TECHNICAL FIELD

The present invention relates to a novel graft polymer that is useful in biology, medicine and other fields, as well as a temperature-responsive substrate for cell culture using the polymer and a method for producing the substrate. The present invention also relates to a carrier that can be prepared by a simple method and which is suitable for use in liquid chromatography that is carried out by controlling the interactions on a solid surface to separate valuables such as pharmaceuticals, bio-related substances (e.g. proteins, DNA, and glycolipids) and cells, as well as a method of liquid chromatography using the carrier.

BACKGROUND ART

With recent remarkable advances in the animal cell culturing technology, R&D activities targeting animal cells are spreading in a wider range of fields. The way of using the target animal cells has also changed; in the early stage of development, attempts were made to commercialize animal cells per se or their products, but now, it is becoming possible to design valuable pharmaceuticals by analyzing cells or their outer surface proteins or perform therapies with cells that have been taken out of a patient, then either proliferated in number or enhanced in their functions, and returned into the patient's body. Currently, the techniques of culturing animal cells constitute one area that is attracting the attention of many researchers.

One basic feature of animal cells including human cells is that many of them are adhesion-dependent. In other words, animal cells must be adhered to a certain place before they are cultured ex vivo. Given this background, many researchers have so far designed or invented substrate surfaces that would be more favorable to cells but all proposals have been directed to the phase during cell culture. When cultured adhesion-dependent cells adhere to something else, they will produce adhesive proteins on their own. Hence, in order to exfoliate the cells, the prior art requires disrupting the adhesive proteins and an enzymatic treatment is usually performed. In the process, outer surface proteins that the cells have produced during culture and which are inherent in the respective cells are also disrupted; in spite of its seriousness, no effective means have been available to solve the problem, with no particular studies made. Solving this problem involved in the phase during cell recovery would be a requirement which indeed must be satisfied before dramatic advances can be seen in future R&D activities targeting animal cells.

High performance liquid chromatography (HPLC) has a wide variety of combinations of the mobile-phase liquid and the stationary phase, from which a suitable combination can be selected depending upon the sample; hence, in recent years, HPLC is utilized to separate and purify various substances. However, in the conventionally used chromatography, interactions between the solute in the mobile phase and the surface of the stationary phase are driven not by changing the surface structure of the stationary phase but mainly by changing the solvent in the mobile phase. For example, in HPLC finding use in a lot of areas, normal-phase columns in which a carrier such as silica gel is used as stationary phase employ a mobile phase comprising an organic solvent such as hexane, acetonitrile or chloroform, and reversed-phase columns for separating highly water-soluble substances with a silica gel derivative being used as a carrier employ an organic solvent such as methanol or acetonitrile.

In ion-exchange chromatography which uses an anion exchanger or a cation exchanger as stationary phase, the concentration or type of the external ion is altered to separate substances. With recent rapid advances in genetic engineering and like fields, use of physiologically active peptides, proteins, DNA, etc. is desired in various fields including pharmaceuticals, and their separation and purification is an extremely important issue. In particular, the need for techniques capable of separating and purifying physiologically active substances without impairing their activities is growing.

Unfortunately, however, organic solvents, acids, alkalis and surfactants that are used in the conventional mobile phase not only impair the activity of physiologically active substances, they are also foreign substances, so improvements of the separation and purification system are desired. It is also necessary to avoid the environmental pollution by these substances and this is another reason why a separation and purification system that does not use them is required.

Given this background, various studies have heretofore been made. Patent Document No. 1 discloses a novel cell culture method which comprises providing a cell culture support having a substrate surface coated with a polymer the upper or lower critical solution temperature of which with respect to water is 0-80° C., culturing cells on the support at a temperature either below the upper critical solution temperature or above the lower critical solution temperature, and subsequently adjusting the temperature either to a point equal to or above the upper critical solution temperature or to a point equal to below the lower critical solution temperature, whereby the cultured cells are exfoliated without enzymatic treatment.

Patent Document No. 2 discloses that using this temperature-responsive cell culture substrate, skin cells are cultured at a temperature either below the upper critical solution temperature or above the lower critical solution temperature, and subsequently adjusting the temperature either to a point equal to or above the upper critical solution temperature or to a point equal to below the lower critical solution temperature, whereby the cultured skin cells are exfoliated while suffering low damage.

Patent Document No. 3 discloses a method of repairing outer surface proteins on cultured cells using this temperature-responsive cell culture substrate. By utilizing the temperature-responsive cell culture substrate, it has become possible to modify the conventional culture technology in various novel ways.

By utilizing the temperature-responsive cell culture substrate, it has now become possible to modify the conventional culture technology in various novel ways. However, the technology here mentioned involves coating the surfaces of chemically inactive engineering plastics using high energy radiations such as an electron beam and to this end, expensive equipment such as an electron beam exposure unit is required and this has caused the problem of an inevitable increase in the cost of the culture substrate.

With a view to solving this problem, several techniques have been developed to date. Representative among them are methods of coating a substrate surface with synthesized polymers having the particular molecular structures disclosed in Non-Patent Document Nos. 1 and 2. But neither method is of such a technical level that it is capable of culturing cells in a comparable way to the conventional substrate for cell culture, that it enables the cells to be exfoliated by simply changing the temperature in a comparable way to the above-described temperature-responsive substrate for cell culture which has been prepared using an electron beam, and that the cultured cells are exfoliated as a cell sheet once they have become confluent, and an improvement in their technical level has been required. Considering this point, Patent Document No. 4 discloses that a substrate surface coated with a temperature-responsive polymer joined to a block copolymer would allow cultured cells to be exfoliated in a cell sheet form; as it turned out, however, the substrate was simply coated with a uniform layer of the temperature-responsive polymer and, hence, the ability to exfoliate the cultured cells was limited.

Among the techniques of temperature-responsive chromatography, the one disclosed in Patent Document No. 1 notably provides a platform technology. Described in this document is a technique which comprises providing a cell culture support comprising a substrate surface coated with a polymer the upper or lower critical solution temperature of which with respect to water is 0-80° C., culturing cells on the support at a temperature either below the upper critical solution temperature or above the lower critical solution temperature, and subsequently adjusting the temperature either to a point equal to or above the upper critical solution temperature or to a point equal to below the lower critical solution temperature, whereby the cultured cells are exfoliated. While this was the first case of using the temperature-responsive polymer as a cell culture material in the field of bio-medicine, the fact is that when cells are adhering to the substrate surface, they will secrete adhesive proteins on their own and use the secreted proteins as a linker for adhesion to the substrate surface. Therefore, the phenomenon here contemplated of cells exfoliating from the substrate surface implies that the adhesive proteins secreted from the cells are also exfoliated from the substrate surface. As a matter of fact, when cells obtained by this technique are re-seeded or transplanted into a biological tissue, the cells exfoliated from the substrate will efficiently adhere to the substrate or tissue. This means that the exfoliated cells retain the adhesive proteins as such after they were secreted during culture. In other words, the technique of interest is exactly the concept of the temperature-responsive chromatographic technique as referred to in the present invention which involves eliminating adsorbed proteins through temperature changes.

Patent Document No. 5 made a study about immobilization on silica gel or polymer gel that is commonly used as chromatographic carriers. However, even considering Examples, the document shows no results (as separation charts) of solute separation in the case where those carriers were actually used, and details as to what substances could be separated using these carriers and what was the specific issue to address were unknown.

In Patent Document No. 6, a temperature-responsive polymer was immobilized on the surface of silica gel and cases are shown where various steroids and even lymphocytes were actually separated using the resulting carrier. It is definitely shown that the various steroids and even lymphocytes were actually separated by the characteristics of the temperature-responsive polymer immobilized on the surface of the carrier silica gel. However, considering the exemplary results of separation given in the document, the number of theoretical stages at the separation needs to be increased further and there has been a need to provide an innovative technique that is improved over the prior art so greatly as to solve this problem.

PRIOR ART LITERATURE

Patent Documents

Patent Document No. 1: JP H 02-211865 A

Patent Document No. 2: JP H 05-192138 A

Patent Document No. 3: JP 2008-220354 A

Patent Document No. 4: Japanese Patent Application No. 2010-208506

Patent Document No. 5: JP H 05-133947 A

Patent Document No. 6: JP H 07-318661 A

Non-Patent Documents

Non-Patent Document No. 1: Soft Matter, 5, 2937-2946 (2009)

Non-Patent Document No. 2: Interface, 4, 1151-1157 (2007)

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

The present invention has been accomplished with the aim of solving the aforementioned problems of the prior art. Briefly, the present invention has as its primary objective providing a novel graft polymer having better cell exfoliating functions based on a concept totally different from the prior art.

Another object of the present invention is to provide a novel temperature-responsive surface having better cell exfoliating functions based on a concept totally different from the prior art and a method of suing this surface.

Yet another object of the present invention is to provide a novel liquid chromatographic carrier prepared on the basis of a concept totally different from the prior art. A further object of the present invention is to provide a liquid chromatographic method using this carrier.

Means for Solving the Problems

In order to solve the aforementioned problems with cell culture, the present inventors performed research and development making studies from various angles and eventually came up with a novel graft polymer. Surprisingly, they found that when cells were cultured on a temperature-responsive substrate for cell culture having a substrate surface coated with a graft polymer comprising a dendritic polymer with a styrene skeleton and a temperature-responsive polymer grafted at a terminal thereof, the cultured cells could be efficiently exfoliated by simply cooling the substrate for a short period of time. From the early stage of their research activities, the present inventors focused on the special stereoregularity and morphology on substrate surface of the graft copolymer in which the dendritic polymer with a styrene skeleton was more compact in structure than the linear styrene and of such a structure that the temperature-responsive polymer was concentrated at a terminal of the dendritic polymer, and they were in great hopes that the substrate for cell culture formed from this graft polymer would have higher performance than the prior art temperature-responsive substrate for cell culture. It was also found that the graft polymer that comprised the dendritic polymer with a styrene skeleton and the temperature-responsive polymer grafted at a terminal thereof and which was applied as a coating to the surface of the substrate of the present invention was insoluble in water and, hence, would neither dissolve out into a culture medium during cell culture nor contaminate the cultured cells during their recovery. It was also found that because of these advantages, the temperature-responsive substrate for cell culture of the present invention can be used over and over again. The present invention has been accomplished on the basis of these findings.

In order to solve the aforementioned problems with chromatographic carriers, the present inventors also performed research and development making studies from various angles. As a result and surprisingly enough, they found that the dendritic polymer with a styrene skeleton could be conveniently immobilized in a thin layer on the surface of a resin-based chromatographic carrier and that when the temperature-responsive polymer was grafted to this dendritic polymer, the carrier's surface displayed such a phenomenon that its chromatographic resolution would abruptly change at a certain temperature. At the early stage of their research activities, the present inventors focused on the stereoregularity of the dendritic polymer and the functional groups occurring at high density on the outside of the molecular chain and speculated that if this dendritic polymer was immobilized on the surface of a chromatographic carrier, the functional groups could be provided at high density on the carrier's surface and that if the above-described temperature-responsive polymer was joined to the dendritic polymer, the product would have higher performance than the prior art temperature-responsive chromatographic carrier. The technique offered by the present invention is totally unexpected from the prior art and if combined with the stereoregularity of the dendritic polymer structure per se, it is expected to evolve to a novel chromatographic system that has never existed in the prior art. The present invention has been accomplished on the basis of these findings.

Thus, the present invention provides a graft polymer as a novel material comprising a dendritic polymer with a styrene skeleton and a temperature-responsive polymer grafted at a terminal thereof.

Further, the present invention provides a temperature-responsive substrate for cell culture that has a substrate surface coated with a graft polymer comprising a dendritic polymer with a styrene skeleton and a temperature-responsive polymer grafted at a terminal thereof. The present invention also provides a method for producing the temperature-responsive substrate for cell culture.

Additionally, the present invention provides a liquid chromatographic carrier on which is immobilized a graft polymer comprising a dendritic polymer with a styrene skeleton and a polymer of different composition grafted at a terminal thereof. The present invention also provides a liquid chromatographic method using the liquid chromatographic carrier.

The present invention is briefly described below.[1] A novel graft polymer comprising a dendritic polymer with a styrene skeleton and a hydrophilic polymer grafted at a terminal thereof.[2] The novel graft polymer as recited in [1], which has electric charges at a terminal of the dendritic polymer.[3] The novel graft polymer as recited in any one of [1] and [2], wherein the hydrophilic polymer is one having temperature response.[4] The novel graft polymer as recited in [3], wherein the polymer having temperature response comprises any one or more of a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, polyvinyl methyl ether, and partially acetylated polyvinyl alcohol, or a copolymer thereof with another monomer.[5] The novel graft polymer as recited in [4], wherein the polymer having temperature response is poly-N-isopropylacrylamide.[6] The novel graft polymer as recited in [4], wherein the another monomer is a monomer having electric charges and/or a hydrophobic monomer.[7] The novel graft polymer as recited in any one of [1] and [2], wherein the hydrophilic polymer comprises any one or more of polyacrylamide, poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate having polyethylene oxide in side chains, and methacrylate having polyethylene oxide in side chains.[8] The novel graft polymer as recited in any one of [1] and [2], wherein the hydrophilic polymer is a mixture of the polymers as recited in any one of [3] to [7].[9] A temperature-responsive substrate for cell culture comprising a substrate coated with a dendritic polymer with a styrene skeleton and a temperature-sensitive polymer grafted at a terminal thereof.[10] The temperature-responsive substrate for cell culture as recited in [8], which has electric charges at a terminal of the dendritic polymer.[11] The temperature-responsive substrate for cell culture as recited in any one of [9] and [10], wherein the polymer having temperature response comprises any one or more of a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, polyvinyl methyl ether, and partially acetylated polyvinyl alcohol, or a copolymer thereof with another monomer.[12] The temperature-responsive substrate for cell culture as recited in any one of [9] to [11], wherein the polymer having temperature response is poly-N-isopropylacrylamide.[13] The temperature-responsive substrate for cell culture as recited in [11], wherein the another monomer is a monomer having electric charges and/or a hydrophobic monomer.[14] The temperature-responsive substrate for cell culture as recited in any one of [9] to [13], wherein any one or more of polyacrylamide, poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate having polyethylene oxide in side chains, and methacrylate having polyethylene oxide in side chains are grafted to part of a terminal of the dendritic polymer with a styrene skeleton.[15] The temperature-responsive substrate for cell culture as recited in any one of [9] to [14], which has a coating of 1.0 to 7.0 μg/cm2in terms of the temperature-responsive polymer.[16] The temperature-responsive substrate for cell culture as recited in any one of [9] to [15], wherein the content of the temperature-responsive polymer in the graft polymer ranges from 40 to 99.5 wt %.[17] The temperature-responsive substrate for cell culture as recited in any one of [9] to [16], wherein the molecular weight of the temperature-responsive polymer in the graft polymer is 5000 or more.[18] The temperature-responsive substrate for cell culture as recited in any one of [9] to [17], wherein a substrate is in the form of a particle, a filament or a plate either individually or in combination of two or more.[19] The temperature-responsive substrate for cell culture as recited in any one of [9] to [18], wherein a substrate is composed of polystyrene either alone or combined with another material.[20] A method for producing a temperature-responsive substrate for cell culture by dissolving or dispersing a graft polymer in an organic solvent, applying a solution of the graft polymer uniformly onto a substrate surface, and drying the same.[21] The method for producing a temperature-responsive substrate for cell culture as recited in [20], wherein the organic solvent is a liquid mixture of tetrahydrofuran and methanol.[22] The method for producing a temperature-responsive substrate for cell culture as recited in [21], wherein the mixing ratio of tetrahydrofuran and methanol in the mixed solvent is 1:4.[23] A liquid chromatographic carrier comprising a carrier surface on which a graft polymer is immobilized, wherein the graft polymer comprises a dendritic polymer with a styrene skeleton and a polymer of a different composition joined to a terminal thereof.[24] The liquid chromatographic carrier as recited in [23], wherein the polymer grafted to the dendritic polymer with a styrene skeleton comprises a polymer having temperature response, and any one or two of a hydrophilic polymer, and a hydrophobic polymer.[25] The liquid chromatographic carrier as recited in [24], wherein the polymer having temperature response comprises any one or more of a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, polyvinyl methyl ether, and partially acetylated polyvinyl alcohol, or a copolymer thereof with another monomer.[26] The liquid chromatographic carrier as recited in [25], wherein the polymer having temperature response is poly-N-isopropylacrylamide.[27] The temperature-responsive surface as recited in any one of [25] and [26], wherein the molecular weight of the polymer having temperature response is 5000 or more.[28] The temperature-responsive surface as recited in any one of [25] to [27], wherein the content of the polymer having temperature response in the graft polymer ranges from 40 to 99.5 wt %.[29] The liquid chromatographic carrier as recited in [24], wherein the hydrophilic polymer comprises any one or more of polyacrylamide, poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate having polyethylene oxide in side chains, and methacrylate having polyethylene oxide in side chains.[30] The liquid chromatographic carrier as recited in any one of [23] to [29] which has electric charges at a terminal of the dendritic polymer.[31] The liquid chromatographic carrier as recited in any one of [23] to [30] which has electric charges on the polymer grafted to the dendritic polymer with a styrene skeleton.[32] The liquid chromatographic carrier as recited in any one of [23] to [31], wherein the graft polymer is immobilized on the surface of the carrier at a dose of 1.0 to 7.0 μg/cm2.[33] The liquid chromatographic carrier as recited in any one of [23] to [32], wherein a carrier is composed of polystyrene either alone or combined with another material.[34] The liquid chromatographic carrier as recited in any one of [23] to [33], wherein the carrier is in the form of a particle, a filament or a plate either individually or in combination of two or more.[35] A method for producing a liquid chromatographic carrier by dissolving or dispersing a graft polymer in an organic solvent, applying a solution of the polymer uniformly onto a substrate surface, and drying the same.[36] The method for producing a liquid chromatographic carrier as recited in [35], wherein the organic solvent is a liquid mixture of tetrahydrofuran and methanol.[37] The method for producing a liquid chromatographic carrier as recited in [36], wherein the mixing ratio of tetrahydrofuran and methanol in the mixed solvent is 1:4.[38] A liquid chromatographic method using the liquid chromatographic carrier as recited in any one of [23] to [34].[39] The liquid chromatographic method as recited in [38] which comprises separating a solute under specified temperature conditions.[40] The liquid chromatographic method as recited in [38] which comprises separating a solute while varying the temperature such that it crosses the level at which the properties of the carrier surface will change.[41] The liquid chromatographic method as recited in [38], wherein the temperature variation is either in an intermittent or continuous way or in both intermittent and continuous ways.[42] The liquid chromatographic method as recited in [38] which comprises adsorbing the solute on the liquid chromatographic carrier and subsequently varying the temperature to change the properties of the carrier surface so that the adsorbed solute becomes free.[43] The liquid chromatographic method as recited in [38] which comprises separating the solute with two or more types of the liquid chromatographic carrier packed in the same column by varying the temperature such that it crosses the level at which the properties of the carrier surface will change.[44] The liquid chromatographic method as recited in [38] which comprises separating the solute with the liquid chromatographic carrier in a column the inlet and outlet temperatures of which are so set that the temperature level at which the properties of the carrier surface will change is in between said inlet and outlet temperatures and that a temperature gradient is provided within the column from the inlet to the outlet end.[45] The liquid chromatographic method as recited in any one of [38] to [44], wherein the mobile phase is an aqueous system.[46] The liquid chromatographic method as recited in any one of [38] to [45] which is for separating pharmaceuticals, metabolites thereof, agrochemicals, peptides or proteins.

Effects Of The Invention

By using substrates coated on their surface with the novel graft polymer to be described herein, cells can be cultured efficiently and, what is more, the cultured cells can be exfoliated efficiently within a short period of time by simply changing the temperature of the substrate surface. And substrates having such functional surface can be conveniently prepared in accordance with the production method of the present invention.

Additionally, a novel separation system is offered by the liquid chromatographic carrier described herein. Given this system, peptides and proteins can be separated over a wide range. And the stereoregularity of the dendritic polymer itself may be utilized to enable solutes to be separated depending on the differences in their molecular structures.

