JP2008167695A - Cell culture substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cell culture substrate that has a quick specific cellular adhesiveness via cell adhesion molecules, immediately after irradiation that is specific to an irradiated part by light irradiation. <P>SOLUTION: The cell culture substrate is obtained by bonding a caged peptide, in which a photodissociating protective group is introduced into a cell adhesive peptide part, to a substrate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell culture substrate and a cell culture method capable of adhering cells in an arbitrary pattern, a specific position, or a specific timing.

  From basic research on intracellular and extracellular signal transduction in relation to surrounding cells, to applied research such as drug discovery and environmental analysis using cell arrays as an evaluation system in the field of regenerative medicine such as rapidly expanding cell therapy in recent years However, there is an increasing need for a technique for finely manipulating and analyzing individual cells, and so-called pattern culture techniques for culturing cells at an arbitrary site or time are required.

Pattern culture methods include microprints that immobilize fibronectin, which can bind cell adhesion factors of cells, at any site on the substrate (Non-patent Document 1), and pattern culture using light, heat, and electricity as a trigger. Technology has been developed. In the microprint method, since the procedure for culturing cells on a substrate after determining the cell adhesion region is taken, control during culturing cannot be performed and time resolution is not provided. Moreover, site-specific control is difficult in the method of controlling cell adhesion using a temperature-responsive polymer membrane (Patent Documents 1 and 2 and Non-Patent Documents 2-4). In the method using a monomolecular film having electrochemical activity (Non-patent Documents 5-7), the binding property of the cell adhesion factor to the ligand is not directly controlled, and therefore, nonspecific adhesion cannot be completely excluded. .

On the other hand, pattern culture technology that can be adjusted with spatio-temporal resolution using light as a trigger
Development is also progressing for (Patent Documents 3-5, Non-Patent Documents 8-14), but the following problems remain for individual technologies.
1) Methods for controlling protein adsorption and subsequent cell adhesion by changing the physicochemical properties such as hydrophilicity of the substrate by light irradiation (Patent Documents 3-5 and Non-Patent Documents 8-10) Non-specific adhesion cannot be ruled out completely because the binding of the cell adhesion factor to the ligand is not directly controlled.
2) In the method of immobilizing peptides, which are ligands for cell adhesion factors, on the substrate via an azobenzene dye (Non-patent Document 13), the control is based on a slight difference in length due to photoisomerization of azobenzene, which is completely Cell adhesion cannot be controlled.
3) In the method for controlling cell adhesion by irradiating the site to which the ligand of the cell adhesion factor is to be bound, reacting and immobilizing the peptide of the ligand to the irradiated site (Non-patent Documents 11-12), the above-mentioned microcontact print Like, it cannot be light-controlled during culture experiments in the presence of cells.
W02002 / 010349 JP 11-349643 A JP 3-7577 JP2006-6214 JP2005-210936 Chen et al. Science 276, 1425-8 Stile et al. Biomacromalecules2, 185-94 (2001) Yamato et al. Tissue Engineering 7, 473-80 (2001) Yamato et al. Biomaterials 23, 561-7 (2002) Harimoto et al. Journal of Biomedical Materials Research 62, 464-70 (2002) Yousaf et al. Proc Natl. Acad. Sci. USA 98, 5992-6 (2001) Yeo et al. J. Amer. Chem. Soc. 125, 14994-5 (2003) Nakayama et al. Macromolecules 29, 8622-30 (1996) Nakanishi et al. J. Am. Chem. Soc. 126, 16314-5 (2004) Edahiro et al. Biomolecules 6, 970-4 (2005) Luo et al. Nature Materials 3, 249-53 (2004) Luo et al. Biomalecules 5, 2315-23 (2004) Auernheimer et al. J. Am. Chem. Soc. 127, 163107-10 (2005)

  Therefore, the present invention creates a substrate having specific cell adhesion properties via cell adhesion molecules immediately after irradiation in a specific manner by irradiation with light under both conditions before or under cell culture. The purpose is to do.

The present invention provides the following cell culture substrate and cell culture method.
1. A cell culture substrate obtained by binding a caged peptide having a photolabile protecting group introduced into a cell adhesion peptide portion onto the substrate.
2. Item 2. The cell culture substrate according to Item 1, wherein the cell adhesion peptide is RGD.
3. Item 3. The cell culture substrate according to Item 2, wherein the photolabile protecting group is an N-2-nitrobenzyl group introduced into the amino group of G of RGD.
4). The cell culture substrate of Item 1, 2, or 3 is irradiated with light at a specific timing and / or in a specific pattern to remove the photolabile protecting group, and the cell adhesion peptide portion is placed on the substrate at a specific timing and / or A method for culturing cells in a state where cells are adhered in a specific pattern and / or at a specific timing on a cell culture substrate, wherein the cells are formed in a specific pattern and cultured on the substrate.

  In the cell adhesion substrate of the present invention, since a photolabile protecting group is introduced into the cell adhesion peptide portion, the cells hardly adhere to the substrate even if the cells are cultured on the substrate before light irradiation. However, by irradiating light at a specific position, a specific pattern or at a specific timing on the substrate, the cell adhesion peptide can be exposed only at the irradiated position, and the cells can be adhered thereto. Therefore, the cell adhesion pattern or the timing of adhesion can be controlled very precisely. In addition, by repeating light irradiation and adhesion of various cells, it is possible to determine a region in which cells are to be proliferated after cell seeding with an arbitrary two-dimensional pattern, so that the relationship between cells can be examined.

Introduction of the caged peptide into the cell culture substrate of the present invention can be performed, for example, via a thiol group of a Cys residue. When immobilizing a caged peptide via a thiol group, an amino group is introduced into the substrate surface in advance, and a heterobifunctional crosslinking agent having an N-hydroxysuccinimide (NHS) group and a maleimide (MAL) group is used. It can be preferably carried out by forming a maleimide surface and reacting this MAL group with the thiol group of the Cys residue. The means for introducing an amino group on the substrate is not particularly limited, and a self-organized surface method for aligning molecules on the substrate surface, a method for introducing using a reaction reagent, a substance having a functional group (for example, poly-L- And a method of coating lysine) on the substrate. Moreover, you may use the method of making a crosslinking agent react with the functional group introduce | transduced on the substrate surface, and introduce | transducing an amino group.

There are various commercially available heterobifunctional crosslinking agents and are not particularly limited. For example, N- (4-maleimidobutyryloxy) succinimide (GMBS)
N- (6-maleimidocaproyloxy) succinimide (EMCS), N- (8-maleimidocapryloxy) succinimide (HMCS), N- (11-maleimidoundecanoyloxy) succinimide (KMUS), N-(( 4- (2-maleimidoethoxy) succinyl) oxy) succinimide (MESS), N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt (sulfo-GMBS), N- (6-maleimidocaproyloxy) sulfosuccinimide sodium Salt (sulfo-EMCS), N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt (sulfo-HMCS) and the like. Moreover, what has a succinimide (NHS) group and a maleimide (MAL) group in the terminal of polymer | macromolecule like PEG (polyethylene glycol) can also be used as a heterobifunctional type crosslinking agent.

  The caged peptide of the present invention contains one or more (preferably one) cell adhesion peptides. Cell adhesion peptides include RGD, REDV, P15 (GTPGPQGIAGQRGVV; SEQ ID NO: 1), C / HV (WQPPPARI; SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), SIKVAV (SEQ ID NO: 4), F-9 ( RYVVLPPVCFGGMNYTVR; SEQ ID NO: 5), HEP-III (GEFYFDLRLKGGDK; SEQ ID NO: 6 and the like are preferable, and RGD is preferable.

The caged peptide has about 3 to 30 amino acids, preferably about 4 to 20 amino acids. The cell adhesion peptide contained in the caged peptide is located at a position far from the substrate (at least 2 amino acids or more, preferably 3 amino acids or more, more preferably 5 amino acids or more away from the substrate) in terms of adhesion to cells. Is preferable.

  The photolabile protecting group is not particularly limited as long as it is cleaved by irradiation with ultraviolet rays. For example, the following formula (II)

〔式中、Ｒ¹、Ｒ²、Ｒ³は、同一又は異なって水素原子、低級アルキル基、低級アルコキ
シ基、アミノ基、ハロゲン原子、水酸基またはシアノ基を示すか、あるいはＲ¹、Ｒ²及びＲ³のいずれか２つが一緒になってメチレンジオキシ基を示す。Ｒは、水素原子又はメチ
ル基を示す。〕で表される
基が挙げられる。

[Wherein R ¹ , R ² and R ³ are the same or different and each represents a hydrogen atom, a lower alkyl group, a lower alkoxy group, an amino group, a halogen atom, a hydroxyl group or a cyano group, or R ¹ , R ² and Any two of R ³ together represent a methylenedioxy group. R represents a hydrogen atom or a methyl group. The group represented by this is mentioned.

In the formula (I), examples of the lower alkyl group represented by R ¹ , R ² , and R ³ include carbon such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. The alkyl group which has a linear or branched number 1-4 is mentioned.

  Examples of the lower alkoxy group include linear or branched alkoxy groups having 1 to 4 carbon atoms such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like. .

  Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In the preferred group of formula (II), any one of R ¹ , R ² and R ³ is a hydrogen atom, the remaining one is a hydrogen atom, a lower alkyl group or a lower alkoxy group, and R is a hydrogen atom. It is a group.

  The photolabile protecting group is removed by ultraviolet irradiation. The ultraviolet ray to be irradiated is not particularly limited as long as the photolabile protecting group can be removed, and an ordinary ultraviolet lamp or the like is used. Although the conditions for ultraviolet irradiation are not particularly limited, for example, it may be processed for about 1 hour with an ultraviolet hand lamp for detection of TLC (Topcon, PU-2).

The caged peptide of the present invention in which a photolabile protecting group is introduced into the main chain, a dipeptide having a photolabile protecting group of the following formula (I) is synthesized, and then an N-protected dipeptide compound represented by the formula (I) is synthesized. Can be obtained by using in place of N-protected amino acids in solid phase synthesis.
Formula (I)

（式中、Ｒ^１はアミノ基の保護基を示す。ＡはＰｒｏ以外のペプチドを構成するαアミノ酸の側鎖を示す。Ｂはαアミノ酸の側鎖を示す。Ｒ^ａは光解離性保護基を示す。）。
光解離性保護基を側鎖に有するケージドペプチドは、Ｎ保護アミノ酸の代わりに光解離性保護基を側鎖に有するＮ保護アミノ酸を使用して、固相合成法により製造することができる。

(In the formula, R ¹ represents a protecting group for an amino group. A represents a side chain of an α-amino acid constituting a peptide other than Pro. B represents a side chain of an α-amino acid. R ^a represents a photolabile protecting group. Is shown.)
A caged peptide having a photolabile protecting group in the side chain can be produced by a solid phase synthesis method using an N-protected amino acid having a photolabile protecting group in the side chain instead of the N-protected amino acid.

The synthesis of the dipeptide of formula (I) involves the amino acid (Aaa having a photolabile protecting group at the α-amino group.
) And an N-protected amino acid (Baa) acid fluoride can be reacted to synthesize the desired dipeptide of general formula (I). The reaction is performed by using N-protection-Baa-F in an excess amount from 1 mole to 1 mole of Aaa to which a photolabile protecting group is bonded, and reacting at a temperature of about 0 ° C. to 100 ° C. for about 1 minute to 24 hours. , Proceed advantageously.

  The number of photolabile protecting groups of the caged peptide is not particularly limited as long as the cell adhesion activity of the caged peptide is substantially lost, but is, for example, 1 to 3, preferably 1 to 2, particularly 1.

  The caged peptide of the present invention has a function in which the cell-dissociating protecting group is bound to the cell adhesion peptide portion, the cell adhesion activity is substantially lost, and the cell is released when the photo-dissociable protecting group is detached by light irradiation. This means that the activity of the adhesion peptide is restored.

In the general formula (I), the group represented by “A” includes Gly, Ala, Ser, Thr, Tyr, Phe, Trp, Asp, Glu, Met, Cys, Leu, Ile, Val, Lys, Arg, Examples include side chains of natural amino acids other than Pro, such as His, Asn, Gln, Orn (Ornithine), and groups represented by “B” include Ala, Ser, Thr, Tyr, Phe, Trp, Asp, Glu , Met, Cys, Leu, Ile, Val, Lys, Arg, His, Asn, Gln, Gly, Pro, and Orn (ornithine) side chains. In addition, when the side chain of the amino acid represented by A, B has a functional group such as NH ₂ , SH, imidazolyl, guanidino, OH, COOH), peptide synthesis (especially solid phase synthesis) may be performed as necessary. Protected as necessary with commonly used protecting groups.

As the resin for solid phase synthesis, known resins such as polystyrene-divinylbenzene copolymer can be widely used.

  In addition, peptide chain elongation, protecting group removal, peptide cleavage from the resin, and the like can be performed according to conventional methods.

  The material of the cell culture substrate of the present invention is not particularly limited as long as nonspecific cell adhesion does not occur, and examples thereof include glass, plastic, metal, ceramic and the like, preferably glass and plastic.

Formation of the pattern from which the photolabile protecting group is removed on the cell culture substrate is not particularly limited, and a known method is widely used. For example, a specific position / region on the substrate through a mask having a specific pattern It can be carried out using an optical lithography technique such as irradiating light at a specific timing. The specific timing includes a specific cycle of a clock gene, a specific cell cycle, cell differentiation, migration, etc. before or after the addition of a physiologically active substance.

  There are no particular limitations on the type and culture conditions of the cells cultured on the substrate, and conventionally known cells and culture conditions can be widely used.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example and a reference example, it cannot be overemphasized that this invention is not limited to these Examples.
Reference Example 1 Production of Peptide for Immobilization The following three peptides were produced according to the method described above.
intact peptide-Cys
Sequence: Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Cys-amide (SEQ ID NO: 7)
Mass analysis value, observed value: 1369.11, calculated value: 1369.48
reverse peptide-Cys
Sequence: Cys-Ser-Ser-Ala-Pro-Ser-Asp-Gly-Arg-Gly-Thr-Val-Ala-Tyr-amide (SEQ ID NO: 8)
Mass analysis value, observed value: 1369.67, calculated value: 1369.48
caged peptide-Cys
Sequence: Tyr-Ala-Val-Thr-Gly-Arg-cGly-Asp-Ser-Pro-Ala-Ser-Ser-Cys-amide (SEQ ID NO: 9) Mass spectrometry, observed value: 1503.58, calculated value: 1504.6
However, cGly represents N-2-nitrobenzylglycine.

(Reference Example 2) Solid phase of peptide Material: Polyethylene glycol in which methyl group and N-hydroxysuccimidyl activated ester are bonded at both ends (hereinafter abbreviated as m-PEG-NHS: trade name SUNBRIGHT ME-050CS, Japan) Yushi Co., Ltd., Shibuya, Tokyo) and polyethylene glycol with maleimide and N-hydroxysuccimidyl activated ester bonded to both ends (hereinafter abbreviated as MA-PEG-NHS: trade name SUNBRIGHT MA-034HS, Nippon Oil & Fats Co., Ltd.) Company, Shibuya, Tokyo).

Method: Prepare phosphate buffered saline (pH 7.4, hereinafter abbreviated as PBS) in which m-PEG-NHS is dissolved at a concentration of 25 mg / ml and MA-PEG-NHS is dissolved at a concentration of 0.25 mg / ml. 150 μl of the solution was dropped onto a glass bottom dish (PLL-Coat, 35 mm dish, Hole: 10 mm No.0, Cat.No. D110110, MATSUNAMI, Kishiwada, Osaka) coated with poly-L resin. The plate was allowed to stand at 4 ° C. for about 24 hours and then washed 3 times with 2 ml of PBS to prepare a substrate on which m-PEG-NHS and MA-PEG-NHS were immobilized at a ratio of 100: 1.
To this substrate, 150 μl of a peptide solution dissolved in PBS at 1 mg / ml was added dropwise, allowed to stand at 4 degrees for about 24 hours, and then washed 3 times with 2 ml of PBS to prepare a substrate on which the peptide was immobilized. did.

(Reference Example 3) Control experiment
In the HeLa cell culture experiment, the effect of the presence or absence of immobilization of the peptide on the substrate on which m-PEG-NHS and MA-PEG-NHS were immobilized was examined (Fig. 2). Poly L-lysine-immobilized substrate (PLL-immobilized substrate), a substrate further immobilized with a polyethylene glycol derivative (substrate immobilizing m-PEG-NHS and MA-PEG-NHS) by the method described in Reference Experiment 2, A substrate (reverse peptide-Cys) further immobilized with a peptide to which a cell adhesion factor does not bind and a substrate (intact peptide-Cys) further immobilized with a peptide capable of binding to a cell adhesion factor were prepared. HeLa cells were also cultured using this substrate. HeLa cells at a cell density of 2 x 10 ⁴ cells / dish in serum-free medium (Delbecco's modified Eagles medium (Cat. No. 12430-047, Invitrogen) containing 500 IU / ml penicillin, 5 mg / ml streptomycin) Cultured. After 30 minutes from the start of the culture, the substrate was washed with PBS, and the cells were fixed with 4% paraformaldehyde diluted with PBS, and differential interference observation (Olympus IX70) was performed.
Almost no cell adhesion was observed on the poly-L-lysine-immobilized substrate, the polyethylene glycol derivative-immobilized substrate, and the peptide-immobilized substrate to which no cell adhesion factor was bound. However, a marked increase in the number of adherent cells was observed on the peptide-immobilized substrate to which the cell adhesion factor can bind (FIG. 2). In addition, nonspecific cell adhesion was observed on the substrate in which the maleimide group contained a hydrophobic site before modification of the peptide (FIG. 3, second stage).

Moreover, the influence of ultraviolet irradiation on these modified substrates was investigated. Ultraviolet rays (wavelength 365 nm, light intensity 16 mW / cm ² ) were irradiated for 4 minutes from below the substrate containing 2 ml of PBS using an ultraviolet lamp (UV-100A type, Hamamatsu Photonics Co., Ltd.). When this ultraviolet irradiation substrate was used to perform a culture experiment in the same manner as described above, no significant influence of ultraviolet irradiation on cell adhesion was observed.

Example 1 Cell Culture Experiment on Substrate Immobilized with Caged Peptide
In a HeLa cell culture experiment, the effect of UV irradiation on adhesion to a substrate on which caged peptide-Cys was immobilized was examined. Prepare a substrate on which caged peptide-Cys is immobilized by the method described in Reference Experiment 2, add 2 ml of PBS to this substrate, and place an ultraviolet lamp (UV-100A type, Hamamatsu Photonics) from the bottom of the substrate. Using ultraviolet rays (wavelength 365 nm, light intensity 16 mW / cm ² ) 0, 0.5,
Irradiated for 1, 2, 4 minutes. HeLa cells were also cultured using this ultraviolet irradiation substrate. HeLa cells at a cell density of 2 x 10 ⁴ cells / dish in serum-free medium (Delbecco's modified Eagles medium (Cat. No. 12430-047, Invitrogen) containing 500 IU / ml penicillin, 5 mg / ml streptomycin) Cultured. After 30 minutes from the start of the culture, the substrate was washed with PBS, and the cells were fixed with 4% paraformaldehyde diluted with PBS, and differential interference observation (Olympus IX70) was performed.
Before UV irradiation, the cells hardly adhered, but on the irradiated substrate, the number of adherent cells increased in a dose-dependent manner (Fig. 3).

(Example 2) Site-specific cell culture experiment
In a HeLa cell culture experiment, site-specific cell adhesion control was performed using a substrate on which caged peptide-Cys was immobilized. Prepare a substrate on which caged peptide-Cys is immobilized by the method described in Reference Experiment 2, place 2 ml of PBS on this substrate, place a mask that shields half of the substrate under the substrate, and place an ultraviolet lamp from below the substrate. (UV-100A type, Hamamatsu Photonics Co., Ltd.) was used for irradiation for 2 minutes with ultraviolet rays (wavelength 365 nm, light intensity 30 mW / cm ² ). Using this UV-irradiated substrate, HeLa cells were cultured at a cell density of 2 x 10 ⁵ cells / dish at a serum-free medium (Delbecco's modified Eagles medium (Cat. No. 5 containing 500 IU / ml penicillin, 5 mg / ml streptomycin). 12
430-047, Invitrogen)). After 30 minutes from the start of the culture, the substrate was washed with PBS, and the cells were fixed with 4% paraformaldehyde diluted with PBS, and differential interference observation (Olympus IX70) was performed.
Almost no adherent cells were observed at the light-shielded site, but adherent cells were observed at the irradiated site, indicating that cells can adhere specifically to the UV-irradiated site (FIG. 4).

(Example 3) Site-specific cell culture experiment by irradiating light during cell culture
In a HeLa cell culture experiment, site-specific cell adhesion control was performed using a substrate on which caged peptide-Cys was immobilized. Prepare a substrate with caged peptide-Cys immobilized by the method described in Reference Experiment 2, and use this substrate to prepare HeLa cells at a cell density of 2 x 10 ⁵ cells / dish and serum-free medium (500 IU / ml Cultured in Delbecco's modified Eagles medium (Cat. No. 12430-047, Invitrogen)) containing penicillin and 5 mg / ml streptomycin. 20 minutes after the start of culture,
A mask that shields half of the substrate is placed under the substrate, and an ultraviolet lamp (UV-100A) is placed under the substrate.
Type, Hamamatsu Photonics Co., Ltd.), ultraviolet light (wavelength 365 nm, light intensity 38.5 mW / cm ² )
Was irradiated for 2 minutes. Then, after further culturing for 20 minutes, the substrate was washed with PBS, then fixed with 4% paraformaldehyde diluted with PBS, and differential interference observation (Olympus IX70) was performed.
Almost no adherent cells were observed at the light-shielded site, but adherent cells were observed at the irradiated site (Fig. 5).
(Example 4) Cell culture experiment in which cell adhesion is induced by light irradiation
In a HeLa cell culture experiment, a cell adhesion control specific to the light irradiation time was performed using a substrate on which caged peptide-Cys was immobilized. Prepare a substrate on which caged peptide-Cys is immobilized by the method described in Reference Experiment 2, and use this substrate to prepare HeLa cells at a cell density of 4 x 10 ⁵ cells / dish and serum-free medium (500 IU / ml The cells were cultured in Delbecco's modified Eagles medium (Sigma-Aldrich D2902) containing penicillin, 5 mg / ml streptomycin, 25 mM HEPES (pH 7.4), and cultured in a microscope stage chamber maintained at 37 degrees every 30 seconds. About 15 minutes after the start of culture, UV light (wavelength 365 nm, light intensity 33 mW / cm ² ) was used from above the substrate using an ultraviolet lamp (UV-100A type, Hamamatsu Photonics). ) For 2 minutes.
The cells were spherical until the start of the culture and immediately before the irradiation with ultraviolet light, but immediately after the irradiation with ultraviolet light, rapid cell expansion was observed due to the initiation of binding between the base RGD fragment site and the integrin on the cell membrane. (Left column in FIG. 6). However, in the case of no light irradiation, the cells remained spherical even after about 1 hour ((right column in FIG. 6). From this experiment, if a substrate on which caged peptide-Cys is immobilized is used, any cell can be obtained. It was shown that at the timing, cell attachment to the substrate can be initiated.

Conclusion
(i) From Reference Experiment 3, it was shown that a substrate on which a substance serving as a ligand of a cell adhesion factor is immobilized is necessary for specific adhesion via a cell adhesion factor in a serum-free medium.
(ii) From Examples 1, 2, and 3, it was shown that the use of caged peptides makes it possible to adjust a substrate that can control specific cell adhesion via cell adhesion factors by ultraviolet irradiation.
(iii) Peptides containing RGD are integrin binding sequences and are involved in intercellular / intracellular signal transduction and cell adhesion, and thus have been studied for a long time in cell biology and biocompatible materials. Since this caged peptide can be solid-phased, it is possible to perform cell pattern culture by photolithography, etc. in a site-specific, time-specific, and non-invasive manner by light irradiation. It is considered industrially useful for use as a research and development reagent for elucidating the mechanism of action, and for the development of biocompatible materials.
(iv) In this pattern culture method, the ligand itself involved in cell adhesion is inactivated, and the same cell adhesion control can be performed by caged peptide sequences involved in cell adhesion other than RGD.
(v) Preparation of a peptide-immobilized substrate is possible by other methods of immobilizing caged peptides, regardless of poly-L-lysine-immobilized substrates or polyethylene glycol derivatives.

Scheme of substrate control and light control experiment of cell adhesion in Reference Experiment 3 and Examples 1 and 2. A culture substrate coated with poly-L-lysine (PLL) (i) is reacted with maleimide and a bi-crosslinkable polyethylene glycol with succimidyl ester (ii), and then the peptide is reacted to coat the peptide. (Iii). When light is irradiated to the site where the cells are to be attached, the cells adhere (iv). (Reference Experiment 3) Adhesion of HeLa cells to various substrates Scale bar = 200 μm (Example 1) The number of HeLa cells adhering to the caged peptide-Cys solid-phase substrate increased in an ultraviolet irradiation time-dependent manner. Scale bar = 200 μm (Example 2) As a result of culturing HeLa cells on the substrate after ultraviolet irradiation only on the right half of the caged peptide-Cys solid-phase substrate, HeLa cells adhered only to the site irradiated with ultraviolet rays. Scale bar = 200 μm (Example 3) HeLa cells were cultured on a caged peptide-Cys solid-phase substrate for 20 minutes, and then only the left half of the substrate was irradiated with ultraviolet rays. As a result of further culturing the HeLa cells on the substrate for 20 minutes, the HeLa cells adhered only to the site irradiated with ultraviolet rays. Scale bar = 200 μm Under a differential interference microscope, HeLa cells were cultured on a caged peptide-Cys solid phase substrate. When light irradiation was not performed, cell adhesion hardly occurred (right series of photographs), but it was observed that cell irradiation was rapidly induced by light irradiation (left series of photographs). Scale bar = 50 μm

Claims (4)

A cell culture substrate obtained by binding a caged peptide having a photolabile protecting group introduced into a cell adhesion peptide portion onto the substrate. The cell culture substrate according to claim 1, wherein the cell adhesion peptide is RGD. The cell culture substrate according to claim 2, wherein the photolabile protecting group is an N-2-nitrobenzyl group introduced into the amino group of G of RGD. 4. The cell culture substrate of claim 1, 2 or 3 is irradiated with light at a specific timing and / or in a specific pattern to remove the photolabile protecting group, and the cell adhesion peptide portion is placed on the substrate at a specific timing and / or Alternatively, a method of culturing cells in a specific pattern and / or at a specific timing on a cell culture substrate, wherein the cells are formed in a specific pattern and cultured on the substrate .

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