polymerized to form the target temperature-responsive polymer and a solvent that can dissolve the monomer. In addition, according to a conventional method, the coating liquid is applied to the surface of the base material to form a coating film, and the monomer in the coating film is polymerized by an appropriate means such as irradiation to form a polymer. It can be formed by causing a grafting reaction between the surface of the substrate and the polymer.

The monomer content in the temperature-responsive polymer layer forming coating solution is preferably 5 to 70% by weight. In addition to the monomer, the coating composition may contain an oligomer or prepolymer obtained by polymerizing a plurality of monomers. The coating amount of the temperature-responsive polymer layer may be a coating amount necessary for the grafted polymer to exhibit temperature responsiveness, for example, 5 to 80 μg / cm ² , and 10 to 50 μg / cm ^2. Is preferred.

  Examples of the radiation used for polymerizing the monomer include α rays, β rays, γ rays, electron beams, and ultraviolet rays. After the irradiation step, the coating film is dried as necessary to remove the solvent.

[Method of determining washing conditions for cell culture support]
The method of the present invention is a method for determining the washing conditions for the cell culture support. The cell culture support produced as described above undergoes a washing step from the viewpoint of assuring its quality. On the surface of the temperature-responsive polymer layer after graft polymerization, there are not only polymers immobilized by covalent bonds, but also free polymers that are not immobilized, unreacted monomers, oligomers, prepolymers, etc. It is thought that. These free polymers and unreacted substances (hereinafter referred to as non-immobilized substances) may have some influence on cell growth and are preferably removed. For example, N-isopropylacrylamide, which is generally selected as a monomer capable of forming a temperature-responsive polymer, is known to be toxic and its residue should be avoided as much as possible. Therefore, the non-immobilized substance is removed by washing. According to the method of the present invention, appropriate washing conditions for the cell culture support can be easily determined.

  The method of the present invention includes a washing step, an immersion step, an absorbance measurement step, and a comparison step, and selects appropriate washing conditions for the cell culture support based on the comparison result in the comparison step. is there.

<Washing process>
The washing step is a step of washing the cell culture support. In this washing step, one or more washing conditions that can be candidates are selected, and the cell culture support is washed. The washing method in this washing step is not particularly limited, and for example, a method usually performed in the washing step of the cell culture support may be selected. For example, immersion cleaning, rocking cleaning, shower cleaning, spray cleaning, ultrasonic cleaning, and the like can be given. As the cleaning liquid, typically, various water-based, alcohol-based, hydrocarbon-based, chlorine-based, acid / alkali cleaning liquids are used. The combination of the washing method and the washing solution is not particularly limited, and may be appropriately selected according to the cell culture support. An example is a method of ultrasonic cleaning using deionized water at a temperature sufficient to swell the polymer coated on the substrate.

<Immersion process>
The dipping step is a step of immersing the cell culture support after washing in the washing step in a liquid under conditions that allow extraction of a non-immobilized substance that is not immobilized on the substrate surface. In this dipping process, non-immobilized substances remaining on the cell culture support that could not be removed under the cleaning conditions selected in the above-described cleaning process are extracted. Examples of the liquid to be immersed include water, methanol, tetrahydrofuran, toluene, dimethylacetamide, N-methylpyrrolidone, and the like, and may be appropriately selected from these. The liquid temperature is not particularly limited, but is preferably not higher than the transition temperature of the immobilized temperature-responsive polymer. This is because the temperature-responsive polymer becomes hydrophilic below the transition temperature, and thus it is considered that the temperature-responsive polymer that is not immobilized is likely to be eluted in the solvent. Although the immersion time is not particularly limited, it is preferably 4 hours or longer when immersed in water. If the immersion time in water is less than 4 hours, extraction of the non-immobilized substance may be insufficient.

<Absorbance measurement process>
The absorbance measurement step is a step of measuring the absorbance of the liquid after the immersion step. In this absorbance measurement step, the absorbance of the liquid after the cell culture support is immersed in the immersion step is measured. For the measurement, ultraviolet / visible spectroscopy is used. The measurement apparatus typically uses an ultraviolet / visible spectrophotometer. The ultraviolet / visible spectroscopic analysis method is a method of measuring absorption of light when an electron circulating around a nucleus or molecule transitions from a low energy activation to a higher energy orbit. Since the ultraviolet / visible spectroscopic analysis method is a known method, it will be briefly described here. A light source having a wavelength in the visible light to ultraviolet light region is placed, the light emitted from the light source is wavelength-resolved by a spectrometer, and the light absorption amount of the sample and the light absorption amount of the sample are sampled by beam splitting or the like. Then, it divides into. Light that samples the amount of light absorption passes through a transparent cell such as glass or quartz containing an analysis sample. Finally, the amount of light is measured by the light receiving element. An absorption spectrum is obtained by measuring the amount of light absorption for each wavelength. The measurement wavelength of absorbance may be appropriately selected according to the type of temperature-responsive polymer, that is, according to the type of non-immobilized substance that can remain. Preferably, the vicinity of the maximum absorption wavelength of the non-immobilized substance that can remain may be selected. This is because the comparison in the comparison process is easy.

<Comparison process>
The comparison step calculates the amount of the non-immobilized substance from the absorbance measurement value obtained in the absorbance measurement step, and calculates the amount of the non-immobilized substance and a preset threshold of the amount of the non-immobilized substance. And a step of comparing the above. There is a correlation between the concentration of the non-immobilized substance and the absorbance. Therefore, in the method of the present invention, for example, the absorbance is first measured for a sample containing a supposed non-immobilized substance at a predetermined concentration to obtain one or more absorption spectra. Next, the fitting coefficient of the one or more absorption spectra is set so that the one or more absorption spectra are fitted to the absorption spectrum obtained in the absorbance measurement step. The amount of non-immobilized substance contained in the liquid subjected to the absorbance measurement step is calculated from the fitting coefficient and the predetermined concentration value. Next, based on experimental results such as cytotoxicity, the allowable limit concentration of the non-immobilized substance is examined. Then, based on the permissible limit concentration, the threshold value of the non-immobilized substance is determined, and the threshold value is compared with the amount of the non-immobilized substance contained in the liquid subjected to the absorbance measurement process, thereby cleaning step It is possible to determine whether or not the cleaning conditions are good.

  For example, when the amount of the non-immobilized substance calculated from the measured value of the absorbance of the liquid after the dipping process is less than a predetermined threshold, it can be determined that the cleaning conditions in the cleaning process are appropriate. If it exceeds, it can be determined that the cleaning conditions in the cleaning step are not appropriate. In the method of the present invention, appropriate washing conditions for the cell culture support can thus be selected.

[Method for producing cell culture support]
The method for producing a cell culture support of the present invention comprises an immobilization step of immobilizing a temperature-responsive polymer on a substrate surface, and a washing step of washing the substrate surface on which the temperature-responsive polymer is immobilized. In the washing step, the cell culture support is washed under the conditions determined by the above method. Since the method for immobilizing the temperature-responsive polymer on the substrate surface in the immobilization step has already been described above, a description thereof will be omitted. In the method for producing a cell culture support of the present invention, it is determined that the method of the present invention (method for determining the washing conditions for the cell culture support) is appropriate, and the conditions determined as the washing conditions for the washing step are as follows: The prepared cell support is washed.

  According to the method for producing a cell culture support of the present invention, the cell culture support after production is prepared under appropriate washing conditions that are determined in advance by the method of the present invention and the non-immobilized substance is less than the allowable limit concentration. Since the washing step is included, it is possible to obtain a cell culture support having a quality in which the amount of non-immobilized substances such as unreacted monomers, oligomers and prepolymers is less than the allowable limit value.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention does not receive a restriction | limiting at all by these description.

[Production Example 1]
(Manufacture of cell culture support)
N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final concentration of 40% by weight to prepare an N-isopropylacrylamide solution. As a base material, a polystyrene film sheet (OPS sheet, manufactured by SUNDIC) was prepared. The N-isopropylacrylamide solution was developed on this polystyrene film sheet, and Miyabar NO. 4 coated. Electron beam irradiation was performed using an electron beam irradiation apparatus (manufactured by Iwasaki Electric Co., Ltd.), and poly-N-isopropylacrylamide was formed on the polystyrene film sheet surface by graft polymerization. The electron beam irradiation dose at this time was 300 kGy. And the polystyrene film sheet | seat with which poly-N-isopropylacrylamide was couple | bonded was cut out circularly, and it was set as the cell culture support body for a test.

<Test 1>
The cell culture support (surface area: 84.6 cm ² ) obtained in Production Example 1 was affixed to the bottom of a dish (tissue culture dish, diameter: 15 cm, manufactured by IWAKI), added with pure water, and immersed for 3 days. did. Immersion was performed in an incubator at 37 ° C. And the liquid under immersion was extract | collected with time, The light absorbency was measured with the ultraviolet and visible spectrophotometer (UV-3100PC, a measurement wavelength: 190-220 nm, Shimadzu Corp. make). The result is shown in FIG.

  As shown in FIG. 1, the absorbance of the solution in which the cell culture support is immersed tends to increase with the lapse of the immersion time, and the tendency is strong particularly in the measurement range of 190 to 220 nm in the wavelength range of 190 nm. It was. FIG. 2 shows changes with time in absorbance at a wavelength of 190 nm. When the cell culture support was immersed in water, it was confirmed that the absorbance at a wavelength of 190 nm increased with the lapse of immersion time. From this, it was speculated that the non-immobilized substance extracted from the cell culture support had strong absorption at least near the wavelength of 190 nm.

<Test 2>
N-isopropylacrylamide (monomer) was dissolved in pure water to prepare liquids having various concentrations (1 ppm, 10 ppm, 100 ppm, 0.1 mass%, 1 mass%, 5 mass%). The absorbance of these solutions was measured with an ultraviolet / visible spectrophotometer (UV-3100PC, measurement wavelength: 190 to 220 nm, manufactured by Shimadzu Corporation). The result is shown in FIG. In addition, poly-N-isopropylacrylamide (polymer) was dissolved in pure water to prepare liquids having various concentrations (100 ppm, 0.1 mass%, 1 mass%, 5 mass%). The absorbance of these solutions was measured in the same manner. The result is shown in FIG.

  As shown in FIGS. 3A and 3B, both the monomer and the polymer had strong absorption near a wavelength of 190 nm. In addition, a correlation was observed between the absorbance and concentration of the monomer and polymer in the vicinity of a wavelength of 190 nm.

<Test 3>
The cell culture support (surface area: 150 cm ² ) obtained in Production Example 1 was attached to the bottom of a dish (tissue culture dish, diameter: 15 cm, manufactured by IWAKI), pure water was added, and the mixture was immersed for 24 hours. Immersion was performed in an incubator at 37 ° C. And the liquid after immersion was extract | collected and the light absorbency was measured with the ultraviolet and visible spectrophotometer (UV-3100PC, a measurement wavelength: 190-220 nm, Shimadzu Corp. make). The absorbance of the liquid after immersion is due to the non-immobilized substance, and the non-immobilized substances estimated from the reaction system are N-isopropylacrylamide (monomer) and poly-N-isopropylacrylamide (polymer). . On the other hand, according to Lambert-Beer's law, the absorbance is proportional to the solute concentration in the solution, and the result according to this law was also obtained in Test 2 above. Therefore, based on the results of Test 2, the absorption spectra at various concentrations of the monomer and polymer were calculated, and the concentrations that coincided with the absorption spectra of the liquid after immersion were examined. The fitting results are shown in FIG.

  As a result, the absorption spectrum of the liquid after the immersion showed a tendency to agree well with the absorption spectrum of the polymer (not shown). This suggested that the main component of the liquid after immersion was the polymer. Next, when an absorption spectrum was calculated when a very small amount of the monomer was added to the polymer, the absorption spectra were more consistent as shown in FIG.

<Test 4>
N-isopropylacrylamide was dissolved in Dulbecco's modified Eagle medium manufactured by Sigma and various concentrations (10 ⁻⁶ , 10 ⁻⁵ , 10 ⁻⁴ , 10 ⁻³ , 10 ⁻² , 10 ⁻¹ , ¹ , 10 mg / ml) A liquid was prepared. Using the obtained solution, bovine vascular endothelial cells were cultured to confluence in a 35 mm dish manufactured by BD falcon and observed.

  As a result, abnormalities were observed in the cell culture form at 1 mg / ml or more, and it became clear that there was a toxicity threshold in the range of 0.1 mg / ml to 1 mg / ml. However, since the sensitivity to toxicity varies greatly depending on the cell type, in consideration of safety, the threshold is 1 μg / ml, which is two orders of magnitude lower than the lower limit of the concentration obtained in Test 4 (0.1 mg / ml). The allowable value of the residual value of N-isopropylacrylamide in the culture support was set.

<Test 5>
The cell culture support obtained in Production Example 1 was washed with running water at 20 ° C. Washing was performed under three conditions of 10 seconds, 30 seconds, and 2 minutes. Each cell culture support after washing was dried, and then attached to the bottom of a dish (tissue culture dish, diameter: 15 cm, manufactured by IWAKI), 20 ml of pure water was added, and immersed for 3 days. Immersion was performed in an incubator at 37 ° C. The liquid after immersion was collected, and the absorbance was measured with an ultraviolet / visible spectrophotometer (UV-3100PC, measurement wavelength: 190 to 220 nm, manufactured by Shimadzu Corporation). In addition, the cell culture support obtained in Production Example 1 was affixed to the bottom of a dish (tissue culture dish, diameter: 15 cm, manufactured by IWAKI) without washing, and 20 ml of pure water was added for 3 days. Absorbance was also measured for the soaked one by the same method. The result is shown in FIG.

The concentration of N-isopropylacrylamide calculated from the absorbance of a solution obtained by immersing a cell culture support washed with running tap water at 20 ° C. for 10 seconds or more for 3 days was 0.5 μg / ml. Based on the isopropyl acrylamide tolerance, it was below the preset threshold (1 μg / ml). From this, it was confirmed that N-isopropylacrylamide could be removed to below the threshold when the cell culture support was washed with tap water at 20 ° C. for 10 seconds or more.

Claims (3)

A method for determining washing conditions for a cell culture support in which a temperature-responsive polymer formed by a polymerization reaction of a polymer-forming material is covalently immobilized on a substrate surface,
A washing step of washing the cell culture support;
A dipping step of immersing the cell culture support after washing in a liquid under conditions that allow extraction of a non-immobilized substance that is not immobilized on the substrate surface,
An absorbance measurement step of measuring the absorbance of the liquid after the immersion step to obtain an absorption spectrum ,
The absorption spectrum obtained in the absorbance measurement step is fitted with an absorption spectrum of a sample contained at a predetermined concentration for each of two or more assumed non-immobilized substances, and the two or more non-immobilized substances are obtained. A fitting coefficient is obtained for each of the absorption spectra of the substance, and the amount of the two or more non-immobilized substances is calculated from each fitting coefficient and the value of the predetermined concentration, and the calculated amount of the non-immobilized substance and A comparison step for comparing a preset threshold value for the amount of non-immobilized substance,
The method according to claim 1, wherein an appropriate washing condition for the cell culture support is selected based on a comparison result in the comparison step.   The method of claim 1, wherein the polymer-forming material comprises at least N-isopropylacrylamide monomer. The method according to claim 1 or 2, wherein the two or more non-immobilized substances comprise the polymer-forming material and the temperature-responsive polymer.

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