JP2002191353A - Cell culture support and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture support that can efficiently culture cells and can collect the cultured cells by peeling off them in a tissue-like form from the support in high yield (high yield and high viability). SOLUTION: The objective cell culture support is characteristically coated on its surface with (A) a polypeptide that includes a minimum amino acid sequence that expresses a cell adhesion signal and (B) a polymer having a critical solution temperature to water at 0-80 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a cell culture support and a method for producing the same.

[0002]

2. Description of the Related Art Cells that have been cultured and grown are separated and recovered from the surface of a support simply by changing the environmental temperature without being treated with a proteolytic enzyme such as trypsin or a chelating agent (eg, EDTA). As a cell culture support material capable of being coated, a first layer is coated with a natural substance such as collagen or a synthetic substance such as sodium polyacrylate, and has thereon a critical dissolution temperature for water as a second layer. One coated with a specific water-soluble polymer has been proposed (JP-A-5-38278).

[0003]

A natural substance such as collagen or a synthetic substance such as sodium polyacrylate has an effect on cell attachment and growth when a medium containing serum is used, but is not effective in serum. Contains proteins with unknown components, and in the case of drug production using cells, the purification process becomes complicated and costly, and furthermore, there is the danger of virus infection. Due to concerns about safety in engineering, a so-called serum-free medium without serum tends to be preferred. However, in the case of a serum-free medium, the cell culture support described above has a low effect on cell adhesion and growth, and sufficient cell growth cannot be obtained. That is, an object of the present invention is to efficiently culture cells, and to separate and recover cultured cells from a cell culture support in a high yield (high recovery rate and high survival rate) as a tissue by simply changing the ambient temperature. It is to provide a support for cell culture.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the above object can be achieved by using a specific polypeptide and a specific polymer, and have reached the present invention. That is, a feature of the cell culture support of the present invention is that a polypeptide (A) having at least one minimal amino acid sequence in one molecule which represents a cell adhesion signal and a polymer having a critical solubility temperature in water of 0 to 80 ° C ( B).

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, (A) will be described.
In the present invention, as the minimum amino acid sequence that expresses a cell adhesion signal, any amino acid sequence that functions as an adhesion signal can be used. For example, "Pathophysiology", Vol. 9, No. 7 (1990) at page 527. RGD sequence, LDV sequence, R
EDV array (1), YIGSR array (2), PDSGR
Sequence (3), RYVVLPR sequence (4), LGTIPG
Sequence (5), RNIAEIIKDI sequence (6), IKV
At least one sequence selected from the group consisting of the AV sequence (7), the LRE sequence, the DGEA sequence (8) and the HAV sequence is preferable, and the RGD sequence and the YI sequence are more preferable.
GSR sequence (2), PDSGR sequence (3), LGTIP
G sequence (5), IKVAV sequence (7) and at least one sequence selected from the group consisting of HAV sequences, most preferably RGD sequence, IKVAV sequence (7) and HA
It is at least one kind of sequence selected from the group consisting of V sequences. In addition, the sequence number corresponding to the amino acid sequence listing is added in parentheses.

It is necessary that the polypeptide (A) contains at least one of the above minimum amino acid sequences in one molecule. If the minimum amino acid sequence is not contained, the cell adhesion is reduced, resulting in insufficient cell growth, especially when a serum-free medium is used. The content of the minimum amino acid sequence is preferably 3 to 50, more preferably 5 to 40, and particularly preferably 10 to 30 in one molecule from the viewpoint of cell adhesion and proliferation.

The number average molecular weight of the polypeptide (A) is low in toxicity to cells and high in adhesion performance.
5,000 to 5,000,000 is preferred, and 10,000
00 to 1,000,000 are more preferable, and 50,0
00 to 500,000 is particularly preferred. The number average molecular weight of the polypeptide (A) was determined by SDS-PAGE (N
a Dodecyl sulfate-polyacrylamide gel electrophoresis), (A) is developed in water, and the migration distance is determined by comparing with a standard substance.

[0008] The polypeptide (A) has, besides the minimum amino acid sequence representing a cell adhesion signal, two or more amino acid sequences which enhance the heat stability of (A), for example, a GAGAGS sequence (9) derived from silk fibroin. The amino acid sequence is more preferably three or more, and particularly preferably 5 to 30. Preferred specific examples of the polypeptide (A) include (GAGAG
S) a peptide having ₉ sequences (10) and an RGD sequence;
(GAGAGS) Peptide having ₉ sequence (10) and YIGSR sequence (2), peptide having (GAP (GPP) ₄ ) ₂ sequence (11) and RGD sequence, (GAP
(GPP) ₄ ) a peptide having _two sequences (11) and a YIGSR sequence (2), and a (GAGAGS) ₉ sequence (1
0) and an IKVAV sequence (7) (JP-A-3-502935).

Commercially available products include Pronectin F (produced by genetically modified Escherichia coli) manufactured by Sanyo Chemical Industries, Ltd., and a cell adhesion signal RGD sequence and a (GAGAGS) ₉ sequence (10) in one molecule. Polynectin F-plus (a polypeptide obtained by reacting Pronectin F with dimeraminoethyl chloride to make it water-soluble), and Pronectin L (produced by genetically modified Escherichia coli) It is produced and the cell adhesion signal IKVAV sequence (7) and (GAGA
GS) a polypeptide having a number average molecular weight of about 90,000 having about 7 each of the ₉ sequences (10)). Also, RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. ｛CS1 signal which is a human fibronectin cell adhesion signal, cell adhesion domain type III and heparin binding domain II
RGDS-Protein A {polypeptide having a number average molecular weight of about 30,000 in which the RGD sequence is inserted into Protein A (IgG binding domain)}.

The method for producing the polypeptide (A) is not particularly limited, and it can be produced in the same manner as a conventionally known method for synthesizing a peptide. For example, an organic synthesis method (solid phase synthesis method, liquid phase synthesis method) Etc.) and biochemical synthesis methods [genetically modified microorganisms (yeast, bacteria, Escherichia coli, etc.)] and the like. Regarding the organic synthesis method, see, for example, “Sequence Chemistry Experimental Course 2 Chemistry of Proteins,” edited by the Biochemical Society of Japan, pp. 641-694 (May 20, 1987).
JP; Tokyo Chemical Co., Ltd.). For biochemical synthesis methods, for example,
The method described in JP-T-7-501443 is used. From the viewpoint that the high molecular weight polypeptide (A) can be easily synthesized, a biochemical synthesis method using a recombinant microorganism is preferable, and a method using a recombinant E. coli is particularly preferable.

Next, the polymer (B) will be described.
Generally, the temperature of cell culture depends on the type of cells,
The temperature is preferably from 90 to 90 ° C, more preferably from 10 to 50 ° C. If the temperature is lower than 0 ° C., the cells tend to be dormant and the proliferative power tends to decrease. If the temperature exceeds 90 ° C., the cell function is destroyed and the cells tend to die. In the cell culture using the cell culture support of the present invention, the cells are cultured on a polymer (B) exhibiting hydrophobicity at a constant temperature,
When the temperature of (B) is changed in the cell detachment / recovery step after completion of the culture, (B) changes to hydrophilic, and the cells grown on (B) are detached, and subsequently washed with an isotonic solution or the like. Proliferating cells can be recovered by simply performing the above steps. The temperature corresponding to the hydrophobic-hydrophilic inflection point is referred to as the critical dissolution temperature, that is, the critical temperature for water means the temperature at which a uniformly dissolved aqueous solution becomes a non-uniform aqueous solution due to a change in temperature. I do.

For example, when the polymer (B) exhibits hydrophilicity below the critical dissolution temperature and shows hydrophobicity above the critical dissolution temperature, the cell culture is usually carried out at a temperature higher than the critical dissolution temperature of (B). The cells grown on (B) are detached and collected by lowering the temperature of (B) below the critical lysis temperature by lowering the ambient temperature or replacing the medium with a low-temperature medium. It is. The critical dissolution temperature of the polymer (B) in water is generally 0 to 80 ° C, preferably 0 to 50 ° C, more preferably 20 to 45 ° C, and particularly preferably 25 to 35 ° C. The critical dissolution temperature of the polymer (B) in water is preferably lower than the temperature of cell culture.

The critical solution temperature of the polymer (B) in water is as follows: (B) is dissolved in water (ion-exchanged water or distilled water) so as to have a concentration of 1% by weight (at a dissolvable temperature). In addition to the method of visually confirming two-phase separation (white turbidity) by raising or lowering the temperature, the method of obtaining the observed value of the critical protein light on the vertical axis and the temperature on the horizontal axis, and the measured value of the scattered light intensity Any method that is generally known, such as a method of obtaining a temperature from a graph having a horizontal axis, a method of obtaining an observed value of transmitted laser light on a vertical axis, and a method of obtaining a temperature from a graph having a horizontal axis. And in combination.

The polymer (B) is not particularly limited as long as it has a critical solution temperature in water of 0 to 80 ° C., and a known (co) polymer of a vinyl monomer can be used.
For example, (meth) acrylic acid derivatives {(meth) acrylic acid amide and (meth) acrylic acid ester etc.} and / or (co) polymers containing vinyl alkyl ether or the like as a structural unit can be used.

Examples of the (meth) acrylic amide include (a) (meth) acrylic amides of monoalkylamines or monoalkoxyalkylamines having 3 to 6 carbon atoms, (b) dialkylamines and dialkoxyalkylamines. Or (meth) acrylamide of a cyclic amine (2-8 carbon atoms), (c) a vinyl monomer having a polyiminoethylene group (degree of polymerization of 2 to 50), and (d) a cyclic amino group or an alkylamino group ( A (meth) acrylic amide having 5 to 50 carbon atoms) is used.

As the (meth) acrylate monomer,
For example, (e) a polyoxyalkylene having a carbon number of 2 to 4 (degree of polymerization: 3 to 40) monool or a diol (meth) acrylic acid monoester, or (f) an alkylene of a cyclic amine (carbon number of 2 to 2) 4) oxides 1-4
(M) acrylic acid ester of 0 mole adduct and (g)
(Meth) acrylic acid ester of an adduct of an acyclic amine (C5-50) alkylene (C2-4) oxide 1-40 mol is used.

Examples of the vinyl alkyl ether monomer include, for example, (h) vinyl alkyl (alkyl having 1 to 6 carbon atoms) ether and (li) alkylene group having 2 carbon atoms.
For example, polyoxyalkylene (degree of polymerization: 3 to 40) monool or diol monovinylphenyl ether is used.

(A) Examples of the (meth) acrylamide of monoalkylamine or alkoxyalkylamine (C3-6) include, for example, N-isopropyl (meth) acrylamide and N-methoxypropyl (meth) acryl. Acid amide and the like. Examples of the (meth) acrylamide of (b) dialkylamine, dialkoxyalkylamine or cyclic amine (2-8 carbon atoms) include, for example, N, N-diethyl (meth) acrylamide and N- (meth) acryloyl And pyrrolidine.

(C) Polyiminoethylene groups (polymerization degree: 2 to 2)
Examples of the vinyl monomer having 50) include monomers described in JP-A-9-12781, such as tetraethyleneimine mono (meth) acrylamide. (D) a cyclic amino group or an alkylamino group (having 5 to 5 carbon atoms)
Examples of the (meth) acrylamide having 50) include N-morpholinoethyl (meth) acrylamide.

(E) The polyoxyalkylene (polymerization degree: 3 to 40) monool having 2 to 4 carbon atoms in the alkylene group or the (meth) acrylic acid monoester of diol includes, for example, tetraethylene glycol monoethyl ether monoester. (Meth) acrylate, pentaethylene glycol monobutyl ether mono (meth) acrylate, methoxytrioxypropylene tetraoxyethylene (meth) acrylate, (meth) acrylic acid monoester of ethylene oxide 6 mol adduct of tetrapropylene glycol, and ethylanol of butanol (Meth) acrylic acid ester of propylene oxide 20 mol adduct of onoxide 20 mol adduct and the like. (F) Examples of (meth) acrylic acid esters of adducts of 1 to 40 moles of alkylene (C2-4) oxides of cyclic amines are described in JP-A-6-9848 such as morpholinoethyl (meth) acrylate. And the like.

(G) Examples of the (meth) acrylic acid ester of an adduct of 1 to 40 moles of an alkylene (2 to 4 carbon atoms) oxide of an acyclic amine (5 to 50 carbon atoms) include, for example, diisopropylaminoethyl (meth) Examples include monomers such as acrylates described in JP-A-6-9848. (H) Vinyl alkyl (C1-6) ether includes, for example, vinyl methyl ether, vinyl ethyl ether and vinyl hexyl ether. (Ii) Examples of the monovinylphenyl ether of a polyoxyalkylene (polymerization degree: 3 to 40) monool or diol having an alkylene group having 2 to 4 carbon atoms include tetraethylene glycol monomethyl ether monovinyl phenyl ether, pentaethylene glycol monoethyl Examples thereof include ether monovinyl phenyl ether, methoxypentaoxypropylene tetraoxyethylene vinyl phenyl ether, and monovinyl phenyl ether of an ethylene oxide 8 mol adduct of tetrapropylene glycol.

Of these, (meth) acrylamide monomers and (meth) acrylic esters are preferable in that the change in hydrophilicity / hydrophobicity around the critical solution temperature is sharp, and cell detachment due to temperature change is preferred. It is more preferable that (A), (B), and (F)
(G) and (g) are particularly preferred in that they have a high affinity for cells due to their ionicity, and the cell proliferation is increased in combination with the effect of (A). . That is, as the polymer (B), a (co) polymer containing (meth) acrylamide and / or (meth) acrylate as an essential monomer is preferable, and (A), (B), and (B) are more preferable. (Co) polymer having at least one (meth) acrylic acid derivative selected from the group consisting of (f) and (g) as an essential monomer, particularly preferably a monomer having (he) and / or (g) as an essential monomer (Co) polymer represented by the following formula:

The critical solution temperature of the polymer (B) in water changes depending on the balance between hydrophilicity and hydrophobicity of (B). For example, the higher the hydrophilicity of (B), the higher the critical solution temperature, and conversely. The higher the hydrophobicity, the lower the critical dissolution temperature. That is, a monomer having high hydrophobicity may be (co) polymerized to obtain a polymer (B) having a low critical solution temperature, and a monomer having high hydrophilicity may be obtained to obtain a polymer (B) having a high critical solution temperature. May be (co) polymerized. When it is desired to repeatedly use the support of the present invention, (B) can be made into a crosslinked body by copolymerizing a crosslinkable monomer.

Examples of the polymer (B) include poly (N-isopropylacrylamide); critical solution temperature of 32 ° C., poly (N, N-diethylacrylamide);
(32 ° C), poly (morpholinoethyl methacrylate); (34 ° C), poly (dipropylaminoethyl acrylate); (25 ° C), poly (morpholinoethyl acrylate); (38 ° C), poly (morpholino) (Butyl methacrylate); (20 ° C.), 99.8 / 0.2 weight ratio copolymer of morpholinoethyl methacrylate and acrylic acid; (35 ° C.), and 96/4 weight ratio of morpholinoethyl methacrylate and methacrylic acid. Polymer;
(45 ° C.). The polymer (B) may be either a crosslinked polymer or a linear polymer, and the weight average molecular weight is not particularly limited, but may be 5,000 to 5,000.
It is preferably 0000 or more, more preferably 10,000 to 1,000,000 or more, particularly preferably 50,000 to 1,000,000 or more. Within this range, cell culture and peeling after culture can be performed more suitably.

Next, the method for producing the cell culture support of the present invention will be described. The material of the substrate to be coated with the polypeptide (A) and the polymer (B) may be any of those commonly used for cell culture, and is not particularly limited. And polymer compounds such as polymethyl methacrylate, glass, modified glass, ceramics, metals and the like. The surface of the base material may be subjected to a hydrophilic treatment using a treatment technique such as ozone treatment, plasma treatment, or sputtering. The shape of the base material is not limited to a petri dish, and may be a plate, a fiber, a (porous) particle, or a container (a flask or the like) generally used for cell culture or the like.

The cell culture support of the present invention has a substrate surface coated with the polypeptide (A) and the polymer (B). In this case, the substrate surface is coated over the entire surface of the substrate to be coated. Alternatively, the coating may partially cover, for example, 50 to 100% of the substrate surface.

In the present invention, the total preferable coating amount of the polypeptide (A) and the polymer (B) is from 1 to
A 1,000,000μg / cm ^2, more preferably 10~100,000μg / cm ^2, particularly preferably 100~10,000μg / cm ^2. 1 μg /
When it is less than cm ² , the cell adhesion and proliferation and the cell detachment due to a temperature change after the cell proliferation tend to be insufficient. On the other hand, if it exceeds 1,000, it tends to be economically disadvantageous. The preferred amount of the polypeptide (A) relative to 100 parts by weight of the polymer (B) is 0.001 to 0.001.
100 parts by weight, more preferably 0.01 to 10 parts by weight.
Parts by weight, particularly preferably 0.1 to 1 part by weight. Within this range, the adhesive growth of the cells and the ease of detachment due to the temperature change after the cell culture can be exhibited more sufficiently.

The method of coating the polypeptide (A) and the polymer (B) is not particularly limited. For example, (1) a method of simultaneously coating (A) and (B), and (2) a method of (A) and (B) Can be used separately. (1) When (A) and (B) are simultaneously coated, (1-
1) A method of coating a mixture of (A) and (B), a method of (1-2) coating a mixture of a monomer constituting (B) and (A), and then polymerizing the monomer. Is mentioned. (2) When coating (A) and (B) separately, (2-
1) A method of first coating (A) on the surface of the base material, and then further coating (B). (2-2) A method of first coating (B) on the surface of the base material, and further (A) How to coat the
(2-3) a method of coating (A), further coating a monomer constituting (B), and then polymerizing the monomer;
-4) A method of coating the monomer constituting (B) on the surface of the base material, polymerizing the monomer, and further coating (A).

In the method (1-1), a mixture of (A) and (B) and, if necessary, various solvents is applied on a substrate, and when a solvent is used, the solvent is distilled off or washed with water. By removing, the support of the present invention is obtained. As the solvent, for example, water, an aqueous solution of various salts (eg, sodium chloride, lithium chloride, lithium perchlorate, etc.),
Hydrophilic solvents (eg, methanol, isopropanol, ethylene glycol, acetone, ethyl acetate, acetonitrile, etc.), lipophilic solvents (eg, benzene,
And mixtures thereof. Among them, water, aqueous solutions of various salts and hydrophilic solvents are preferable, and aqueous solutions of various salts and hydrophilic solvents are more preferable, and hydrophilic solvents are particularly preferable.

In the method (1-2), a mixture of the monomers constituting (A) and (B) and, if necessary, the above-mentioned various solvents, a polymerization initiator, a chain transfer agent and / or a crosslinkable monomer is used as a base material. After coating on top and polymerizing the monomer, if a solvent is used, the support of the present invention can be obtained by distilling off the solvent or removing it by washing with water. The polymerization initiator is not particularly limited, but for example, an azo initiator, an organic or inorganic peroxide, or the like is used.
The chain transfer agent is not particularly limited, but for example, a mercaptan compound or the like is used. The crosslinking monomer is not particularly limited, and for example, ethylene glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, divinylbenzene, and the like are used. As a method of polymerizing, for example, electron beam irradiation (E
B), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, heating (far infrared rays, near infrared rays, hot air, etc.) are used.

In the method (2-1), as a method for coating (A) on the surface of the base material, a method of directly applying (A), a method of applying a mixture with various solvents of (A), and Examples of the method include removal by distillation or washing with water. Further, the support of the present invention can be obtained by coating (B) in the same manner.

In the method (2-2), similarly to the above method, the support of the present invention can be obtained by coating (A) after coating with (B). In the method of (2-3), similarly to the above method, (A)
After coating, a mixture of the monomer constituting (B) and, if necessary, the above-mentioned various solvents, a polymerization initiator, a chain transfer agent and / or a crosslinkable monomer was applied on a substrate, and the monomer was polymerized. Thereafter, when a solvent is used, the support of the present invention can be obtained by distilling off the solvent or removing it by washing with water. In the method of (2-4), similarly to the above method, (B)
When a mixture of a monomer constituting the above and, if necessary, the above-mentioned various solvents, a polymerization initiator, a chain transfer agent and / or a crosslinkable monomer is applied to a substrate, and the monomer is polymerized, the solvent is used. The solvent of the present invention is removed by distilling off or washing with water, if necessary, followed by coating with (A) and, if necessary, by distilling off the solvent or removing by washing with water, followed by coating to obtain the support of the present invention. Can be Among these methods, (1-1), (1-2), (2-2) and (2-
4) is preferable, and (1-2) and (2-) are more preferable.
2) and (2-4), particularly preferably (1-2) and (2-4), and most preferably (2-4).

Next, a method for culturing cells using the cell culture support of the present invention will be described. The method of cell culture is not particularly limited, but a method of adding a mixture of cells and a medium on a support, leaving the mixture at a predetermined temperature and a predetermined temperature, or the like can be used. The type of cell is not particularly limited, and human,
Known cells used in cell culture experiments, such as primary cultured cells and cell lines of monkeys, mice, hamsters, rats and the like can be used.

Examples of the cells include, for example, CHO (Chinese hamster ovary) cells and WI38 (human fetal lung) cells in the case of producing pharmaceuticals, such as skin, cartilage, bone, liver, kidney and cornea. When used for regenerative medicine, human stem cells, endothelial cells, epithelial cells, parenchymal cells, fibroblasts, keratinocytes and the like can be mentioned. In addition, in the case of drug screening as an alternative to animal experiments or evaluation of irritation to eyes and skin, human skin keratinocytes, human and bovine vascular endothelial cells, rat hepatocytes, human corneal cells, and the like are mentioned. Among these cells, human-derived cells are preferable in that sheet-shaped cells (cell sheets) obtained only by using the support of the present invention are particularly suitably used for regenerative medicine.

The medium is not particularly limited, and either a serum medium or a serum-free medium can be used. Depending on the type of polypeptide (A) used, the serum medium
EM medium, BME medium, DME medium, αMEM medium, I
MEM medium, ES medium, DM-160 medium, Fishe
r medium, F12 medium, WE medium, RPMI medium, and the like, and a medium obtained by adding serum to a basal medium described in “Tissue Culture Techniques, Third Edition, edited by The Japanese Society for Tissue Culture”, page 581, published by Asakura Shoten.

As serum, for example, fetal calf serum, calf serum, horse serum, human serum, chicken serum and the like can be used. Examples of the serum-free medium include a commercially available serum-free medium [Serum-free medium ASF103, manufactured by Ajinomoto Co., Inc .;
F104, ASF301 and Gibco's serum-free medium C
HO-SFM, VP-SFM, etc.) and the above-mentioned basal medium contain cell growth factors (fibroblast growth factor, transforming growth factor, insulin-like growth factor, hepatocyte growth factor, vascular endothelial growth factor, heparin-binding epithelium). Cell growth factor, connective tissue growth factor, etc.). When a serum medium is used, proteins with unknown components are contained in the serum, and in the case of drug production using cells, the purification process becomes complicated and costly, and there is a risk of virus infection. For the reason, regenerative medical engineering, which has recently attracted attention, has a concern about safety, and so-called serum-free medium without serum tends to be preferred.However, the support of the present invention has excellent cell adhesion. Because
The use of a serum-free medium is particularly preferred.

There are no particular restrictions on the atmosphere for cell culture. For example, a mixture of carbon dioxide and air (volume mixing ratio of carbon dioxide: air = 0.1-40: 99.9-6)
An atmosphere such as 0) can be used. The culture temperature of the cell culture using the support of the present invention is preferably 1 to 30 ° C higher than the critical dissolution temperature of the polymer (B) coated on the support, more preferably 5 to 20 ° C higher. It is. If the temperature is lower than the critical temperature, cell adhesion / proliferation becomes insufficient. A specific cell culture temperature is preferably 1 to 90 ° C. from the viewpoint that cell growth can be actively performed,
A temperature of 10 to 50C is particularly preferred. Using the support obtained according to the above method, the cells cultured by the above method are detached from the support and recovered by lowering the ambient temperature or the temperature of the substrate or replacing the medium with a low-temperature medium. For example, it is only necessary to lower the temperature of the medium, that is, the temperature of the polymer (B), below the critical dissolution temperature.

[0038]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. <Example 1> Becton Dickinson Labware was used as a base material.
are) using two FALCON Petri dishes (35 mm in diameter) manufactured by Sanyo Chemical Industry Co., Ltd.
Pronectin F plus 1 mg dissolved in ion-exchanged water 1 ml, and morpholinoethyl acrylate (critical temperature of homopolymer: 38 ° C.) (acrylic acid ester of morpholine ethylene oxide 1 mol adduct)
Was dissolved in 0.6 g of isopropyl alcohol, and 0.2 ml each of the mixture was added to a Petri dish, spread over the entire dish, and irradiated with 20 Mrad of an electron beam to produce Pronectin F + on the surface of the Petri dish. And polymorpholinoethyl acrylate. After the electron beam irradiation, the petri dish is washed with ion-exchanged water to remove residual monomers and free polymorpholinoethyl acrylate, dried at room temperature in a clean bench for one week, and further dried overnight in an ethylene oxide (EO) gas atmosphere. After exposure and sterilization, degassing was performed at room temperature under reduced pressure of 1 to 5 mmHg for one day to obtain two cell culture supports (dish 1) of the present invention.

<Comparative Example 1> Two cell culture supports (dish 2) for comparison were obtained in the same manner as in Example 1 except that Pronectin F Plus was not used.

Comparative Example 2 Two cell culture supports (dish 3) for comparison were obtained in the same manner as in Example 1 except that morpholinoethyl acrylate was not used.

<Example 2> In two petri dishes as in Example 1, 0.4 g of morpholinoethyl acrylate (critical temperature of homopolymer: 38 ° C.) dissolved in 0.6 g of isopropyl alcohol was added. .1
After spreading the whole dish, add 2 ml of electron beam.
Polymorpholinoethyl acrylate was coated on the Petri dish surface by irradiation with 0 Mrad. After the electron beam irradiation, the petri dish was washed with ion-exchanged water to remove residual monomers and free polymorpholinoethyl acrylate, and dried in a clean bench.
Then, Pronectin F0.5m manufactured by Sanyo Chemical Industries, Ltd.
g was dissolved in 0.5 ml of the same pronectin FDient (a saturated solution of lithium perchlorate), further diluted 10-fold with water, and 3 ml of the solution kept at 50 ° C was spread over the entire dish kept at 50 ° C. By allowing to stand at 50 ° C. for 1 hour, Pronectin F was physically adsorbed.
Then, after washing twice with a phosphate buffer kept at 50 ° C.,
Drying in a clean bench at room temperature for 1 week, exposure to ethylene oxide (EO) gas atmosphere for 24 hours, sterilization, and degassing at 1 to 5 mmHg under reduced pressure at room temperature for 24 hours. 2 cell culture supports (dish 4) were obtained.

Comparative Example 3 Collagen type I instead of solution of Pronectin F;
Rat (Becton, Code # 354236)
In the same manner as in Example 2 except for using, two cell culture supports (dish 5) for comparison were obtained.

Comparative Example 4 A cell culture support (dish 6) for comparison was prepared in the same manner as in Example 2 except that Pronectin F was not used.
I got one.

Comparative Example 5 Two comparative cell culture supports (dish 7) were obtained in the same manner as in Example 2 except that morpholinoethyl acrylate was not used.

Example 3 A cell culture support of the present invention was prepared in the same manner as in Example 2 except that morpholinoethyl acrylate was replaced with N-isopropylacrylamide (critical temperature of homopolymer: 32 ° C.). (Dish 8) were obtained.

Example 4 Instead of morpholinoethyl acrylate, 99.8 / 0.2 of morpholinoethyl methacrylate and acrylic acid
Two cell culture supports (dish 9) of the present invention were obtained in the same manner as in Example 2 except that the mixture at a weight ratio (critical dissolution temperature of the copolymer: 35 ° C) was used.

Example 5 In the same manner as in Example 2 except that dipropylaminoethyl acrylate (critical temperature for homopolymer dissolution: 25 ° C.) was used instead of morpholinoethyl acrylate, the cell culture support of the present invention ( Two dishes 10) were obtained.

<Cell culture> Obtained dishes 1 to 10
Was used to conduct serum-free cell culture experiments. Chinese hamster ovary cells CHO-K1 (ATCC strain number CCL
-61) was dispersed in a serum-free medium ASF104 manufactured by Ajinomoto Co., Inc. at a cell concentration of 1.0 × 10 ³ cells / mL, and 3 ml was added to each of dishes 1 to 10 to obtain a mixture of carbon dioxide and air (carbon dioxide). / Air = 5/95 volume ratio) at 37 ° C. for 3 days. Next, the adherent growth of the cells on the dish (proliferation) was observed with a microscope, and the results are shown in Table 1.

The criteria for judging the cell adhesion and proliferation (proliferation) are as follows. ◎: 90% or more of the entire area of the dish is covered with cells, and a very good growth state. Good: 50-89% of the whole dish area is covered with cells, and the cells are in a good growth state. Δ: 10 to 50% of the area of the entire dish is covered with cells, and the growth state is poor. ×: The cells were covered by less than 10% of the area of the entire dish, and the proliferation state was completely poor.

The viability of the trypsin method (conventional method), the recovery rate of the cooling method, and the survival rate of the cooling method were determined for each of the dishes 1 to 10 according to the following methods. <Viability of trypsin method> Trypsin-EDTA manufactured by Dainippon Pharmaceutical Co., Ltd.
0.25 mL of the solution was added, and after leaving still for 10 minutes, the cells were detached by pipetting. Dainippon Pharmaceutical Co., Ltd.
A cell suspension was obtained by adding 0.5 mL of a 10% by weight sterilized aqueous solution of fetal bovine serum and pipetting.
Then, a part of this cell suspension was taken and treated with trypan blue, and the number of dead cells per 1 ml was colored, and the number (dead cells) and the total number of cells were counted visually under a microscope using a hemocytometer. The trypsin survival rate was determined according to the formula. Trypsin viability (%) = ｛(total cell count−dead cell count)
/ (Total cell number)｝ × 100 For the dishes 1 to 10, the trypsin method viability was 60 to 80% in each case.

<Cooling Method Recovery Rate, Cooling Method Survival Rate> Out of the two dishes, another dish was cooled to 5 ° C.
The cells were allowed to stand for 15 minutes to detach the adhered cultured cells, followed by pipetting to obtain a cell suspension. Next, a portion of this cell suspension is taken, treated with trypan blue, and the dead cells are colored. The number of cells (dead cells) and the total number of cells are visually observed under a microscope, and the cooling method is used according to the following formula. I asked. Cooling method viability (%) = {(total cell number−dead cell number) / (total cell number)} × 100 In each case, the cooling method viability was 90% or more.
From the total number of cells collected by the trypsin method and the total number of cells obtained by the cooling method, the recovery rate by the cooling method (recovery rate by the cooling method)
Was determined in accordance with the following equation, and the results are shown in Table 1. Cooling method recovery rate (%) = {(total number of cells detached and collected by cooling method) / (total number of cells collected by conventional method)} × 100

[0052]

[Table 1]

From the above Examples and Comparative Examples, when the cell culture support of the present invention was used, cell growth was promoted even in serum-free culture, and simply by lowering the temperature,
It can be seen that the cells can be detached and recovered in high yield (higher recovery rate than the conventional trypsin method and higher cell viability), indicating that the usefulness as a cell culture support is extremely high.
On the other hand, in the case of Comparative Examples 1, 3 and 4 not using the polypeptide (A) of the present invention, the cell proliferation was insufficient, and Comparative Examples 2 and 5 not using the polymer (B) of the present invention. In the case of (1), the cells are not detached even when cooled, so that only the conventional trypsin method can be used to recover the cells. However, in this case, a high survival rate cannot be obtained, and the yield is low. That is, only the cell culture support of the present invention enables high-proliferation and high-yield (high recovery and high viability) cell recovery.

[0054]

According to the support for cell culture of the present invention,
Cells can be cultured efficiently even without serum, and detached and recovered cultured cells with high yield (high recovery and high viability) by simple operation of low temperature treatment (keeping the ambient temperature below the critical temperature) it can. Therefore, cells may not be damaged,
Cultured cells can be obtained extremely easily and efficiently without contamination or contamination of components. Further, the support for cell culture of the present invention is an attempt to obtain tissue cells in vitro in tissue engineering, various hybrid artificial organs,
It can be effectively used for bio-simulators and safety evaluation cells.

[0055]

[Sequence list] <110> Sanyo Chemical Industries, Ltd .; SANYO CHEMICAL INDUSTRIES, LTD. <120> Cell culture support and method for producing the same <160> 11 <210> 1 <211> 4 <212> PRT <213> Homo sapiens <400> 1 Arg Glu Asp Val 1 <210> 2 <211> 5 <213> PRT <213> Homo sapiens <400> 2 Tyr Ile Gly Ser Arg 1 5 <210> 3 <211> 5 <213> PRT <213> Homo sapiens <400> 3 Pro Asp Ser Gly Arg 1 5 <210> 4 <211> 7 <213> PRT <213> Homo sapiens <400> 4 Arg Tyr Val Val Leu Pro Arg 1 5 <210> 5 <211> 6 <213> PRT <213> Homo sapiens <400> 5 Leu Gly Thr Ile Pro Gly 1 5 <210> 6 <211> 10 <213> PRT <213> Homo sapiens <400> 6 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile 1 5 10 <210> 7 <211> 5 <213> PRT <213> Homo sapiens <400> 7 Ile Lys Val Ala Val 1 5 <210> 8 <211> 4 <213> PRT < 213> Homo sapiens <400> 8 Asp Gly Glu Ala 1 <210> 9 <211> 6 <213> PRT <213> Bombyx mori <400> 9 Gly Ala Gly Ala Gly Ser 1 5 <210> 10 <211> 54 <213> PRT <213> Bombyx mori <400> 10 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser G ly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser 50 <210> 11 <211> 30 <213> PRT <213> Homo sapiens <400 > 11 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 1 5 10 15 Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 20 25 30

Claims (11)

[Claims] 1. A polypeptide (A) having at least one minimum amino acid sequence in a molecule that represents a cell adhesion signal, and a polymer (B) having a critical solubility temperature in water of 0 to 80 ° C. A cell culture support, characterized in that: 2. The method according to claim 1, wherein the number of the minimum amino acid sequences representing a cell adhesion signal is 3 to 50 per molecule (A).
A cell culture support as described. 3. The RGD sequence, L, wherein the minimum amino acid sequence representing a cell adhesion signal is represented by one-letter amino acid code.
DV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3), RYVVLPR sequence (4), LGTIPG sequence (5), RNIAEIIKD
I sequence (6), IKVAV sequence (7), LRE sequence, D
3. The cell culture support according to claim 1, which is at least one kind of sequence selected from the group consisting of a GEA sequence (8) and a HAV sequence. 4. The cell culture support according to claim 1, wherein (A) is synthesized by a genetically modified microorganism. 5. The cell culture support according to claim 1, wherein (A) further comprises two or more GAGAGS sequences (9) represented by one-letter amino acid code. 6. The cell culture support according to claim 1, wherein (B) is a (co) polymer comprising a (meth) acrylamide monomer and / or a (meth) acrylate monomer as an essential monomer. . 7. (B) is (meth) acrylic acid amide of monoalkylamine, monoalkoxyalkylamine, dialkylamine, dialkoxyalkylamine or cyclic amine, or (meth) acrylic acid of alkylene oxide adduct of cyclic amine. 2. A (co) polymer comprising at least one (meth) acrylic acid derivative selected from the group consisting of an ester and a (meth) acrylic acid ester of an alkylene oxide adduct of an acyclic amine as an essential monomer. 7. The cell culture support according to any one of claims 6 to 6. 8. A polypeptide (A) having at least one minimal amino acid sequence in one molecule which shows a cell adhesion signal on a surface of a substrate and a critical solution temperature of 0 to 80 for water.
A method for producing a cell culture support, comprising a step of coating a mixture comprising a monomer constituting the polymer (B) at a temperature of 0 ° C. and then polymerizing the monomer. 9. A polymer (B) having a critical solution temperature in water of 0 to 80 ° C. coated on a surface of a base material as a first layer, and a second amino acid sequence exhibiting a cell adhesion signal is further formed on a second layer. A method for producing a cell culture support, comprising a step of coating a polypeptide (A) having at least one in a molecule. 10. A method for producing a cell sheet, comprising a step of culturing cells on the cell culture support according to claim 1 using a serum-free medium. 11. The method according to claim 8, wherein a serum-free medium is used.
A method for producing a cell sheet, comprising a step of culturing cells on a cell culture support produced by the production method described above.