JP6313822B2 - Temperature-responsive cell culture beads and method for producing the same - Google Patents

Description

  The present invention relates to a cell culture substrate useful in fields such as biology and medicine.

  Today, animal cell culture technology has made significant progress, and research and development on animal cells has been extended to various fields. The target animal cells can be used not only to commercialize the original cells, but also to produce useful products by analyzing the cells and their surface proteins. In addition, the patient's own cells are being grown in vitro, or the function of the cells is increased and returned to the living body for treatment. Currently, the technique of culturing animal cells is one field that many researchers are paying attention to.

  By the way, many animal cells including human cells are adhesion-dependent. That is, when cultivating animal cells in vitro, it is necessary to attach them once somewhere. Against this background, more researchers have designed and devised a device surface that is more favorable for cells than before, but all of these techniques have been related to cell culture. When an adhesion-dependent cultured cell attaches to something, it produces an adhesive protein by itself. Therefore, when the cells are detached, the adhesive protein must be destroyed in the prior art, and usually an enzyme treatment is performed. At that time, although there was a serious problem that the cell surface proteins inherent to various cells produced by the cells during the culture were destroyed at the same time, there was no means to solve the problem at all, and no particular investigation was made. It was. It is thought that the solution to the problem at the time of cell recovery will be strongly demanded for the dramatic development of research and development for animal cells.

  Under such a background, Patent Document 1 discloses that the upper critical solution temperature of cells is covered on a cell culture support whose surface is covered with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. A novel cell culturing method is described in which culture is performed at or below the lower critical lysis temperature or higher and then the upper critical lysis temperature or higher or the lower critical lysis temperature or lower to detach the cultured cells without enzyme treatment. Further, in Patent Document 2, skin cells are cultured at a temperature lower than the upper critical solution temperature or lower than the lower critical solution temperature by using this temperature-responsive cell culture device, and then the upper critical solution temperature or lower or the lower critical solution temperature. By doing this, it is described that cultured skin cells are detached with low damage. Furthermore, Patent Document 3 describes a method for repairing surface protein of cultured cells using this temperature-responsive cell culture equipment. By using temperature-responsive cell culture equipment, various new developments have been made for conventional culture techniques.

  By using temperature-responsive cell culture equipment, various new developments can be made to conventional culture techniques. However, the technique here is mainly a technique for a petri dish-like base material, and further improvement has been demanded in designing a bead-like base material surface capable of culturing a large amount of cells.

  Under such circumstances, Patent Document 4 also illustrates an example of separation when the temperature-responsive polymer is immobilized at a high density on the silica gel surface by using the atom transfer radical polymerization method, and the beads are actually used. Yes. However, in any of the examples, it was only used as a method for separating a physiologically active substance, and no knowledge about cell culture technology was shown.

  Non-Patent Document 1 describes a technique in which atom transfer radical polymerization of N-isopropylacrylamide is carried out in various solvents including isopropyl alcohol (IPA), and a comparison of reaction rates depending on the reaction solvent is also described. ing. However, the technique here relates to a polymerization reaction in solution, and there is no description of immobilizing a temperature-responsive polymer on solid cell culture beads, and there is no description that suggests that. . Furthermore, there was no description used for the atom transfer radical polymerization reaction in the copolymerization of the monomer that is the raw material of the temperature-responsive polymer and the monomer having a functional group that exhibits electric charge, and there was no description that suggested that. .

JP-A-2-21865 JP 05-192138 A JP 2008-220354 A JP 2007-69183 A

Macromolecules, 38, 5937-5943 (2005).

  The present invention has been made with the intention of solving the problems of the prior art relating to the above-described beads for cell culture. further. It was made with the intention of solving the aggregation of the cell culture beads generated during the cell culture, which was found in the process. That is, an object of the present invention is to provide a novel bead for temperature-responsive cell culture from an idea completely different from the prior art. Another object of the present invention is to provide a method for producing the temperature-responsive cell culture bead.

  In order to solve the above problems, the present inventors have studied and developed from various angles. As a result, surprisingly, a polymer having a charge and a change in hydration power within a temperature range of 0 to 80 ° C. is immobilized on the surface of the granule made of a material having a specific gravity of 1.2 or less. It has been found that when temperature-responsive cell culture beads are used, cells can be efficiently cultured, and can be efficiently detached simply by changing the temperature of the substrate surface. Moreover, aggregation of the beads for temperature-responsive cell culture could be suppressed during the culture period. If this is used as a cell culture substrate, it is highly expected that low-damage cells different from conventional cells can be efficiently cultured in large quantities. The present invention has been completed based on such findings.

  That is, the present invention is a temperature responsiveness in which a polymer having a charge and a hydration power changing within a temperature range of 0 to 80 ° C. is immobilized on a particle surface made of a material having a specific gravity of 1.2 or less. A cell culture bead is provided.

  The present invention also provides a method for producing the temperature-responsive cell culture bead.

  With the cell culture beads described in the present invention, a large amount of cells can be efficiently cultured, and the cells can be efficiently separated simply by changing the temperature of the substrate surface. Moreover, the base material having such a functional surface can be easily produced according to the production method of the present invention.

2 is a table showing conditions for preparing the thermoresponsive cell culture beads of Examples 1 and 2. It is a figure which shows the result of having cultured the cell using the bead for temperature-responsive cell culture obtained in Example 1. FIG. It is a figure which shows the result of having cultured the cell using the bead for temperature-responsive cell culture obtained in Example 2. FIG. It is the figure which put together the result obtained in Example 1,2.

  As a result of various studies to solve the above problems, the inventors of the present invention hydrated within a temperature range of 0 to 80 ° C. if a charge is applied to the particle surface made of a material having a specific gravity of 1.2 or less. The present invention was completed by finding that cells can be cultured without aggregating between beads even in temperature-responsive cell culture beads to which a polymer that changes force is immobilized. An object of the present invention is to provide a temperature-responsive cell obtained by a growth reaction of a polymer having a charge and changing hydration power within a temperature range of 0 to 80 ° C. under a specific condition by an atom transfer radical method. A culture bead and a method for producing the bead are provided.

  The present invention provides a method for immobilizing a polymer having electric charge and changing hydration power within a temperature range of 0 to 80 ° C. on beads for cell culture. In the case of immobilizing a temperature-responsive polymer on a cell culture bead, controlling the amount of immobilization is an extremely important technique because it affects the cell culture function of the bead. In addition, the immobilization is desirably performed so that a polymer having a uniform molecular weight exists evenly on the bead surface. The shape of the beads used in the present invention is not particularly limited. For example, the beads may be in the form of particles (spherical, oval, or any other shape as long as the shape of grains), flat, flat There are a finely cut shape, a tubular shape, a finely cut shape of a tubular shape, and the like. In the present specification, particles and particulates have the same meaning, and those obtained by finely cutting a flat plate, those obtained by finely cutting a tubular one, and those obtained by finely cutting other shapes. Collectively called strips. In particular, when the beads of the present invention are used as a cell culture bead, the beads preferably have a specific gravity of 1.2 or less, preferably 1.05 or less, and more preferably 1.0 or less. When the specific gravity is larger than 1.2, the cell culture beads are precipitated during the cell culture, and it is not preferable because strong stirring is required to disperse the beads in the culture medium. Specifically, the material for the cell culture beads is preferably made of plastic or polysaccharide. Such beads are not particularly limited, and examples thereof include polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, or a mixture of two or more thereof. In that case, the bead for cell culture may be porous, and may not be porous. When the cell culture beads are porous, the pore diameter is not particularly limited. The particle diameter of the beads is not particularly limited, but the longest dimension of the beads is preferably 20 to 300 μm, preferably 50 to 200 μm, and more preferably 80 to 120 μm. If it is smaller than 20 μm, it is not preferable because it is too small as a cell culture bead to be dispersed in a medium and the operability is deteriorated. On the contrary, if it is larger than 300 μm, the operability is deteriorated.

In the present invention, a temperature-responsive polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized on the beads. As the immobilization method, beads having chloromethyl groups are used as the starting point of atom transfer radical polymerization on the substrate surface, and a temperature-responsive polymer is grown from the start point by the atom transfer radical method in the presence of a catalyst. Or a method in which an atom transfer radical polymerization initiator is immobilized and a temperature responsive polymer is grown from the initiator by the atom transfer radical method in the presence of a catalyst. The initiator used in this case is not particularly limited. For example, 1-trichlorosilyl-2- (m-chloromethylphenyl) ethane, 1-trichlorosilyl-2- (p-chloromethylphenyl) Examples include ethane, 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane, (3- (2-bromoisobutyryl) propyl) dimethylethoxysilane, and the like. In the present invention, polymer chains are grown from this initiator. The catalyst at that time is not particularly limited. However, when an N-alkyl-substituted (meth) acrylamide derivative is selected as a polymer whose hydration power changes, Cu ^I Cl, Cu ^I as copper halide (Cu ^I X) is selected. Br and the like. The ligand complex for the copper halide is not particularly limited, but tris (2- (dimethylamino) ethyl) amine (Me ₆ TREN), N, N, N ″, N ″ -pentamethyl. Diethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (HMTETA), 1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me) ₄ Cyclam), bipyridine and the like.

  As the solvent used in the polymerization in the present invention, isopropyl alcohol (IPA) is suitable. As a result of various studies, the inventors first selected N-isopropylacrylamide as a raw material for the temperature-responsive polymer, and in the case where the atom transfer radical polymerization reaction was performed in solution at room temperature, It was found that the reaction rate was almost the same regardless of the choice of formamide (DMF), water, or IPA. However, in the case of a solid-phase reaction in which N-isopropylacrylamide is immobilized on the surface of a solid cell culture bead as in the present invention, if the reaction solvent is IPA, the other two are selected. It was found that the reaction rate was remarkably slower than that. In addition, t-butyl alcohol shown in the above Macromolecules 38, 5937-5943 (2005) may solidify at room temperature, and therefore the reaction temperature must be increased to room temperature or higher, resulting in an increase in the reaction rate. It was found that this was inappropriate for the present invention. The knowledge here is not known at all in the prior art, and according to the present invention, the molecular weight of the polymer chain gradually increases when IPA is selected as the reaction solvent in the immobilization polymerization to the beads, The amount of polymer chains immobilized on the bead surface will also gradually increase. Therefore, according to the method of the present invention, the polymer chain on the bead surface can be fixed uniformly. Furthermore, by stopping the reaction at a predetermined time, it becomes possible to produce beads having an immobilized state at the time of stopping the reaction with good reproducibility.

  The present invention is a method of growing and reacting a polymer having a charge and changing hydration power within a temperature range of 0 to 80 ° C. by an atom transfer radical method under a polymerization catalyst from the starting point of the bead surface. Other initiator concentrations, copper halide concentrations, ligand complex concentrations, reaction temperatures, reaction times, and the like during polymerization are not particularly limited, and may be changed according to the purpose. Further, the state of the reaction solution may be allowed to stand or be stirred, but the latter is preferable in view of uniform fixation on the bead surface.

  Examples of the temperature-responsive polymer used in the present invention include a polymer having a lower critical solution temperature (LCST) and a polymer having an upper critical solution temperature (UCST), and any of those homopolymers, copolymers, and mixtures thereof. May be. Examples of such a polymer include polymers described in Japanese Patent Publication No. 06-104061. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and polyvinyl alcohol partial acetylates. Of these, any two or more of them can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. At that time, since the substance to be separated is a biological substance, the separation is performed in the range of 5 ° C. to 50 ° C. Therefore, as the temperature-responsive polymer, poly-Nn-propylacrylamide (lower limit of homopolymer) is used. Critical dissolution temperature 21 ° C), poly-Nn-propylmethacrylamide (27 ° C), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N -Cyclopropylacrylamide (at about 45 ° C), poly-N-ethoxyethylacrylamide (at about 35 ° C), poly-N-ethoxyethylmethacrylamide (at about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (at about 45 ° C) 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (about 35 ° C.), poly-N, N-ethylmethylacrylate Amide (the same 56 ° C.), poly -N, N-diethyl acrylamide (the 32 ° C.), and the like.

  Among these, poly (N-isopropylacrylamide) has a lower critical temperature at 32 ° C., and therefore the bead surface chemically modified with this polymer greatly changes the hydrophilic / hydrophobic surface properties at this critical temperature. Is used after being immobilized on the surface of a cell culture bead, the holding force for the sample can be changed depending on the temperature. As a result, the cultured cells can be detached only at the culture temperature without using a hydrolase or the like. In order to set the lower critical temperature to 32 ° C. or higher, hydrophilic monomers such as acrylamide, methacrylic acid, acrylic acid, dimethylacrylamide, and vinylpyrrolidone, which are more hydrophilic than isopropylacrylamide, are copolymerized with N-isopropylacrylamide. It is possible to make adjustments. Further, when the lower critical temperature is desired to be 32 ° C. or lower, it can be adjusted by copolymerization with hydrophobic monomers such as styrene, alkyl methacrylate, alkyl acrylate and the like which are hydrophobic monomers.

  Further, the lower critical temperature of polydiethylacrylamide is about 30 ° C. to 32 ° C., and the surface physical property changes to hydrophilic / hydrophobic with this temperature as a boundary, as in the case of poly (N-isopropylacrylamide) described above. In addition, cell attachment and detachment can be adjusted by temperature. The novel beads for cell culture used in the present invention are produced by chemical modification or polymer coating.

  In the present invention, the polymer coated on the surface of the cell culture bead is hydrated and dehydrated by changing the temperature. The temperature range is 0 ° C. to 80 ° C., preferably 10 ° C. to 50 ° C. More preferably, it is 20 degreeC-45 degreeC. If it exceeds 80 ° C., it is not preferable for cells. On the other hand, if it is lower than 0 ° C., the cells may be excessively cooled, which is not preferable.

  In the present invention, the polymer coated on the surface of the cell culture bead is charged. The method for imparting the charge is not particularly limited, but usually, when synthesizing the temperature-responsive polymer chain to be coated on the bead surface, there is a method of copolymerization including an ionic monomer having a functional group that generates a charge. . Examples of the ionic monomer include dialkylaminoalkyl (meth) acrylamide, dialkylaminoalkyl (meth) acrylate, aminoalkyl (meth) acrylate, aminostyrene, aminoalkylstyrene, aminoalkyl (meth ) Acrylamide, alkyloxyalkyltrimethylammonium salt, (meth) acrylamide alkyltrimethylammonium salt 3-acrylamidopropyltrimethylammonium chloride, dimethylaminopropylacrylamide having a quaternary amino group, etc., and a polymer having a carboxyl group (Meth) acrylamide as a structural unit of a polymer having acrylic acid, methacrylic acid or sulfonic acid as a structural unit Etc. Rusuruhon acid, and the like, but not limited to the present invention. Considering that cells are generally negatively charged, cations are preferred on the bead surface side.

  In the present invention, when a surface-initiated radical polymerization method is used with an RAFT (Reversible Addition-Fragmentation Chain Transfer) agent as an initiator, a dithioester functional group that is a part of the structure of the RAFT agent Will remain at the end of the produced polymer. This is a characteristic phenomenon in the RAFT polymerization method, and after the polymerization reaction is completed, the polymerization reaction can be started from the end. By utilizing this feature, the surface of the block copolymer can be produced unlike the existing copolymer. At that time, the dithioester functional group present at the terminal of the temperature-responsive polymer is easily substituted with a thiol group by adding 2-ethanolamine or the like. This reaction does not need to be carried out under special conditions, is simple and proceeds in a short time. As a result, since a polymer chain having a highly reactive thiol group can be obtained, a functional molecule having a functional group such as a maleimide group or a thiol group can be selectively and efficiently modified at the end of the polymer chain. Therefore, new functionality can be imparted to the surface of the temperature-responsive culture bead. At that time, the type of the functional group is not particularly limited, and examples thereof include a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, an aldehyde group, and a sulfonic acid group. In addition, a peptide or protein that promotes cell adhesion may be immobilized at the end of the polymer chain. And since the lower critical solution temperature (LCST) of poly-N-isopropylacrylamide changes depending on the hydrophilicity / hydrophobicity of the terminal functional group, the functional group introduction to the polymer chain end as in the present invention is It is also expected as a new method for controlling the temperature response of the bead surface from another viewpoint.

In the present invention, the polymer is fixed with high density and uniformity. The degree of immobilization is preferably 0.08 molecular chain / nm ² or more, preferably 0.10 molecular chain / nm ² or more, more preferably 0.12 molecular chain in terms of the number of molecular chains per unit area. / Nm ² or higher, and most preferably 0.15 molecular chain / nm ² or higher. When the degree of immobilization of the polymer on the substrate surface is less than 0.08 molecular chain / nm ² , the present invention is achieved only by developing the characteristics of the individual polymer chains in the same manner as the polymer immobilization on the substrate surface by the conventional method. It is not preferable as a bead. The calculation method of the numerical value indicating the degree of immobilization is not particularly limited, but for example, a molecular weight obtained by preparing a polymer that is not immobilized on the substrate surface under similar reaction conditions and analyzing the polymer chain And the amount of polymer immobilized obtained from elemental analysis of beads on which the polymer is immobilized.

  The molecular weight of the polymer to be coated is not particularly limited as long as the molecular weight is sufficiently large to cause a change in hydration power within a temperature range of 0 to 80 ° C., but the polymer molecular weight is preferably 1000 or more, preferably Is 2,000 or more, more preferably 5,000 or more. If the molecular weight is less than 1000, the molecular weight is too low, and thus a change in hydration power cannot be expressed, which is not preferable. However, if the molecular weight exceeds 50,000, the molecular weight of the polymer is too high, so that the molecule itself becomes bulky and the temperature responsiveness decreases, which is not preferable.

Furthermore, immobilization of the polymer onto the substrate at which in this invention may have a range of 0.8~10.0mg / ^{m 2,} preferably from 0.9~8.0mg / ^{m 2,} further The range of 1.0 to 6.0 mg / m ² is preferable. If it is less than 0.8 mg / m ² , the temperature responsiveness is not recognized, and even if the value is higher than 10.0 mg / m ^{2, the} temperature responsiveness decreases due to the bulk of the polymer, which is not preferable. . The amount of immobilization may be measured according to a conventional method, and examples thereof include FT-IR-ATR method, elemental analysis, ESCA amount, and any method may be used. The state of the polymer to be immobilized in the present invention is not particularly limited, and may be linear or cross-linked. In order to achieve immobilization, the former linear one is preferred.

  For the temperature-responsive cell culture beads obtained in the present invention, there are no particular limitations on the cells used, their sources, and production methods. Examples of cells that can be cultured using the temperature-responsive cell culture beads of the present invention include cells such as animals, insects, and plants, and bacteria. In particular, the origin of animal cells includes humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cattle, marmoset, African green monkeys, etc. is not. The medium used in the present invention is not particularly limited, and examples thereof include serum-free medium and serum-containing medium as long as animal cells are cultured. Such a medium may further contain a differentiation inducer such as retinoic acid or ascorbic acid. The seeding density on the surface of the substrate is not particularly limited as long as it follows conventional methods in the art.

  In addition, in the case of the temperature-responsive cell culture bead of the present invention, the cultured cells are treated with an enzyme by setting the temperature of the culture substrate to the upper critical solution temperature of the coating polymer on the culture substrate or higher or lower than the lower critical solution temperature. Can be peeled off. In that case, it can be performed in a culture solution or in another isotonic solution, and can be selected according to the purpose. For the purpose of detaching and recovering cells faster and more efficiently, use a method of tapping or shaking the substrate, or a method of stirring the medium using a pipette, alone or in combination. May be.

  By using the temperature-responsive cell culture beads described in the present invention, cells obtained from each tissue can be cultured efficiently. If this culturing method is used, it can be efficiently peeled off without being damaged only by changing the temperature. Conventionally, such work has required labor and operator's skill, but according to the present invention, this need is eliminated and a large amount of cells can be processed. In the present invention, it is clarified that such a culture substrate surface is produced by using a living radical polymerization method. In addition, according to the present invention, the surface of the culture substrate can be easily and precisely designed, and as described above, if the reaction is continued with respect to the molecular chain terminal, a functional group can be easily added, which is extremely advantageous for cell culture. It is.

Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
[Examples 1 and 2]

(Preparation of cationic temperature-responsive polymer-modified beads)
FIG. 1 summarizes the charged amounts of monomers, catalysts and the like when preparing cationic temperature-responsive polymer-modified beads. Monomer (N-isopropylacrylamide (IPAAm) / butyl methacrylamide (BMA) / quaternary amino grouped dimethylaminopropylacrylamide (DMAPAAm-Q) = 89/5/6 (IB5DQ6 beads, Example 1), IPAAm / tBAAm / DMAPAAm-Q = 65/25/10 (ItB25DQ10 beads, Example 2)) was dissolved in isopropanol at various concentrations (0.1, 0.5, 1, 2 mol / L), and this solution was bubbled with nitrogen for 30 minutes with stirring. . CuCl, CuCl2, and Me6TREN were added to this solution, and a copper catalyst complex was formed by stirring. This reaction solution and polystyrene beads (particle size 100 μm, non-porous particles) were mixed in a glove bag filled with nitrogen, and reacted at 25 ° C. for 16 hours. After the reaction, the solution was stopped by opening it to the atmosphere. Thereafter, the prepared beads were shaken in methanol and 50 mM EDTA solution, and then the operation of removing the supernatant was repeated to purify the unreacted monomer and the copper catalyst. Further, the same operation was repeated in pure water (Mili-Q water) to remove EDTA. The obtained beads were collected by suction filtration and dried under reduced pressure to obtain the target polymer-modified beads.

(Cell culture)
Each dried bead was dispersed in a PBS solution and autoclaved. Thereafter, 0.5 μl was dropped on a hemocytometer, and the bead concentration was calculated. The beads were added to Hydrocell (35 mmφ) at 1 × 10 ⁴ beads / ml and CHO cells at 100 cells / bead, and cultured at 37 ° C. After culturing for 24 hours, the culture solution was transferred to a tube, 2 ml of the culture solution was added, allowed to stand for 10 minutes, and then the supernatant was discarded to remove unadherent floating cells and cell aggregates. Thereafter, the cells were transferred to an incubator at 20 ° C, and after pipetting, the desorption behavior of the cells as the temperature decreased was observed with a phase contrast microscope. In addition, cell desorption experiments were performed when low-temperature treatment was performed in a 0.02% EDTA solution, and cell desorption behavior was compared by microscopic observation.

(Cell recovery from temperature-responsive beads)
For cell recovery from temperature-responsive beads, after culturing for 24 hours, transfer the culture solution to a tube, add 2 mL of the culture solution, allow to stand for 10 minutes, and discard the supernatant. The lump was removed. Further, the floating cells were filtered with a cell strainer (mesh size 40 μm), and the filtrate was redispersed in 2 mL of the medium. Thereafter, the cells were incubated in an incubator at 20 ° C. for 2 hours. After pipetting, the detached cells were filtered with a cell strainer (mesh size 40 μm), and the number of cells was counted using a hemocytometer. In addition, as control experiments, the number of cell desorptions when a low-temperature treatment was performed in a 0.02% EDTA solution, and the number of cells recovered only with a trypsin-EDTA solution were counted.

(result)
In each cell culture bead, no aggregation of beads was observed during the culture. These cell culture beads have been found to meet the objectives of the present invention. Phase contrast microscopic images of the cells on each bead are shown in FIGS. In IB5DQ6 beads, cell adhesion was observed with beads having monomer concentrations of 0.1 and 0.5 mol / L. When the temperature was lowered to 20 ° C, cell detachment was not observed with 0.1 mol / L beads, but some cells were detached with 0.5 mol / L beads, and the temperature was lowered in pipetting or EDTA solution. The treatment promoted cell detachment. However, some cells remained attached. With ItB25DQ10 beads, cell adhesion was observed with beads having a monomer concentration of 1 mol / L or less. In addition, with 0.5 and 1 mol / L beads, some cells were detached by low-temperature treatment, and almost all cells were detached by pipetting. It can be seen that ItB25DQ10 beads are particularly preferred as temperature responsive beads. The ItB25DQ10 beads were used to quantitatively analyze the cells that were detached due to the temperature drop to 20 ° C. (FIG. 4). As shown in FIG. 4, the number of cells recovered by the temperature drop + EDTA solution treatment with 1 mol / L beads was almost the same as the number of cells collected by the trypsin treatment. Therefore, it can be seen that by using ItB25DQ10 beads prepared at a monomer concentration of 1 mol / L, a temperature-responsive bead culturing system can be obtained in which almost 100% of cells can be recovered by temperature change.

Comparative Example 1

  In Example 1, when preparing temperature-responsive polymer-modified beads, beads for cell culture having no charge were prepared with the monomer charge (IPAAm / BMA = 92/8). Thereafter, the cells were cultured in the same manner as in Example 1. As a result, it was found that the cell culture beads aggregated during the cell culture, which was not preferable for the present invention.

  By using the temperature-responsive cell culture substrate described in the present invention, cells obtained from each tissue can be efficiently cultured in large quantities. If this culturing method is used, the cultured cells can be efficiently peeled off without being damaged only by changing the temperature.

Claims (6)

Immobilization of 0.8 to 10.0 mg / m ² of a polymer having a specific gravity of 1.2 or less and having a charge and a hydration power changing within a temperature range of 0 to 80 ° C. Temperature-responsive cell culture beads that are fixed in quantity and can be detached by temperature change.   The bead for temperature-responsive cell culture according to claim 1, wherein the charged polymer is a copolymer of polyalkylacrylamide. The bead for temperature-responsive cell culture according to claim 1 or 2 , wherein the polyalkylacrylamide is poly- (N-isopropylacrylamide) and / or poly- (N, N-diethylacrylamide).   The temperature-responsive cell culture bead according to any one of claims 1 to 3, wherein the charge is a cation.   The bead for temperature-responsive cell culture according to any one of claims 1 to 4, wherein the beads are polystyrene particles or polystyrene strips.   The cell culture method using the bead for temperature-responsive cell cultures of any one of Claims 1-5.