WO2013002311A1

WO2013002311A1 - Substrate for stem cell culture, and culture method using same

Info

Publication number: WO2013002311A1
Application number: PCT/JP2012/066491
Authority: WO
Inventors: 谷原　正夫; 匠徳佐藤; 友美幸得; 賀彰柴崎
Original assignee: 国立大学法人奈良先端科学技術大学院大学
Priority date: 2011-06-28
Filing date: 2012-06-28
Publication date: 2013-01-03
Also published as: JPWO2013002311A1

Abstract

Provided are a substrate for stem cell cultures having no risk of pathogen infection or undesirable side effects, and a method for using the same. The present invention comprises a substrate for stem cell cultures in which a cell adhesion peptide having a cyclic skeleton structure is bonded to a polymer substrate, and a stem cell culture method using the substrate for stem cell cultures. A substance containing the Arg-Gly-Asp sequence found in fibronectin can be used as the cell adhesion peptide with a cyclic skeleton structure. A polypeptide and/or a polysaccharide can be used for the polymer substrate.

Description

Stem cell culture substrate and culture method using the same

The present invention relates to a stem cell culture substrate that does not have the risk of pathogen infection and undesirable side effects, and a method for using the same. More specifically, the present invention relates to a culture substrate that is useful for the proliferation and differentiation of stem cells and has high safety, and a culture method thereof.

Stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) reconstruct tissues and organs inside and outside the body, and realize regenerative medicine that transplants in place of lost tissues and organs Indispensable for. Currently, in order to proliferate stem cells while maintaining pluripotency, fibroblasts derived from animals or humans are used as feeder cells, or culture containers coated with animal-derived materials such as gelatin and matrigel are used. These techniques have a risk of contaminating animal or human-derived fibroblasts or animal-derived materials with pathogens such as prions or unknown viruses, which is one factor that hinders the realization of regenerative medicine.

The inventors have conducted intensive research aimed at creating highly safe biocompatible materials that can be used as alternatives to materials derived from animals and humans, and have so far fully synthesized chemically synthesized polypeptides. Has completed the synthesis technology. For example, Patent Document 1 discloses a novel polypeptide useful as a carrier for various physiologically active substances and apatites without the risk of causing infection of pathogens and transmission of pathogenic factors and undesirable side effects. A peptide unit having an amino acid sequence represented by -Pro-X-Gly- (X represents Pro or Hyp), and -Pro-Hyp (OYZ) -Gly- (Y has a carbonyl group or a carbonyl group) A saturated or unsaturated hydrocarbon group with or without, or a saturated or unsaturated hydrocarbon group with or without a carbonyl group, including an aromatic group, and Z represents a carboxyl group) A polypeptide comprising a peptide unit having an amino acid sequence is disclosed.

On the other hand, Non-Patent Document 2 discloses a material obtained by binding a cell adhesion sequence Gly-Arg-Gly-Asp-Ser and Pro-His-Ser-Arg-Asn found in fibronectin to a polypeptide poly (Pro-Hyp-Gly). Have been reported to promote cell adhesion and migration of the mouse fibroblast cell line NIH3T3 and to promote stratification of the rabbit corneal epithelial cell line.

Patent Document 2 discloses a human pluripotent stem cell culture substrate coated with a human laminin fragment in order to maintain human pluripotent stem cells while maintaining differentiation pluripotency in a feeder-free culture environment. A culture method using this has been proposed.

Patent Document 3 proposes a substrate for culturing embryonic stem cells mainly composed of a sponge-like cross-linked polysaccharide for the purpose of providing a method and material for efficiently inducing differentiation of hepatocytes from embryonic stem cells. Has been.

Patent Document 4 discloses a porous substrate such as a nonwoven fabric for the purpose of providing a culture substrate and a culture method for maintaining undifferentiated embryonic stem cells in large quantities and safely in the absence of feeder cells and feeder cell-derived components. A culture substrate composed of a solid material is proposed.

Patent Document 5 describes a stem cell culture characterized by containing a cell adhesion active substance for the purpose of providing a culture substrate for efficiently proliferating stem cells while maintaining their differentiation ability. Substrates have been proposed.

Non-Patent Document 3 reports a method for binding a cell adhesion sequence Arg-Gly-Asp to a polymer surface containing polylactic acid and a method for constructing a bone tissue using this.

International Patent Publication WO2009 / 035092 Patent Publication 2011-78370 Patent Publication 2006-42758 International Patent Publication WO2003 / 038070 Patent Publication 2002-315567

Under such circumstances, the present invention is excellent in adhesion to stem cells, has no risk of causing pathogen infection or transmission of pathogenic factors, and there is no risk of undesirable side effects, and stem cells using the same It is an object of the present invention to provide a culture method of Another object of the present invention is to provide a culture substrate capable of safely and simply separating stem cells adhered to the culture substrate from the substrate, and a method for culturing stem cells using the same. .

The present inventor has intensively studied to solve the above-mentioned problems, and in order to improve the cell adhesion between the culture substrate obtained by chemical synthesis and the stem cells, the concept of binding the cell adhesion peptide to the synthetic culture substrate is proposed. It came. However, as shown in Test Example 1 to be described later, Gly-Arg-Gly-Asp-Ser (SEQ ID NO: 1) known as a cell adhesion peptide was actually bound to a synthetic culture substrate, and stem cells were cultured. However, although adhesion improvement has been reported in cells other than stem cells (Patent Document 1), no improvement was observed in the adhesion of stem cells to a synthetic culture substrate. This result showed that the integrin of stem cells, unlike cells other than stem cells, did not recognize a peptide sequence known as a cell adhesion peptide. Despite such knowledge, the present inventors made further efforts to study day and night, and tried to culture stem cells by binding a cell adhesion peptide having a cyclic structure to a culture substrate. However, it was surprisingly found that the adhesiveness was dramatically improved and the stem cells can be cultured in an undifferentiated state.

In this way, the present inventors improved the adhesion of stem cells to the culture substrate by binding the cell adhesion peptide having a cyclic skeleton structure to the synthetic culture substrate, and more efficiently differentiated stem cells. It was found that the cells can be cultured while maintaining the performance. Based on these findings, the present inventors have further studied stem cell culture and improvement of its use. In order to differentiate cultured stem cells according to their intended use, the cultured cells are used as a base material. Although it is necessary to liberate it, it has been found that there is a problem that the conventional method using an enzyme or the like causes a large damage to the cells and causes a decrease in the survival rate. As a means for solving this problem, the present inventors use a cytotoxic reagent such as an enzyme by binding a cell adhesive peptide having a cyclic skeleton structure via a photodegradable linker. It was found that the cells can be easily released from the substrate by irradiation with light.

The present inventors have completed the present invention by further studying and improving the above knowledge. The representative present invention is as follows.
Item 1. A stem cell culture substrate in which a cell adhesion peptide having a cyclic skeleton structure is bound to a polymer substrate.
Item 2. Item 2. The stem cell culture substrate according to Item 1, wherein the cell adhesion peptide having a cyclic skeleton structure comprises an Arg-Gly-Asp sequence.
Item 3. Item 2. The stem cell culture substrate according to Item 1, wherein the cell adhesion peptide having a cyclic skeleton structure is cyclo (Arg-Gly-Asp- (D) Phe-Lys) (SEQ ID NO: 2).
Item 4. Item 2. The stem cell culture substrate according to Item 1, wherein the polymer substrate is a polypeptide and / or a polysaccharide.
Item 5. A polypeptide in which the polymer substrate comprises a peptide unit (1) having an amino acid sequence represented by the following formula (1) and a peptide unit (2) having an amino acid sequence represented by the following formula (2): The stem cell culture substrate according to Item 1.
-Pro-X-Gly- (1)
-Pro-Hyp (OYZ) -Gly- (2)
(In the formula, X represents Pro or Hyp, Y represents a carbonyl group, or a saturated or unsaturated hydrocarbon group with or without a carbonyl group, and Z represents a carboxyl group)
Item 6. Y is, - (C = O) - (CH 2) n- (n in the formula is an integer of 0 or 1 ~ 18) ;-( C = O ) - (CH 2) n- (CH = CH) m- (CH ₂ ) k- (wherein n and k independently represent 0 or an integer of 1 to 18, and m represents an integer of 1 to 18); and-(C = O)-(CH ₂ ) n- (C ₆ H ₄ )-(CH ₂ ) k- (wherein n and k independently represent 0 or an integer of 1 to 18, and C ₆ H ₄ represents a phenylene group). Item 6. The stem cell culture substrate according to Item 5, which is one or more types.
Item 7. Item 6. The stem cell culture substrate according to Item 5, wherein the ratio (molar ratio) of peptide unit (1) to peptide unit (2) is (1) / (2) = 99.9 / 0.1 to 1/99. .
Item 8. Item 6. The stem cell culture substrate according to Item 5, wherein the polymer substrate exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum.
Item 9. 6. The stem cell culture substrate according to item 5, wherein at least a part of the polypeptide forms a triple helical structure.
Item 10. 6. The stem cell culture substrate, wherein the polymer substrate is a polypeptide according to item 5, which exhibits a peak in a molecular weight range of 5 × 10 ³ to 5 × 10 ⁶ .
Item 11. Item 2. The stem cell culture substrate according to Item 1, wherein the cell adhesive peptide having a cyclic skeleton structure is bound to a polymer substrate via a photodegradable linker.
Item 12. Item 12. The stem cell culture substrate according to Item 11, wherein the photodegradable linker has a 2-nitrobenzene skeleton, a 2-nitrophenol skeleton, a nitroindole skeleton, or a coumarin skeleton.
Item 13. Item 13. A method for culturing a stem cell, comprising a step of culturing a stem cell on the stem cell culture substrate according to any one of Items 1 to 12.
Item 14. Item 14. The culture method according to Item 13, wherein the stem cells are embryonic stem cells and / or induced pluripotent stem cells (iPS cells).
Item 15. The step of culturing stem cells on the stem cell culture substrate according to Item 11 or 12, and
A method for culturing stem cells, comprising a step of separating stem cells from a stem cell culture substrate by irradiating the culture substrate after the culture with light.
Item 16. Item 5. Cell adhesion having a cyclic skeleton structure by an amide bond obtained by dehydration condensation of a carboxyl group of a polypeptide of a polymer substrate according to Item 5 and an amino group of a cell adhesion peptide having a cyclic skeleton structure A method for producing a stem cell culture substrate, comprising binding a peptide and a polymer substrate.

By using the stem cell culture substrate of the present invention, the adhesion rate of stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) to the substrate is dramatically improved. It becomes possible to culture efficiently while maintaining the pluripotency. Moreover, since the stem cell culture substrate of the present invention does not use animal-derived materials, the risk of pathogen infection and other undesirable side effects (for example, allergic reactions due to animal-derived proteins) are reduced, Excellent safety. Furthermore, in a preferred embodiment of the present invention, the stem cells adhered to the culture substrate can be easily and efficiently released from the culture substrate while suppressing damage to the stem cells. Therefore, by using the stem cell culture substrate of the present invention, the cultured stem cells can be efficiently used for differentiation induction, so that the operability of a series of operations from stem cell culture to differentiation induction is greatly improved. Can do.

2 shows a micrograph showing mouse embryonic stem cells (EB3) cultured for 30 hours on the stem cell culture substrate prepared in Test Example 2. In Production Example 4, a procedure in which a photodegradable linker is bonded to a polymer substrate is shown. The pattern irradiated with light in Test Example 5 is shown. The pattern of the pigment | dye dissociated by the light irradiation in Test Example 5 is shown.

The present invention is described in detail below.
[Definition etc.]
In this specification, various amino acid residues are described by the following abbreviations.
Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: glycine residue His: L-histidine residue Hyp: L-hydroxyproline residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue (D) Phe: D-phenylalanine residue Pro: L-proline residue Sar: sarcosine residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue (D) Val: D-valine residue In the present specification, unless otherwise indicated, the N-terminal amino acid residue is left Is positioned, with the amino acid residues of the C-terminal is located on the right side describes the amino acid sequence of the peptide chain.

[Cell-adhesive peptide having a cyclic skeleton structure]
The cell adhesion peptide is a peptide that is involved in adhesion with cells and has a function of promoting cell adhesion with a substrate. The cell adhesive peptide used in the present invention is not particularly limited as long as it has such a function, and a naturally occurring peptide and an artificially designed peptide can be widely used. Preferred cell adhesion peptides include integrin recognition sequences in the extracellular matrix that bind to integrin molecules on the cell membrane. Currently known cell adhesion peptides include, for example, Arg-Gly-Asp, Pro-His-Ser-Arg-Asn (SEQ ID NO: 3), Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 4), Ile -Lys-Val-Ala-Val (SEQ ID NO: 5), Leu-Asp-Val, Arg-Glu-Asp-Val (SEQ ID NO: 6), Leu-Arg-Glu sequences, etc. The peptide containing can be used suitably. Among these, a cell adhesive peptide containing an Arg-Gly-Asp sequence found in fibronectin is preferable.

The cell adhesive peptide used in the present invention has a cyclic skeleton structure. Here, having a cyclic skeleton structure means that the cell-adhesive peptide described above forms a cyclic skeleton structure. The cyclic skeleton structure may be composed only of a cell adhesive peptide or may be composed of a combination with other peptides. The number of amino acids forming the cyclic skeleton structure is not particularly limited as long as the adhesion to stem cells is maintained. For example, the lower limit is 3 or more and 4 or more, and the upper limit is 7 or less or 6 or less. is there. By limiting the number of amino acids forming the cyclic skeleton structure in this way, the three-dimensional structure of the cell adhesion peptide (ie, the angle formed by the constituent amino acids) is caused by the integrin molecule that is a receptor present on the surface of the stem cell. It is thought that the structure is suitable for recognition. Fibronectin is a typical adhesion protein of the extracellular matrix, and is known to have an important role in controlling cell adhesion, proliferation, migration and differentiation. Fibronectin has multiple cell / extracellular matrix binding domains. Among them, III-10 corresponds to a “cell binding domain”, has an Arg-Gly-Asp sequence, and this part binds to an integrin molecule present on the cell membrane to perform cell adhesion.

The cell adhesive peptide having a cyclic skeleton structure may be, for example, a cyclic peptide composed of the above-mentioned cell adhesive peptide alone or a combination thereof with one or more other amino acids. A cell adhesion peptide having a preferred cyclic skeleton structure is a cyclic peptide containing the Arg-Gly-Asp sequence found in fibronectin, for example, the general formula (I): cyclo (Arg-Gly-Asp-BJ) (where , B and J are arbitrary amino acids independent of each other, one of which is a D type), and a cyclic peptide composed of 5 amino acids. In addition, general formula (I) shows that the α-amino group of Arg and the α-carboxyl group of J form a peptide bond and form a ring as a whole. A cell-adhesive peptide having a more preferred cyclic skeleton structure is as follows. In the general formula (1), B is (D) Phe and J is any amino acid, or B is any amino acid and J is (D) Val. is there. More specific examples of the cell adhesion peptide having a suitable cyclic skeleton structure include cyclo (Arg-Gly-Asp- (D) Phe-Lys) (SEQ ID NO: 7), cyclo (Arg-Gly-Asp- ( D) Phe-Val (SEQ ID NO: 8) cyclo (Arg-Gly-Asp-Phe- (D) Val) (SEQ ID NO: 9), cyclo (Arg-Gly-Asp- (D) -Phe-Ser) (SEQ ID NO: 10), and cyclo (Cys-Arg-Gly-Asp- (D) Phe-Cys) (SEQ ID NO: 11), etc. The cell adhesion peptide having a cyclic skeleton structure is a cell adhesion to stem cells. As long as the property is maintained, it may be modified with other peptides or sugar chains.

The peptide having a cyclic skeleton structure used in the present invention can be produced using a known peptide synthesis method. For example, it is prepared by a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in operation [for example, “Sequence Chemistry Laboratory Lecture 2 Protein Chemistry (below)” edited by the Japanese Biochemical Society (Showa) May 20, 62, issued by Tokyo Chemical Co., Ltd.), pages 641-694].

Preparation of a peptide having a cyclic skeleton structure according to the present invention by a solid phase synthesis method can be performed, for example, by the following procedure. First, an amino acid corresponding to the C-terminus of the target peptide is bonded to a polymer that is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer via the α-COOH group that it has. Next, the amino acid or peptide fragment corresponding to the amino acid in the direction of the N-terminal of the target peptide is protected with a functional group such as an α-amino group other than the α-COOH group of the amino acid or peptide fragment. To condense and bond. Then, the peptide chain is elongated by sequentially repeating this operation and the operation of removing the protecting group of the amino group that forms a peptide bond such as an α-amino group in the bound amino acid or peptide fragment. When a peptide chain corresponding to the target peptide is formed, the peptide chain is detached from the polymer and then cyclized. The protecting group is removed from the protected functional group of the resulting cyclic peptide. Finally, the obtained peptide is purified. Here, the elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed using trifluoroacetic acid from the viewpoint of suppressing side reactions. Further, it is effective to purify the obtained peptide by reverse phase liquid chromatography.

[Polymer substrate]
The type of polymer substrate used in the present invention is not particularly limited as long as the risk of pathogen infection and other undesirable side effects are reduced, but is usually a polypeptide and / or polysaccharide. A polypeptide is preferable, and a polypeptide including a peptide unit having an amino acid sequence represented by the formula (1) -Pro-X-Gly- is particularly preferable. The sequence represented by -Pro-X-Gly- contributes to making the entire polypeptide into a stable triple helical structure. Therefore, this unit (1) has a repeating structure (oligo or polypeptide unit structure) represented by-(Pro-X-Gly) n- in the polypeptide from the viewpoint of the stability of the triple helix structure. It may be formed. The repeating number n of this arrangement is, for example, about 1 to 5000, preferably about 2 to 3000. X may be either Pro or Hyp, but is more preferably Hyp in view of the stability of the triple helical structure. Hyp is usually a 4Hyp (eg, trans-4-hydroxy-L-proline) residue.

The polypeptide includes a peptide unit having an amino acid sequence represented by the formula (2) -Pro-Hyp (O-Y-Z) -Gly-. By having such a peptide unit (2), a cell adhesive peptide having a cyclic skeleton structure can be bound to the polypeptide. In the formula (2), Y represents a carbonyl group or an alkyl group with or without a carbonyl group, and Z represents a carboxyl group.

YZ in the formula (2) serves as a linker group for binding a cell adhesion peptide having a cyclic skeleton structure to the polypeptide. Z is a linker terminal and represents a carboxyl group. When Y is — (C═O) — (CH ₂ ) n—, n preferably represents 0 or an integer of 1 to 18, more preferably 1 to 15, and most preferably 2 to 12. In addition, when Y is — (C═O) — (CH ₂ ) n— (CH═CH) m— (CH ₂ ) k—, n and k are independently preferably 0 or 1 to 18, More preferably, it represents an integer of 1 to 15, most preferably 2 to 12, and m preferably represents 0 or an integer of 1 to 18, more preferably 1 to 12, and most preferably 1 to 8. Further, when Y is — (C═O) — (CH ₂ ) n— (C ₆ H ₄ ) — (CH ₂ ) k—, n and k are independently preferably 0 or 1 to 18, More preferably, it represents an integer of 0 to 12, most preferably 0 to 8, and C ₆ H ₄ represents a phenylene group.

The dicarboxylic acid linker represented by (YZ) in formula (2) can be formed by adding dicarboxylic anhydride to the polypeptide main chain. That is, when Y is-(C = O)-(CH ₂ ) n-, oxalic anhydride, malonic anhydride, succinic anhydride, 4-carboxybutyric anhydride, 5-carboxyvaleric anhydride, 6-carboxy anhydride Caproic acid, 7-carboxyheptanoic anhydride, 8-carboxycaprylic anhydride, 9-carboxypelargonic anhydride and the like can be added by reacting with the hydroxyl group of the hydroxyproline residue of the polypeptide main chain. Likewise, Y is - (C = O) - ( CH 2) n- (CH = CH) if m- the (CH ₂₎ k-, maleic anhydride, pent-2-ene dioic acid anhydride, hexa In the case of-(C = O)-(CH ₂ ) n- (C ₆ H ₄ )-(CH ₂ ) k- for -3-enedioic anhydride, citraconic anhydride, etc., phthalic anhydride Etc. can be added by reacting with the hydroxyl group of the hydroxyproline residue of the polypeptide main chain.

In the polypeptide, the ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) is, for example, (1) / (2) = 99.9 / 0.1 to 1/99 The ratio is preferably about 0.5 / 99.5 to 2/98, more preferably about 99/1 to 5/95. The ratio (molar ratio) between the total amount of the peptide unit (1) and the peptide unit (2) and other peptide units is the former / the latter = 100/0 to 50/50, preferably 100/0 to 60 / 40, more preferably about 100/0 to 70/30.

The polypeptide is, for example, a molecular weight of 5 × 10 ³ to 5 × 10 ⁶ , preferably a molecular weight of 1 × 10 ⁴ to 3 × 10 ^{6 in} terms of globular protein measured by aqueous gel permeation chromatography (GPC). The peak is preferably in the range of about 3 × 10 ⁴ to 2 × 10 ⁶ , more preferably about 5 × 10 ⁴ to 1 × 10 ⁶ .

Further, the polypeptide exhibits a positive cotton effect at wavelengths of 220 to 230 nm and a negative cotton effect at wavelengths of 195 to 205 nm in a circular dichroism spectrum. Therefore, at least a part of the polypeptide (that is, part or all) forms a triple helical structure, and forms a collagen-like polypeptide. The cotton effect refers to a phenomenon that occurs because the optical rotation material has different absorption coefficients for left and right circularly polarized light at a specific wavelength.

Since the polypeptide (including the complex) is chemically synthesized from a pure reagent, infection with pathogens or pathogenic factors [eg, proteins converted to pathogenicity (eg, abnormal prions, etc.)] There is no danger of transmission. Therefore, the polypeptide is highly safe. It is also excellent in cell affinity and biocompatibility.

The polypeptide can be produced according to a known method. For example, a polypeptide obtained by condensing an amino acid component or peptide fragment component containing at least an amino acid or peptide fragment corresponding to formula (1) and an amino acid or peptide fragment corresponding to formula (3) below, It is preferable to obtain a polypeptide by reacting the compound represented by 4).
-Pro-Hyp-Gly- (3)
HO-YZ (4)
(Wherein Y represents a carbonyl group or an alkyl group having or not having a carbonyl group, and Z represents a carboxyl group)

The above-mentioned polypeptide suitable for use as a polymer substrate and the production method thereof are described in detail in WO2009 / 035092, and can be produced with reference to them. WO2009 / 035092 is incorporated herein in its entirety with the aid of it.

[Stem cell culture substrate]
The base material for stem cell culture of the present invention is obtained by binding the cell adhesive peptide having the above cyclic skeleton structure to the above polymer base material, and in a culture vessel such as a plastic dish used for normal cell culture. And can be commercialized. Such a culture container provided with the stem cell culture substrate of the present invention is obtained, for example, by binding a cell adhesion peptide having a cyclic skeleton structure after coating a polymer substrate on a culture surface such as a plastic dish. be able to. Alternatively, after the cell substrate peptide having a cyclic skeleton structure is bound to the polymer substrate, the culture vessel may be prepared by coating the culture surface such as a plastic dish using the solution. In such a production process, a cell-adhesive peptide having a cyclic skeleton structure after processing a polymer substrate in various shapes such as a film shape, a sponge shape, a fiber shape, a rod shape, a bead shape, and a gel in advance. May be combined. In addition, a solution in which a polymer substrate and a cell-adhesive peptide having a cyclic skeleton structure are combined may be processed into the above shape, and the surface or inner surface of various forms of processed products such as metals, ceramics, semiconductors, and plastics may be coated. May be.

The polymer substrate of the present invention or the polymer substrate to which the cell-adhesive peptide having a cyclic skeleton structure is bonded can be formed into a desired form by a conventional method depending on the application. For example, a polymer substrate solution or suspension is cast on a peelable substrate (for example, a fluororesin (specifically, polytetrafluoroethylene)) sheet, dried, and the peelable group A polymer substrate in the form of a sheet or film can be obtained by peeling from the material. Also, a fibrous polymer substrate is obtained by extruding a solution or suspension of the polymer substrate from a nozzle in a poor solvent for the polymer substrate (or a solution containing a high concentration of salt, etc.). Can do. Furthermore, a gel-like polymer substrate can be obtained by allowing an aqueous solution or suspension of the polymer substrate to stand, or if necessary, adding a polyvalent crosslinking reagent (glutaraldehyde, etc.) and allowing to stand. it can. A sponge-like porous body can be obtained by freeze-drying the gel-like polymer substrate thus produced. A polymer substrate that is a porous body can be obtained by foaming (foaming) an aqueous solution or suspension of the polymer substrate by stirring or the like and drying. In the above, the polymer substrate may be bound with a cell adhesive peptide having a cyclic skeleton structure.

Furthermore, a polymer substrate to which a cell adhesion peptide having a cyclic skeleton structure is bound can also be used as a coating. For example, the surface of the molded body can be coated with the polymer base material by applying or dispersing the solution or suspension of the polymer base material on the surface of various molded bodies and then drying. Here, examples of the molded body include molded bodies formed of various materials such as metals, ceramics, and polymers (synthetic or natural polymers). In addition, base materials, such as a polymer molded object, may have biodegradability or biodegradability.

The form of the molded body and the polymer substrate is not particularly limited, and is granular, one-dimensional shaped substrate (fibrous or thread-like substrate, linear substrate, rod-shaped substrate, etc.), two-dimensional shape Base materials (film (or sheet) or plate-like base material), and three-dimensional base materials (tube-like base material etc.). Further, the molded body and the polymer substrate may be non-porous bodies (for example, powdered porous bodies, cellulose fiber papers, two-dimensional porous bodies such as nonwoven fabrics and woven fabrics, cylindrical shapes) Or a three-dimensional porous body). In such a porous body, a polymer substrate solution or suspension may be impregnated to hold the polymer substrate. If necessary, the molded body may be surface-treated with a surface treatment agent (for example, a physiologically acceptable surface treatment agent).

[Bonding of cell adhesion peptide and polymer substrate]
The cell adhesion peptide having a polymer substrate and a cyclic skeleton structure is an amide obtained by dehydration condensation of a carboxyl group usually possessed by a polymer substrate and an amino group possessed by a cell adhesion peptide having a cyclic skeleton structure. It can be combined by bonding. The binding method is not particularly limited as long as it can be immobilized without losing the cell adhesion activity of the cell adhesive peptide, and can be immobilized by any method known in the art. The preferred immobilization method may vary depending on the type of cell adhesive peptide, the type of polymer base material, and the like. For example, the cell base peptide having a carboxyl group and a cyclic skeleton structure is amino acid. When it has a group, an active ester method using N-hydroxysuccinimide, a direct condensation method using water-soluble carbodiimide, or the like is preferably employed. The substrate may be used after sterilization or sterilization as necessary. In particular, in medical applications and the like, usually, sterilization or sterilization is often performed. As the sterilization and sterilization method, conventional methods such as wet heat steam sterilization, gamma ray sterilization, ethylene oxide gas sterilization, chemical sterilization, and ultraviolet sterilization can be used. Among these methods, gamma ray sterilization and ethylene oxide gas sterilization are preferable in that they have a small effect on sterilization efficiency and materials. The amount of the peptide having a cyclic skeleton structure bonded to the polymer substrate is not particularly limited, and can be appropriately set according to the type of cells to be used.

The cell adhesive peptide having a cyclic skeleton structure may be directly bonded to the polymer substrate as described above, but can also be bonded via various functional linkers. Suitable linkers include photodegradable linkers. By binding a cell adhesion peptide having a cyclic skeleton structure to a polymer substrate via a photodegradable linker, the stem cells cultured on the substrate are irradiated with light, and the damage to the stem cells is minimized. It can be separated from the stem cell culture substrate in a suppressed manner.

The photodegradable linker that can be used for the above purpose has the property that intramolecular bonds are cleaved by irradiation with light, as long as it does not adversely affect the adhesion of cell adhesion peptides, the growth of stem cells, etc. A photodegradable linker known in the art (for example, one used for producing a caged compound) can be appropriately selected and used. Specific examples include a photodegradable linker having a 2-nitrobenzene skeleton, a 5-nitrophenol skeleton, a nitroindole skeleton, or a coumarin skeleton, and preferably a photolysis having a 5-nitrophenol skeleton and a coumarin skeleton. These are all known linkers. More specific photodegradable linkers include 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butanoic acid, Bis (4- (4- (1- (acryloyloxy) ethyl) -2 Examples include linkers formed using -methoxy-5-nitrophenoxy) butanate) PEG and N- (6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl) -L-glutamate.

As long as the bond between the polymer substrate and the cell-adhesive peptide having a cyclic skeleton structure via the photodegradable linker does not affect the cell-adhesive activity of the cell-adhesive peptide, the structure of the photodegradable linker and cell adhesion It can implement by the arbitrary methods according to the structure of sex peptide. For example, when a hydroxymethyl photolinker is used, the carboxy group and the carboxy group of the polymer substrate of the present invention are linked via Lys-Lys, and then the cell having the hydroxy group of the hydroxymethyl photolinker and a cyclic skeleton structure is used. By binding the amino group or hydroxy group of the adhesive peptide via succinic acid, a stem cell culture substrate in which the cyclic cell adhesive peptide is bound to the polymer substrate via a photodegradable linker can be obtained. . Similarly, when other photodegradable linkers are used, the cell adhesive peptide having a cyclic structure and the polymer substrate can be bonded via the photodegradable linker.

[Stem cell culture method]
In the present specification, stem cells are cells having both undifferentiated and pluripotent properties such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), hematopoietic stem cells, mesenchymal stem cells, neural stem cells, etc. Means. As for the culture of stem cells, optimal culture methods and conditions are known depending on the type of stem cells. Among the culture conditions, the culture substrate, feeder cells, medium, passage time, etc. are considered important for maintaining the cell properties. Conventionally, plastic culture flasks and dishes are usually used as the culture substrate. Depending on the type of stem cells, collagen, gelatin, laminin, matrigel, etc. are coated on the plastic culture surface, or feeder cells such as MEF are simply used. Layer culture is used. In the stem cell culturing method of the present invention, a polymer substrate to which the cell adhesive peptide having the above-described cyclic skeleton structure is bonded instead of the culture substrate using the animal-derived material in the conventional stem cell culturing method is used. Thereby, there is no danger of contamination with a heterologous protein or a pathogen and it can be used safely. Regarding conditions other than using the culture substrate of the present invention as a culture substrate, appropriate conditions known for various types of stem cells to be cultured (for example, an appropriate medium such as a GMEM medium, conditions of 37 ° C. and 5% CO ₂ ) Can be cultured.

[Method of light separation of stem cells]
Stem cells cultured using a stem cell culture substrate in which a cell-adhesive peptide having a cyclic skeleton structure and a polymer substrate are bonded via the photodegradable linker described above can be easily obtained by irradiating light. It can be separated from the material. Therefore, since it is not necessary to perform a toxic treatment such as enzyme treatment, the stem cells can be separated from the substrate without reducing the viability of the cultured stem cells. It is also possible to specifically separate only the cells present at the site irradiated with laser light from the substrate. The stem cells thus separated from the base material can be used as they are, and can be used for further subculture or various differentiation induction treatments as necessary. The light intensity, wavelength, and irradiation time necessary for separating the cells from the substrate can be appropriately set according to the photodegradable linker used. Depending on the type of the photodegradable linker used, the irradiation can be carried out by irradiating light in the vicinity of 350 nm to 410 nm for about 5 minutes. The means for irradiating such light is not particularly limited, but it is preferable to use a confocal laser microscope or the like because irradiation intensity can be controlled and pattern irradiation can be performed.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Production Example 1: Production of polymer substrate and incubator provided with the same H-Pro-Hyp-Gly-OH (manufactured by Peptide Institute, Inc.) 100 mg (0.35 mmol) in 2 mL of 10 mM phosphoric acid Dissolved in salt buffer (pH 7.4). To this mixture, 9.5 mg (0.07 mmol) of 1-hydroxybenzotriazole was added and dissolved by stirring. While the mixture was cooled to 4 ° C. and stirred, 335 mg (1.75 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added, and the mixture was further stirred at 4 ° C. for 90 minutes. Continued. Then, the temperature is raised to 20 ° C., diluted with 4 mL of 10 mM phosphate buffer saline (containing 0.15 M NaCl, pH 7.4), dialyzed against Milli-Q water for 7 days, condensing agent, etc. The reagents and unreacted monomers were removed.

The obtained polypeptide was subjected to gel permeation chromatography (Amersham Biosciences, AKTApurifier system). A Superdex 200 HR GL column was used and the flow rate was 0.5 mL / min. As an eluent, 10 mM phosphate buffered saline (containing 0.15 mM NaCl, pH 7.4) was used. As a result, a polypeptide peak was observed at the elution position where the molecular weight was 120,000 or more. The molecular weight was calculated using a polyethylene glycol standard product manufactured by Waters as a standard substance. When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 197 nm, confirming the formation of a triple helical structure.

The obtained polypeptide was diluted with MilliQ water to give a 1 mg / mL aqueous solution. 1 mL of the aqueous solution of the obtained polypeptide was added to a glass dish having a diameter of 28 mm, and evenly diffused on the bottom surface, and then allowed to stand at room temperature for 48 hours to obtain a dry film. The polypeptide film prepared on the bottom surface of the glass dish was washed twice with a small amount of dimethylformamide (DMF). To the washed film, 3.8 mg (0.038 mmol) of succinic anhydride (Wako Pure Chemical), purified by recrystallization from hot isopropanol, and 6.5µL (0.038 mmol) of diisopropylethylamine were added under ice cooling, and then overnight at room temperature. Shake. After removing the reaction solution, the polypeptide film was washed 5 times with a small amount of DMF, then 5 times with a small amount of methanol, and dried under reduced pressure.

In the infrared absorption spectrum of the resulting succinylated polypeptide, ester absorption appeared at 1735 cm ⁻¹ , confirming succinic oxidation. Further, from the intensity ratio with the absorption of amide at 1639 cm ^−1, the ratio of peptide units (1) and (2) ((1) / (2)) was 25/75 (molar ratio). When the circular dichroism spectrum of the obtained succinylated polypeptide was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm, confirming the formation of a triple helical structure. It was. As described above, a glass dish in which a polymer substrate which is a succinylated polypeptide film was provided on the bottom surface was produced.

Production Example 2: Binding of linear cell adhesion peptide The succinylated polypeptide film obtained in Production Example 1 was washed once with DMF. After adding 4.3 mg (0.038 mmol) of N-hydroxysuccinimide and 7.3 mg (0.038 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride, the mixture was stirred at room temperature overnight. Then, it was washed 5 times with DMF, and the linear cell adhesion peptide Gly-Arg-Gly-Asp-Ser, 1 / 2AcOH, 2H ₂ O 0.053 mg (95 nmol: manufactured by Peptide Institute, Inc.) And 17 nL (95 nmol) of diisopropylethylamine were dissolved in 100 μL of DMF and added. The mixture was then stirred overnight at room temperature. After washing 5 times with DMF and then 3 times with ethanol, it was aseptically dried under reduced pressure. Thus, a glass dish provided with a polymer substrate to which a linear cell adhesive peptide was bound was prepared.

Test Example 1: Stem cell adhesion test 1
A glass dish in which the polymer substrate obtained in Production Examples 1 and 2 is provided on the bottom surface, a glass dish in which a polymer substrate to which a linear cell-adhesive peptide is bound is provided on the bottom surface, and gelatin Mouse embryonic stem cells (CJ7) were dispensed at a rate of 1 × 10 ⁴ cells / cm ² into the plastic dish coated with the above. CJ7 cells used were dispersed in GMEM medium (1% FCS, 0.1 mM MEM-NEAA, 0.1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 10% Knockout SR, 40 U / mL penicillin / streptomycin). The cells were cultured for 2 hours at 37 ° C. and 5% CO ₂ , and the cell adhesion rate was measured. The cell adhesion rate was determined by removing the cells that did not adhere to the culture substrate film by washing once with PBS, counting the number of adhered cells under a microscope, and the number of cells dispensed (1 × 10 ⁴ cells / cm Calculated as the ratio (%) of the number of adherent cells to ² ). As a result, the cell adhesion rate in the gelatin-coated dish was 25%, whereas the cell adhesion rate in the dishes obtained in Production Examples 1 and 2 was 5% or less. This is substantially cell-adhesive to both polymer substrates to which stem cells bind linear cell-adhesive peptides and polymer substrates to which cell-adhesive peptides are not bound. Indicates no. This result is different from the result of the cell adhesion test using NIH3T3 cells which are non-stem cells reported in WO2009 / 035092. This was thought to indicate that, unlike non-stem cells, stem cells do not recognize cell adhesion peptides.

Test Example 2: Stem cell adhesion test 2
A test similar to Test Example 1 was performed using another stem cell, mouse-derived stem cell EB3 (AES0139). GMEM medium (1% FCS, 0.1 mM MEM-NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 10% Knockout SR, 50 U / mL penicillin / streptomycin, 2000 U / mL ESGRO (Chemicon) Registered trademark), 10 μg / mL blasticidin S). After culturing at 37 ° C. and 5% CO ₂ for 12 hours and washing with PBS to remove non-adherent cells, the number of adherent cells was counted by observation with a phase contrast microscope. As a result, as in Test Example 2, the adhesion rate of EB3 cells was lower than that when a gelatin-coated dish was used. This result strongly suggested that stem cells, unlike non-stem cells, did not recognize cell adhesive peptides and did not have adhesiveness.

Production Example 3 Binding of Cyclic Cell Adhesive Peptide In Production Example 2 above, cell adhesive peptide cyclo (Arg-Gly-Asp- (D) Phe-Lys) having a cyclic skeleton structure instead of the linear cellular peptide A glass dish provided with a polymer substrate to which a cell-adhesive peptide having a cyclic skeleton structure was bonded was prepared in the same manner as in Production Example 2 except that 0.057 mg (95 nmol) (AnaSpec) was used. .

Test Example 3: Stem cell adhesion test 3
Instead of the glass dishes obtained in Production Examples 1 and 2, a glass dish in which a polymer substrate to which a cell adhesive peptide having a cyclic structure skeleton obtained in Production Example 3 was bonded was provided on the bottom surface was used. Except for the above, the adhesion rate of mouse-derived stem cells EB3 was measured in the same manner as in Test Example 2. As a comparison target, a plastic dish for culture manufactured by Iwaki Co., Ltd., whose bottom surface was coated with a 0.1% gelatin solution was used. After culturing for 12 hours, the cell adhesion rate was measured. As a result, in the dish to which the cell adhesion peptide having a cyclic structure skeleton was bound, about 1.9 times cell adhesion was recognized as compared with the gelatin-coated dish. This is probably because the cell adhesion peptide has a three-dimensional structure that can be recognized by integrins on the stem cell surface as a result of the cyclic structure. Moreover, the stem cells grown 30 hours after the start of culture formed colonies on both substrates (FIG. 1).

Test Example 4: Confirmation of Undifferentiated Culture Stem cell culture of Test Example 3 was continued for 3 days at 37 ° C. and 5% CO ₂ . The medium was replaced with fresh one day after the start of culture. After culturing for 3 days, the cells were detached from the substrate with 0.25% trypsin and 0.02% EDTA, and EB3 dispersed in the GMEM medium on each substrate was again dispensed at a rate of 1 × 10 ⁴ cells / cm ² . Thereafter, the cells were further cultured for 3 days, and the ALP activity of the cells was measured using NBT / BCIP stock solution manufactured by Roche. As a result, stem cells on both substrates were stained positive for ALP. Thus, it was confirmed that stem cells can be cultured in an undifferentiated state by using a polymer base material to which a cell adhesive peptide having a cyclic skeleton structure of the present invention is bound.

Test Example 5: Effect of binding amount of cell adhesion peptide having cyclic skeleton structure Except that the amount of cyclo (Arg-Gly-Asp- (D) Phe-Lys) and diisopropylethylamine was changed to 380 nmol, 190 nmol, and 48 nmol In the same manner as in Production Example 3, a glass dish was prepared in which a polymer substrate to which a cell adhesion peptide having a cyclic skeleton structure was bound was provided on the bottom surface. Using these, mouse-derived stem cells EB3 were cultured for 12 hours in the same manner as in Test Example 2. Thereafter, the cells were washed with PBS to remove cells not adhered to the substrate, and the number of adherent cells was measured using a phase contrast microscope. As a comparison object, a gelatin-coated dish was used. As a result, compared with the case of using a gelatin-coated culture substrate, culture was performed on a substrate prepared using 380 nmol, 190 nmol, and 48 nmol of cyclo (Arg-Gly-Asp- (D) Phe-Lys). The number of adherent cells was 1.6 times, 1.8 times, and 1.4 times, respectively.

Production Example 4: Preparation of Photodegradable Linker-Linked Polymer Substrate A photodegradable linker was bonded to a polypeptide (polymer substrate) that forms a triple helical structure according to the following procedure. First, a Lys-Lys group was bonded to a polymer substrate. 0.29 ml of an aqueous solution containing a polymer base material confirmed to form a triple helical structure in Production Example 1 at a concentration of 1 mg / ml was applied to a diameter of 18 mm on a cover glass and air-dried. This was washed 3 times with DMF, added with 80 μL of DMF solution containing 5.5 mg of succinic anhydride recrystallized from isopropanol and 9.6 μl of diisopropylethylamine, shaken on ice for 30 minutes, and then stirred at room temperature overnight. After washing 5 times with DMF and 5 times with methanol, it was dried under reduced pressure. The FTIR spectrum of the film thus obtained was measured, and the addition of succinic acid was confirmed by the appearance of a new absorption peak attributed to 1,730 cm ⁻¹ carboxylic acid ester.

The obtained succinic polymer substrate was washed 3 times with DMF, 6.3 mg HOSu and 10.5 mg EDC · HCl dissolved in 80 μL DMF were added, and the mixture was stirred at room temperature overnight. After washing 5 times with DMF and 5 times with methanol, it was dried under reduced pressure. The FTIR spectrum of the obtained film was measured, and the formation of HOSu ester was confirmed by the appearance of two new absorption peaks attributed to the HOSu ester near 1,800 cm ⁻¹ .

The polymer base HOSu ester was washed 3 times with DMF, and then 2.3 mg Lys-Lys · HCl and 3.5 μl diisopropylethylamine dissolved in 80 μL 50% DMF / H ₂ O were added at room temperature. Stir overnight. The extract was washed 5 times with 50% DMF / H ₂ O, washed 5 times with methanol, and then dried under reduced pressure. The FTIR spectrum of the obtained film was measured, and the reaction of the HOSu ester was confirmed by the disappearance of two absorption peaks attributed to the HOSu ester near 1,800 cm ⁻¹ . In this way, a polymer substrate to which Lys-Lys was bonded was obtained.

Next, a photodegradable linker was bonded to the polymer substrate to which the Lys-Lys group was bonded. The subsequent procedure is shown in FIG. 2 (in FIG. 2, R represents a fluorescent dye or a cell adhesion peptide). Dissolve 50 mg of 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butanoic acid (Merck) in 3 ml of dehydrated THF, add 23 mg of HOSu and 41 mg of DCC. Stirred for 1 hour. Thereafter, the mixture was stirred overnight at room temperature under light shielding. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a yellow solid. This was washed with isopropanol and then dried under reduced pressure under light shielding to obtain 57 mg of 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butanoic acid HOSu ester. The FTIR spectrum was measured, and the formation of HOSu ester was confirmed by the appearance of two new absorption peaks attributed to the HOSu ester near 1,800 cm ⁻¹ .

After washing the Lys-Lys-bonded polymer substrate 3 times with DMF, 2.2 mg 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butanoic acid HOSu ester dissolved in 80 μL DMF And 0.5 μl of diisopropylethylamine were added, and the mixture was stirred overnight at room temperature in the dark. Then, after washing 5 times with DMF and 5 times with methanol, it was dried under reduced pressure. The FTIR spectrum of the obtained film was measured, and new appearance of two absorption peaks attributed to 1,280 cm ⁻¹ nitro group and 1,500 cm ⁻¹ phenyl group was confirmed. Moreover, the UV absorption spectrum of the obtained film was measured, and the appearance of a new absorption peak at 350 nm was confirmed. These confirmed the binding of 4- [4- (1-Hydroxyethyl) -2-methoxy-5-nitrophenoxy] butanoic acid to the polymer substrate.

The obtained polymer substrate was washed 3 times with DMF, 5.5 mg of succinic anhydride recrystallized from isopropanol and 80 μL of DMF solution of 9.5 μl of diisopropylethylamine were added, and the mixture was shaken on ice for 30 minutes, and then at room temperature. Stir overnight. After washing 5 times with DMF and 3 times with methanol, it was dried under reduced pressure. The FTIR spectrum of the obtained film was measured, and a marked increase in the absorption peak attributed to the carboxylic acid ester was observed at 1,730 cm ⁻¹ , confirming the addition of succinic acid.

The obtained polymer substrate to which the succinylated photodegradable linker was bonded was washed 3 times with DMF, 3.6 mg HOSu and 6.3 mg EDC · HCl dissolved in 80 μL DMF were added, and the mixture was stirred overnight at room temperature. After washing 5 times with DMF and 3 times with methanol, it was dried under reduced pressure. The FTIR spectrum of the obtained film was measured, and the production of HOSu ester was confirmed by the appearance of two new absorption peaks attributed to the HOSu ester near 1,800 cm ⁻¹ . In this way, a polymer substrate to which a photodegradable linker was bonded was obtained.
Test Example 6: Dissociation of Rhodamine 110 from substrate by light irradiation

The polymer substrate with the photodegradable linker obtained in Production Example 4 was washed 3 times with 10 mM phosphate buffer (pH 7.4) and then dissolved in 200 μL of 10 mM phosphate buffer (pH 7.4). Rhodamine 110 (53.5 mol / ml) was added, and the mixture was stirred overnight at room temperature in the dark. Thereafter, the polymer substrate was washed 5 times with 10 mM phosphate buffer (pH 7.4) to obtain a polymer substrate to which Rhodamine 110 was bonded through a photolabile crosslinking agent. A confocal laser microscope (Zeiss LSM710: manufactured by Carl Zeiss) was used to irradiate the white area of the pattern shown in FIG. Irradiation conditions are as follows: objective lens: 10 times, laser: 405 nm (30 mW), intensity: 5%, 10%, 30%, 、 60%, 90%, irradiation time: 1 minute, 5 minutes, 10 Min. Thereafter, the fluorescence obtained by exciting the dissociation of the fluorescent dye with light irradiation at 488 nm was observed at 491 nm to 740 nm. The conditions at the time of observation are as follows: objective lens: 10 times, laser: 488 nm (250 mW), intensity: 2%, observation wavelength: 491-740 nm. The results are shown in FIG. 4 (the numerical value on the vertical axis indicates the intensity of the irradiation laser, and the numerical value on the horizontal axis indicates the laser irradiation time). From the result of FIG. 4, since a clear pattern corresponding to the irradiation region was obtained, it was confirmed that the photodissociation linker bonded to the culture substrate of the present invention can be dissociated with high accuracy by light irradiation. It was done. In this way, instead of fluorescent dye, a peptide having a cyclic skeleton structure is bound to a polymer substrate via a photodissociation linker, and the stem cells are cultured using the peptide, thereby allowing the cultured stem cells to be easily and easily irradiated with light. Can be released from the culture substrate.

Production Example 5 Binding of Cyclic Peptide to Polymer Substrate via Photodegradable Linker In Test Example 6, Rhodamine 110 (53.5 nmol / ml) dissolved in 200 μL of 10 mM phosphate buffer (pH 7.4) was used. Instead, in the same manner as in Production Example 4, except that 0.057 mg (95 nmol) of the cell adhesion peptide cyclo (Arg-Gly-Asp- (D) Phe-Lys) (AnaSpec) having a cyclic skeleton structure was used. Thus, a culture substrate in which a cell adhesive peptide having a cyclic skeleton structure was bonded to a polymer substrate via a photodegradable linker was obtained.

Test Example 7: Dissociation of Stem Cell after Culture by Light Irradiation from Substrate for Culture in which Cell Adhesive Peptide with Cyclic Skeleton Structure is Bonded to Polymer Substrate via Photodegradable Linker Obtained in Production Example 5 Stem cells are adhered to the substrate in the same manner as in Test Example 2. After confirming the adhesion of the stem cells, a part of the substrate is irradiated with a 405 nm laser (30 mW) at an intensity of 10% for 5 minutes. When observed 1 hour after irradiation, it is confirmed that the stem cells are released specifically in the light irradiation site.

The base material for stem cell culture of the present invention is a safety that does not rely on animal-derived materials to enable proliferation while maintaining the undifferentiation and pluripotency of stem cells, which are indispensable elements for realizing regenerative medicine. It is a base material with high properties. Therefore, the realization of regenerative medicine, which has been delayed in practical use due to the risk of contamination with foreign proteins and pathogens from conventional base materials, is promoted. The substrate for stem cell culture of the present invention is a carrier or support for tissue engineering of tissues / organs such as artificial bone, artificial tooth root, bone repair agent, bone filler, artificial skin, artificial nerve, artificial liver, etc., regenerative medicine It can be used as a carrier or support for use.

Claims

A stem cell culture substrate in which a cell adhesion peptide having a cyclic skeleton structure is bound to a polymer substrate.
The substrate for stem cell culture according to claim 1, wherein the cell adhesive peptide having a cyclic skeleton structure comprises an Arg-Gly-Asp sequence.
2. The stem cell culture substrate according to claim 1, wherein the cell adhesion peptide having a cyclic skeleton structure is cyclo (Arg-Gly-Asp- (D) Phe-Lys).
The stem cell culture substrate according to claim 1, wherein the polymer substrate is a polypeptide and / or a polysaccharide.
A polypeptide in which the polymer substrate comprises a peptide unit (1) having an amino acid sequence represented by the following formula (1) and a peptide unit (2) having an amino acid sequence represented by the following formula (2): The stem cell culture substrate according to claim 1.
-Pro-X-Gly- (1)
-Pro-Hyp (OYZ) -Gly- (2)
(In the formula, X represents Pro or Hyp, Y represents a carbonyl group, or a saturated or unsaturated hydrocarbon group with or without a carbonyl group, and Z represents a carboxyl group)
Y is, - (C = O) - (CH 2) n- (n in the formula is an integer of 0 or 1 ~ 18) ;-( C = O ) - (CH 2) n- (CH = CH) m- (CH 2 ) k- (wherein n and k independently represent 0 or an integer of 1 to 18, and m represents an integer of 1 to 18); and-(C = O)-(CH 2 ) n- (C 6 H 4 )-(CH 2 ) k- (wherein n and k independently represent 0 or an integer of 1 to 18, and C 6 H 4 represents a phenylene group). The stem cell culture substrate according to claim 5, which is at least one of the above.
6. The stem cell culture substrate according to claim 5, wherein the ratio (molar ratio) of the peptide unit (1) to the peptide unit (2) is (1) / (2) = 99.9 / 0.1 to 1/99. Wood.
6. The stem cell culture substrate according to claim 5, wherein the polymer substrate exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum.
The stem cell culture substrate according to claim 5, wherein at least a part of the polypeptide forms a triple helical structure in the polymer substrate.
6. The stem cell culture substrate as a polypeptide according to claim 5, wherein the polymer substrate exhibits a peak in a molecular weight range of 5 × 10 3 to 5 × 10 6 .
The stem cell culture substrate according to claim 1, wherein the cell adhesive peptide having a cyclic skeleton structure is bound to a polymer substrate via a photodegradable linker.
The stem cell culture substrate according to claim 11, wherein the photodegradable linker has a 2-nitrobenzene skeleton, a 2-nitrophenol skeleton, a nitroindole skeleton, or a coumarin skeleton.
A method for culturing a stem cell, comprising a step of culturing a stem cell on the stem cell culture substrate according to any one of claims 1 to 12.
The culture method according to claim 13, wherein the stem cells are embryonic stem cells and / or induced pluripotent stem cells (iPS cells).
Culturing stem cells on the stem cell culture substrate according to claim 11 or 12, and
A method for culturing stem cells, comprising a step of separating stem cells from a stem cell culture substrate by irradiating the culture substrate after the culture with light.
A cell adhesion having a cyclic skeleton structure by an amide bond obtained by dehydration condensation of a carboxyl group of the polypeptide of the polymer substrate according to claim 5 and an amino group of a cell adhesive peptide having a cyclic skeleton structure. A method for producing a stem cell culture substrate, comprising binding a sex peptide and a polymer substrate.