# US 2019/0169567 A1 · Novel graft polymer, temperature-responsive substrate for cell culture using the same and production method therefor, as well as liquid chromatographic carrier having the novel graft polymer immobilized thereon and liquid chromatographic method using the same

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- Number: US 2019/0169567 A1
- Document ID: US20190169567A1
- Status: Published application
- Kind: A1
- Application number: 16/208,163
- Filed: 2018-12-03
- Published: 2019-06-06
- Earliest claimed: 2013-02-28
- Assignees: Hideaki Sakai
- Inventors: Hideaki SAKAI; Masa-aki KAKIMOTO; Yu SUDO
- Web page: https://patentgrep.com/patent/US20190169567A1

## CPC

- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- B01D 15/3876: {modifying the temperature}
- B01J 20/264: {derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers}
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/321: {consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds}
- B01J 20/3278: {Polymers being grafted on the carrier}
- C08F 257/02: on to polymers of styrene or alkyl-substituted styrenes
- C08F 293/005: {using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent}
- C08F 299/00: Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers
- C08L 51/003: {grafted on to macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds (C08L51/04, C08L51/06 take precedence)}
- C08L 101/04: containing halogen atoms
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- B01D 15/305: {Hydrophilic interaction chromatography [HILIC]}
- B01D 15/327: {with hydrophobic interaction}
- C08F 2438/03: Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
- C08L 2203/02: for biomedical use
- C12N 2533/30: Synthetic polymers
- C12N 2539/10: Coating allowing for selective detachment of cells, e.g. thermoreactive coating
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

By using a graft polymer comprising a dendritic polymer with a styrene skeleton and a hydrophilic polymer grafted to a terminal thereof, a temperature-responsive substrate for cell culture having a temperature-responsive surface for cell culture that allows cells to be cultured with high efficiency and which yet allows cultured cells to be exfoliated in a short period of time and with high efficiency by simply changing the temperature of the substrate surface can be prepared conveniently. If this temperature-responsive substrate for cell culture is used, cells obtained from a variety of tissues can be cultured with high efficiency. If this culture method is utilized, cultured cells can be exfoliated intact in a short amount of time with high efficiency. In addition, by using this graft polymer, a wide range of peptides and proteins can also be separated by simply changing the temperature of a chromatographic carrier. This allows for convenient separation procedure and improves the efficiency of separating operations. What is more, the stereoregularity of the dendritic polymer per se may be utilized to enable separation of solutes based on differences in their molecular structures.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, which has electric charges at a terminal of the dendritic polymer.

### Claim 3 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, 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.

### Claim 4 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, wherein the polymer having temperature response is poly-N-isopropylacrylamide.

### Claim 5 (depends on 3)

The temperature-responsive substrate for cell culture according to claim 3, wherein the another monomer is a monomer having electric charges and/or a hydrophobic monomer.

### Claim 6 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, 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.

### Claim 7 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, which has a coating of 1.0 to 7.0 μg/cm2 in terms of the temperature-responsive polymer.

### Claim 8 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, wherein the content of the temperature-responsive polymer in the graft polymer ranges from 40 to 99.5 wt %.

### Claim 9 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, wherein the molecular weight of the temperature-responsive polymer in the graft polymer is 5000 or more.

### Claim 10 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, wherein a substrate is in the form of a particle, a filament or a plate either individually or in combination of two or more.

### Claim 11 (depends on 1)

The temperature-responsive substrate for cell culture according to claim 1, wherein a substrate is composed of polystyrene either alone or combined with another material.