PHOTODEGRADABLE HYDROGEL, CULTURE DEVICE, METHOD FOR FORMING TISSUE, AND METHOD FOR SEPARATING CELLS

Abstract

Provided are a photodegradable hydrogel in which cells can be embedded in the photodegradable gel without causing cytotoxicity when the cells are embedded in the photodegradable gel by allowing the cells to coexist at the time of preparation of the photodegradable gel, and which contains a protein as one of the main components; a culture device using the same; a method for forming tissue; and a method for separating cells. A photodegradable hydrogel is obtained by condensation of an alkyne group contained in a cyclooctyne ring or an azacyclooctyne ring of the following compound A with an azido group of the following compound B. (Compound A) A compound is a photocleavable crosslinker which contains a main chain having a linear type- or a branched type- (of three or more branches) polyethylene glycol structure, a photodegradable nitrobenzyl group disposed at both terminals or a branched terminal of the main chain, and a group having a cyclooctyne ring or an azacyclooctyne ring disposed at a terminal side of the nitrobenzyl group. (Compound B) A compound is an azide-modified protein in which a main chain is a protein and at least some of an amino group present at lysine and arginine side chains of the main chain and an amino group present at a terminal of the main chain are modified with the azido group.

Claims

1. A photodegradable hydrogel of which an alkyne group contained in a cyclooctyne ring or an azacyclooctyne ring of the following compound A is modified with the following compound B through an azido group of the compound B: (compound A) wherein a compound is a photocleavable crosslinker which contains a main chain having a linear type- or a branched type- (of three or more branches) polyethylene glycol structure, a photodegradable nitrobenzyl group disposed at both terminals or a branched terminal of the main chain, and a group having a cyclooctyne ring or an azacyclooctyne ring disposed at a terminal side of the nitrobenzyl group, and (compound B) wherein a compound is an azide-modified protein in which a main chain is a protein and at least some of an amino group present at lysine and arginine side chains of the main chain and an amino group present at a terminal of the main chain are modified with the azido group.

2. The photodegradable hydrogel according to claim 1, wherein the protein of the compound B includes one or more of cell adhesion proteins selected from gelatin, collagen, laminin, and Matrigel.

3. The photodegradable hydrogel according to claim 1, wherein the average number of repeating ethylene glycol units in the polyethylene glycol structure of the compound A is within a range of 30 to 250.

4. The photodegradable hydrogel according to claim 1, wherein the number of branches in the branched type-main chain of the compound A is 4 or 8.

5. The photodegradable hydrogel according to claim 1, wherein the branched type-main chain of the compound A has a neopentyl skeleton on the center thereof.

6. The photodegradable hydrogel according to claim 1, wherein the group having a cyclooctyne ring or an azacyclooctyne ring of the compound A is an azadibenzocyclooctyne (DBCO) group.

7. The photodegradable hydrogel according to claim 1, wherein the photodegradable hydrogel contains a cell growth factor.

8. The photodegradable hydrogel according to claim 1, wherein a modification ratio at which the alkyne group is modified through an azido group is 10% to 100% with respect to the number of the alkyne group.

9. The photodegradable hydrogel according to claim 1, wherein in the compound B, an azido-modification ratio of the amino group in the azide-modified protein is 10% to 100% with respect to the number of the amino group.

10. A culture device, wherein the photodegradable hydrogel according to claim 1 is formed on a bottom surface of a culture vessel.

11. A method for forming tissue using the photodegradable hydrogel according to claim 1, the method comprising: (I) a step of forming the photodegradable hydrogel in which cells are embedded; (II) a step of defining a structure of the photodegradable hydrogel by light irradiation; and (III) a step of culturing the cells to form tissue.

12. The method for forming tissue according to claim 11, wherein in the step of defining a structure of the photodegradable hydrogel, light having a light intensity of 0.001 to 1.0 W/cm.sup.2 is emitted in the light irradiation, and a structure of the photodegradable hydrogel is defined by CBB staining, fluorescent staining, or microscopic observation.

13. A method for separating cells using the photodegradable hydrogel according to claim 1, the method comprising: (I) a step of forming the photodegradable hydrogel in which cells are embedded; (II) a step of dissolving the photodegradable hydrogel in a region containing a specific cell among the cells by light irradiation; and (III) a step of washing the dissolved photodegradable hydrogel to recover the specific cell in the dissolved region.

14. The method for separating cells according to claim 13, wherein in the step of dissolving the photodegradable hydrogel, light having a wavelength of 300 to 500 nm is emitted in the light irradiation and the photodegradable hydrogel is dissolved by pipetting.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0143] FIG. 1A is a schematic diagram of crosslinking formation and hydrogel formation by click reaction, and degradation by light irradiation, and a schematic diagram of a click crosslinking-type photocleavable crosslinker, which is DBCO-PC-4armPEG, and a molecular structure of an azide-modified gelatin. R.sub.2 represents a main chain of the gelatin molecule.

[0144] FIG. 1B shows crosslinking formation between DBCO-PC-4armPEG and the azide-modified gelatin by click reaction. R.sub.1 represents a site on a main chain side of DBCO-PC-4armPEG.

[0145] FIG. 1C is a schematic diagram of degradation of a crosslinking site by light irradiation. R.sub.3 represents a site derived from the main chain side of DBCO-PC-4armPEG, and R.sub.4 represents a site derived from a side chain side of the azide-modified gelatin and DBCO-PC-4armPEG.

[0146] FIG. 1D is a schematic diagram of hydrogel formation by reaction between DBCO-PC-4armPEG and the azide-modified gelatin, and degradation by light irradiation.

[0147] FIG. 1E shows changes in the absorption spectrum of DBCO-PC-4armPEG by light irradiation.

[0148] FIG. 2 is a photograph showing the state 2 hours after reacting of gelatin and DBCO-sulfo-NHS at 37 C., which is the state 2 hours after reacting of gelatin, DBCO-gelatin (25), DBCO-gelatin (50), DBCO-gelatin (75), and DBCO-gelatin (100) from the left in order.

[0149] FIG. 3A is a NMR spectrum diagram of synthesized click crosslinking-type photocleavable crosslinker, DBCO-PC-4armPEG. Shown are a .sup.1H-NMR spectrum of DBCO-PC-4armPEG, and a peak integrated value below.

[0150] FIG. 3B is a comparative diagram of a .sup.1H-NMR spectrum of DBCO-PC-4armPEG (above) and NHS-PC-4armPEG (below).

[0151] FIG. 4A is a diagram showing changes in a storage modulus (G) and a loss modulus (G) over time when forming a gel by click reaction. A point where G exceeds G (crossover point, CP) is a point where gelation occurs. A modulus change when preparing a gel of PD-gelatin (25) is shown. A composition of each gel is shown in Table 7.

[0152] FIG. 4B shows PD-gelatin (50) as same as that of FIG. 4A.

[0153] FIG. 4C shows PD-gelatin (75) as same as that of FIG. 4A.

[0154] FIG. 4D shows PD-gelatin (100) as same as that of FIG. 4A.

[0155] FIG. 5A is a photograph showing the state of micropatterned degradation of a photodegradable gel by micropatterned light irradiation. The state where a gel of PD-gelatin (25) photodegrades is shown. A dark colored portion shows a hydrogel stained with CBB and a light colored portion shows a degrading portion. A composition of each gel is shown in Table 7. A reduction scale is as same as that of FIG. 5D.

[0156] FIG. 5B shows the state where a gel of PD-gelatin (50) photodegrades as same as that of FIG. 5A. A reduction scale is the same as that of FIG. 5D.

[0157] FIG. 5C shows the state where a gel of PD-gelatin (75) photodegrades as same as that of FIG. 5A. A reduction scale is the same as that of FIG. 5D.

[0158] FIG. 5D shows the state where a gel of PD-gelatin (100) photodegrades as same as that of FIG. 5A. A scale bar is 200 m.

[0159] FIG. 5E is an image of an irradiation pattern. A black portion in the irradiation pattern image is an irradiated image.

[0160] FIG. 6A is a photograph showing the state of micropatterned degradation by micropatterned light irradiation when Matrigel is added to produce the photodegradable gel by using a click-type crosslinker. The state where a gel of PD-gelatin (25)_M+ degrades is shown. A dark colored portion shows a hydrogel stained with CBB and a light colored portion shows a degrading portion. A composition of each gel is shown in Table 7. A reduction scale is the same as that of FIG. 6D.

[0161] FIG. 6B shows the state where a gel of PD-gelatin (50)_M+ photodegrades as same as that of FIG. 6A. A reduction scale is the same as that of FIG. 6D.

[0162] FIG. 6C shows the state where a gel of PD-gelatin (75)_M+ photodegrades as same as that of FIG. 6A. A reduction scale is the same as that of FIG. 6D.

[0163] FIG. 6D shows the state where a gel of PD-gelatin (100)_M+ photodegrades as same as that of FIG. 6A. A scale bar is 200 m.

[0164] FIG. 7A is a diagram showing a relationship between a concentration of the crosslinker and cell survival rate when DU145 cells and HeLa cells are embedded in the photodegradable gel by using the click-type crosslinker. A composition (Gelatin, PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), PD-gelatin-gelatin (25)_M+, PD-gelatin (50)_M+, PD-gelatin (75)_M+, and PD-gelatin (100)_M+) of each gel in a case where the click-type crosslinker is used is shown in Table 7. Furthermore, in a case where an active ester-type crosslinker is used, a gelatin at a concentration of 25 mg/L is mixed with the active ester-type crosslinker to preprare the photodegradable gel.

[0165] FIG. 7B is a diagram showing a relationship between a concentration of the crosslinker and cell survival rate measured in the same manner as FIG. 7A by using the click-type crosslinker and adding Matrigel.

[0166] FIG. 7C is a diagram showing a relationship between a concentration of the crosslinker and cell survival rate measured in the same manner as FIG. 7A by using the active ester-type crosslinker.

[0167] FIG. 8A is a photograph showing the state of growth and form change of HeLa cells embedded in the photodegradable gel. The state in a case where the click-type crosslinker is used is shown. Each of the numbers 25, 50, 75, and 100 on a vertical axis in the drawing shows that the gel embedded in the cells is a gel of PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), and PD-gelatin (100) in Table 7. A scale bar is 100 m.

[0168] FIG. 8B is a photograph showing the state of growth and form change of HeLa cells embedded in the photodegradable gel. The state in a case where the click-type crosslinker is used and Matrigel is added is shown. Each of the number 25, 50, 75, and 100 on a vertical axis in the drawing indicates PD-gelatin (25)_M+, PD-gelatin (50)_M+, PD-gelatin (75)_M+, and PD-gelatin (100)_M+ in Table 7. A scale bar is 100 m.

DESCRIPTION OF EMBODIMENTS

Examples

[0169] Hereinafter, the present embodiment will be described in further detail based on examples, but the present invention is not limited to these examples at all, and it is needless to say that various material changes, design changes, setting adjustments, and the like are possible without departing from the gist of the present invention.

[0170] In the following examples, the following materials were used.

[0171] Gelatin (G2500, Sigma-Aldrich Co. LLC., St. Louis, Mo.), Azide-PEG.sub.4-NHS ester (Click Chemistry Tools LLC. Scottsdale, Ariz.), DBCO-PEG.sub.4-amine (Click Chemistry Tools LLC.), DBCO-sulfo-NHS (Click Chemistry Tools LLC.), Matrigel (Corning, Tewksbury, Mass.) were purchased to be used.

[0172] 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was dissolved in MilliQ water (Millipore, Billerica, USA) at a concentration of 300 mM, and a pH was adjusted to 7.4 with 0.1N sodium hydroxide aqueous solution to prepare a HEPES buffer. The prepared solution was used after being filtrated using a filter having a pore size of 0.2 m (Millipore Co., Billerica, Mass.).

Example 1

Synthesis of Azide-Modified Gelatin and Azide-Modified Matrigel

[0173] According to the following procedure, azide-modified Gelatin and azide-modified matrigel were synthesized to gelate Gelatin and Matrigel by click reaction (Formula 1).

[0174] Gelatin is a polymer of which water solubility increases by modifying collagen, which is a cell adhesion protein, with acid, alkali, heat, and the like, and maintains cell adhesiveness while being modified, and therefore is used as a substrate for cell culture as same as collagen. Gelatin is inexpensive compared to collagen, and therefore is frequently used for coating of a culture dish.

[0175] Azide-modified gelatin was synthesized by mixing gelatin and Azide-PEG.sub.4-NHS ester at a ratio shown in Table 1. A synthesis scheme is shown in Formula 1. Gelatin was dissolved in 300 mM HEPES buffer having a pH 7.4, Azide-PEG.sub.4-NHS ester was dissolved in 10 mM phthalic acid buffer (pH 4.0), and these were mixed and stirred at 37 C. for 2 hours. The reaction solution was put in a dialysis membrane (fraction molecular weight 6,000 to 8,000, Spectrum laboratories, Inc., Rancho Domingues, Calif.) and dialyzed against MilliQ water (5L) for 24 hours. MilliQ water was exchanged a total of five times after 30 minutes, 1, 3, 5, and 7 hours. The dialyzed sample was dried using a freeze dryer (FDS-1000, Tokyo RIKAKIKAI Co., LTD., Tokyo, Japan) to obtain azide-modified gelatin. The yield was between 71% and 82%. The obtained azide-modified gelatin was dissolved in 300 mM HEPES of pH 7.4 at 37 C. and stored at 4 C. The azide-modified amount with respect to a reactive amino group of azide-modified gelatin was quantified by using fluorescamine and using a fluorescent labeling method of a reactive amino group described in NPL 9.

##STR00006##

TABLE-US-00001 TABLE 1 Mixed concentration of gelatin and Azide-PEG.sub.4-NHS when synthesizing azide-modified gelatin Feed concentration when synthesizing Ratio.sup.a of azido Azidification group to amino modification ratio.sup.b Type of group in gelatin of reactive amino azide-modified Gelatin Azide-PEG.sub.4-NHS Azide/NH.sub.2 group in gelatin gelatin (mg/mL) (mM) (mol %).sup.a (mol %) Gelatin 25 0 0 0 Azide-gelatin (25) 4.7 25 37 Azide-gelatin (50) 9.4 50 67 Azide-gelatin (75) 14.1 75 87 Azide-gelatin (100) 18.8 100 98 The content of amino group in .sup.agelatin was calculated based on information of NPL 10. The .sup.bazide-modified amount was quantified using fluorescamine and using the method described in NPL 9.

[0176] Matrigel is a cell culture substrate sold by Corning Incoporated, and various growth factors such as transforming growth factor (TG), epithelial cell growth factor (EGF), insulin-like growth factor (IGF), and fibroblast cell growth factor (FGF) are contained in addition to an extracellular matrix such as laminin and collagen which are main components thereof.

[0177] When synthesizing azide-modified matrigel, Matrigel was dialyzed and freeze-dried in advance before use in the same protocol as the above protocol. Azide-modified matrigel was synthesized by mixing Matrigel and Azide-PEG.sub.4-NHS ester at a ratio shown in Table 2 in the same method as the synthesis of azide-modified gelatin. The yield was between 68% and 93%. The obtained azide-modified matrigel was dissolved in 300 mM HEPES of pH 7.4 and dissolved at 4 C. The dissolved azide-modified matrigel was stored at 4 C. until immediately before being used. The azide-modified amount of azide-modified matrigel with respect to the reactive amino group was quantified by using fluorescamine and using the fluorescent labeling method of the reactive amino group described in NPL 9.

TABLE-US-00002 TABLE 2 Mixed concentration of purified Matrigel and Azide- PEG.sub.4-NHS when synthesizing azide-modified matrigel Feed concentration when synthesizing Ratio.sup.a of azido Azidification group to amino modification ratio.sup.b Type of group in matrigel of reactive amino azidemodified Matrigel Azide-PEG.sub.4-NHS Azide/NH.sub.2 group in gelatin matirgel (mg/mL) (mM) (mol %).sup.a (mol %) Gelatin 5 0 0 0 Azide-matrigel (25) 3.8 25 44 Azide-matrigel (50) 7.5 50 67 Azide-matrigel (75) 11.3 75 80 Azide-matrigel (100) 15.0 100 83

[0178] The content of the amino group in .sup.aMatrigel was quantified using fluorescamine and using the method described in NPL 9. The reactive amino group of azidified reaction gelatin was comparatively quantified based on information when the gelatin solution was 100 mol % of the reactive amino group and HEPES was 0 mol % of the reactive amino group. The .sup.bazide-modified amount was quantified using fluorescamine and using the fluorescent labeling method of the reactive amino group described in NPL 9.

Example 2

Examination of Solubility of Azide-Modified Gelatin and Azide-Modified Matrigel

[0179] The solubility of azide-modified Gelatin and azide-modified matrigel was examined in 300 mM HEPES buffer (pH 7.4) (Table 5 and Table 6). It is known that if 25 mg/mL aqueous solution of gelatin is kept at 4 C. and 25 C., gelation occurs, but the solution dissolves at 37 C. On the other hand, it was checked that when gelatin was azide-modified to become azide-modified gelatin, gelation does not occur even when the 25 mg/mL aqueous solution was kept at 25 C. (Table 5). It is considered that this is because hydrophilicity of gelatin increased by the introduction of an azido group, by which water solubility of gelatin was improved. It can be said that a fact that mixing operation of the solution became possible at room temperature (25 C.) due to this improvement in hydrophilicity is an excellent property when preparing the photodegradable gel.

TABLE-US-00003 TABLE 3 Comparison of water solubility of gelatin and azide-modified gelatin. Each was dispersed in 300 mM HEPES buffer (pH 7.4) at a concentration of 25 mg/mL, and the solubility was examined. Gelatin concentration Temperature (mg/mL) ( C.) Solubility Gelatin 25 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Dissolved Azide-gelatin (25) 4 Insoluble (gelation) 25 Dissolved 37 Dissolved Azide-gelatin (50) 4 Insoluble (gelation) 25 Dissolved 37 Dissolved Azide-gelatin (75) 4 Insoluble (gelation) 25 Dissolved 37 Dissolved Azide-gelatin (100) 4 Insoluble (gelation) 25 Dissolved 37 Dissolved

[0180] It is checked that 25 mg/mL aqueous solution of Matrigel can maintain the dissolution state at 4 C., but if the solution is kept at 25 C., gelation occurs (Table 4). On the other hand, it was checked that when Matrigel was azide-modified to become azide-modified matrigel, gelation does not occur even when the 25 mg/mL aqueous solution was kept at 25 C. (Table 4). It is considered that this is because hydrophilicity of Matrigel increased by teh introduction of an azido group, by which water solubility of Matrigel was improved. It can be said that a fact that mixing operation of the solution became possible at room temperature (25 C.) due to this improvement in hydrophilicity is an excellent property when preparing the photodegradable gel.

TABLE-US-00004 TABLE 4 Comparison of water solubility of Matrigel and azide-modified matrigel. Each was dispersed in 300 mM HEPES buffer (pH 7.4) at a concentration of 25 mg/mL, and the solubility was examined. Matrigel concentration Temperature (mg/mL) ( C.) Solubility matrigel 25 4 Dissolved 25 Insoluble (gelation) 37 Insoluble (gelation) Azide-matrigel (25) 4 Dissolved 25 Dissolved Azide-matrigel (50) 4 Dissolved 25 Dissolved Azide-matrigel (75) 4 Dissolved 25 Dissolved Azide-matrigel (100) 4 Dissolved 25 Dissolved

Comparative Example 1

Synthesis of DBCO-Modified Gelatin and Examination of Water Solubility Thereof

[0181] Since the click reaction forms a covalent bond by the reaction between a DBCO group and an azido group, it is considered that the DBCO group may be introduced into gelatin and the azido group may be introduced into the crosslinker in order to form a gel using the click reaction. In order to verify the above, the DBCO group was introduced into gelatin to synthesize DBCO-modified gelatin, and water solubility was examined in 300 mM HEPES buffer (pH 7.4).

[0182] DBCO-modified gelatin was synthesized by mixing gelatin and DBCO-sulfo-NHS ester at a ratio shown in Table 5. The synthesis scheme is shown in Formula 2. Gelatin was dissolved in 300 mM HEPES buffer of pH 7.4 and DBCO-sulfo-NHS ester solution was dissolved in 10 mM phthalic acid buffer (pH 4.0), and these were mixed and reacted at 37 C. for 2 hours.

##STR00007##

TABLE-US-00005 TABLE 5 Mixed concentration of gelatin and DBCO-PEG.sub.4-NHS (after mixing) when synthesizing DBCO-modified gelatin Feed amount.sup.a of DBCO group to amino Gelation DBCO-sulfo-NHS group in gelatin Type of concentration concentration (DBCO/NH.sub.2) DBCO-gelatin (mg/mL) (mM) (mol %).sup.a Gelatin 25 0 0 DBCO-gelatin (25) 4.7 25 DBCO-gelatin (50) 9.4 50 DBCO-gelatin (75) 14.1 75 DBCO-gelatin (100) 18.8 100 The content of amino group in .sup.agelatin was calculated based on the information of NPL 10 with a molar mass of amino group contained in gelatin.

[0183] As a result of examining the water solubility of the synthesized DBCO-modified gelatin in 300 mM HEPES buffer (pH 7.4), it was checked that in the process of synthesizing DBCO-modified gelatin, there was no condition under which DBCO-modified gelatin can be dissolved at 25 C., and even at 37 C., only DBCO-gelatin (25) having a low introduction ratio of the DBCO group is dissolved (Table 6 and FIG. 2).

[0184] From the results, it was checked that azide-modified gelatin and azide-modified matrigel exhibited more high solublility in a HEPES buffer compared to DBCO-modified gelatin, and this is excellent for preparing the photodegradable gel using the click reaction.

TABLE-US-00006 TABLE 6 Comparison of water solubility of gelatin and DBCO-modified gelatin. Each was dispersed in 300 mM HEPES buffer (pH 7.4) at a concentration of 25 mg/mL, and the solubility was examined. Gelatin Concentration Temperature (mg/mL) ( C.) Solubility Gelatin 25 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Dissolved DBCO-gelatin (25) 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Dissolved DBCO-gelatin (50) 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Insoluble (gelation) DBCO-gelatin (75) 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Insoluble (gelation) DBCO-gelatin (100) 4 Insoluble (gelation) 25 Insoluble (gelation) 37 Insoluble (gelation)

Example 3

Synthesis of DBCO-PC-4ArmPEG

[0185] In order to synthesize a click crosslinking-type photocleavable crosslinker, DBCO-PC-4arm PEG, firstly, a photocleavable crosslinker NHS-PC-4arm PEG of an active ester-type was synthesized according to a method for introducing a nitrobenzyl group into an active ester terminal 4arm PEG described in NPL 6.

[0186] DBCO-PC-4armPEG was synthesized by reacting NHS-PC-4armPEG with DBCO-PEG.sub.4-amine. The synthesis scheme is shown in Formula 3. DBCO-PEG.sub.4-amine (600 mg) and NHS-PC-4armPEG (3.018 g) were added to 125 mL of dimethyl sulfoxide (DMSO) and heated at 40 C. and stirred. The reactant was precipitated in diethyl ether to be purified. First, the reaction solution was added dropwise to 1L of diethyl ether and ice-cooled for 30 minutes. After removing the supernatant by decantation, the precipitate was dissolved in 10 mL of THF. Next, the reactant dissolved in THF was added dropwise to 400 mL of diethyl ether to obtain a precipitate. This ether precipitation was repeated three times to obtain 3.4 g of DBCO-PC-4armPEG (yield: 99%).

##STR00008## ##STR00009##

[0187] The molecular structure of the synthesized DBCO-PC-4armPEG was checked by .sup.1H-NMR in deuterated chloroform (FIG. 3A). It was checked that in DBCO-PC-4armPEG, a peak assigned to DBCO was around 7.3 ppm compared to NHS-PC-4armPEG (FIG. 3B).

Example 4

Evaluation on Photocleavability of DBCO-PC-4ArmPEG

[0188] The photocleavage reaction of DBCO-PC-4armPEG was evaluated by changes in the absorption spectrum before and after light irradiation.

[0189] In order to measure the absorption spectrum, 120 M DBCO-PC-4armPEG solution was prepared using 300 mM HEPES of pH 7.4 and 1.5 L thereof was added. The solution in the Eppendorf tube is irradiated with ultraviolet light (365 nm, 25.2 mW/cm.sup.2, 0 to 7.6 J) emitted from an ultraviolet light source (UVE-251S, San-Ei Electric, Osaka, Japan) through a 350 nm long wavelength cut filter and a 385 nm short wavelength cut filter. The absorption spectrum was measured at 10 C. using an absorption photometer (Nanodrop, Thermo Fisher Scientific K. K., Kanagawa, Japan).

[0190] The results are shown in FIG 1e. An increase in absorption at 390 nm due to the cleavage reaction of an o-nitrobenzyl group was checked by light irradiation at 0.4 J/cm.sup.2 or more.

Example 5

Preparation of Photodegradable Gel by Click Reaction between Azide-Modified Gelatin and DBCO-PC-4ArmPEG

[0191] (1) Checking of Gel Formation by Rheology Measurement

[0192] In order to check the sol-gel transition of the click reaction from changes in dynamic viscoelasticity, a storage modulus (G) and a loss modulus (G) was measured by using a rheometer (MCR-302; Anton Paar Ltd., Graz, Austria). Azide-Gelatin and DBCO-PC-4armPEG were dissolved in a HEPES buffer (300 mM, pH 7.4), and then both solutions were mixed in equal amounts, respectively, so that the concentrations thereof become concentrations shown in Table 7 (refer to (2)). Immediately after mixing the two solutions, a device was filled with 500 L of the mixed solution, and measurement (frequency f: 5 Hz/temperature: 25 C./measurement interval: 30 seconds, 1 hour) was immediately started.

[0193] Changes in G and G over time when forming a gel by click reaction are shown in FIG. 4. A point where G exceeds G (crossover point, CP) is a point where gelation occurs. When four kinds of gels of which a composition is shown in Table 7, which are PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), and PD-gelatin (100), were examined (each shown in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D), in every composition, G exceeded Gwithin 5 minutes, and thereafter, an increase in G was checked over several tens of minutes.

[0194] (2) Preparation of Photodegradable Gel Membrane

[0195] Azide-modified Gelatin and DBCO-PC-4armPEG were dissolved in 300 mM HEPES buffer of pH 7.4 to be used. Equal amounts of the azide-modified gelatin solution and the DBCO-PC-4armPEG solution were mixed, respectively, so that the final concentrations thereof become concentrations shown in Table 7. The solutions were interposed between Teflon blocks by using a spacer manufactured by Teflon (registered trademark) so that a thickness becomes 300 m, and incubated at room temperature for 30 minutes to prepare a membrane-like photodegradable gel.

[0196] A HEPES buffer containing 1.0 mg/mL Matrigel was used when preparing the azide-modified gelatin solution, followed by mixing with the DBCO-PC-4armPEG solution and molding based on the same protocol as the above protocol, and therefore a membrane-like photodegradable hydrogel containing Matrigel was prepared.

TABLE-US-00007 TABLE 7 Mixed concentration of azide-modified gelatin and DBCO-PC-4armPEG (and Matrigel) (after mixing) when preparing photodegradable gel DBCO-PC- Type of Type of Azide-gelatin 4amPEG Matirgel hydrogel azide-gelatin (mg/mL) (mM).sup.b (mg/mL) Gelatin Gelatin 12.5 0 0 PD-gelatin (25) Azide-gelatin (25) 12.5 0.6 0 PD-gelatin (50) Azide-gelatin (50) 12.5 1.2 0 PD-gelatin (75) Azide-gelatin (75) 12.5 1.8 0 PD-gelatin (100) Azide-gelatin (100) 12.5 2.3 0 Gelatin_M+ Gelatin 12.5 0 0.5 PD-gelatin Azide-gelatin (25) 12.5 0.6 0.5 (25)_M+ PD-gelatin Azide-gelatin (50) 12.5 1.2 0.5 (50)_M+ PD-gelatin Azide-gelatin (75) 12.5 1.8 0.5 (75)_M+ PD-gelatin Azide-gelatin (100) 12.5 2.3 0.5 (100)_M+

Example 6

Checking of Micropatterned Degradation of Photodegradable Gel by Micropatterned Light Irradiation

[0197] It was checked that the membrane-like hydrogel prepared in Example 5 was irradiated with micropatterned light and the hydrogel photodegraded according to the micropattern.

[0198] Micropatterned light irradiation was carried out using a PC control type microprojection system (DESM-01 Engineering System, Co. Ltd., Nagano, Japan) based on a method of NPL 11.

[0199] The pattern to be irradiated was prepared using Adobe illustrator CS4 (Adobe systems software Ireland Ltd.). Micropatterned light having an intensity of 156 mW/cm.sup.2 and a wavelength of 365 nm was emitted for 30 seconds and the hydrogel was incubated at 37 C. for 1 hour. Thereafter, the hydrogel was stained with Coomassie Brilliant Blue (CBB, 0.01%) and the micropattern formed in the hydrogel was observed with an inverted microscope (IX-71, Olympus Corporation, Tokyo, Japan).

[0200] Regarding the four kinds of gels of which a composition is shown in Table 7, which are PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), and PD-gelatin (100), the results of micropatterned degradation of the photodegradable gel by micropatterned light irradiation are shown in FIGS. 5A to D. After each gel is irradiated with patterned light shown in FIG. 5E, the hydrogel was stained with CBB which is a protein staining reagent. It was checked that the color of the irradiated portion was missing in FIGS. 5A to D, and it was checked that the hydrogel at the irradiated portion degraded according to the irradiation pattern.

[0201] Similarly, the four kinds of gels containing Matrigel, of which a composition is shown in Table 7, which are PD-gelatin (25)_M+, PD-gelatin (50)_M+, PD-gelatin (75)_M+, and PD-gelatin (100)_M+, micropatterned degradation of the photodegradable gel by micropatterned light irradiation was performed. The results are shown in FIGS. 6A to D. After each gel is irradiated with patterned light shown in FIG. 5E, the hydrogel was stained with CBB which is a protein staining reagent. It was checked that the color of the irradiated portion was missing in FIG. 6, and it was checked that the hydrogel at the irradiated portion degraded according to the irradiation pattern.

Example 7

Cytotoxicity Test

[0202] Cells were embedded in a hydrogel and the survivability of the embedded cells was evaluated with respect to cell damage using Live/Dead cell survival rate assay kit (Life Technologies, Carlsbad, USA) according to the following procedure.

[0203] As cells, cells derived from human cervical carcinoma (hereinafter will be referred to as HeLa cells) and cells derived from human prostate cancer (hereinafter will be referred to as DU145 cells) provided by RIKEN BioResource Center were used to be cultured, respectively, at 37 C. under a 5% CO.sub.2 environment using Dulbecco's modification of Eagle's medium (DMEM, Life technologies) in which 10% serum was added as a culture medium.

[0204] Azide-modified Gelatin and DBCO-PC-4armPEG were dissolved in 300 mM HEPES buffer of pH 7.4 to be used. HeLa cells and DU145 cells were dispersed in an azide-modified gelatin solution at a concentration of 1.410.sup.5 cells/mL. 7 L of the azide-modified gelatin solution and 7L of the DBCO-PC-4armPEG solution were mixed, and were interposed between Teflon blocks by using a spacer manufactured by Teflon so that a thickness becomes 300 m, and then incubated at room temperature for 30 minutes. After forming the hydrogel, 500 L of the culture solution was added and cultured at 37 C. for 24 hours under a 5% CO.sub.2 environment.

[0205] A Live/Dead cell survival rate assay test solution was prepared by adding 1 L of calcein AM and 4 L of ethidium homodimer-1 solution to 5 mL of a phosphate buffered saline (PBS). The cells cultured in the hydrogel were washed with PBS, the test solution of the Live/Dead cell survival rate assay was added thereto, followed by incubation at 37 C. for 30 minutes, and then obsevtion was performed using a confocal laser microscope (FV300, Olympus). The living cells and the dead cells were counted so that a total of 100 cells or more was counted for each sample.

[0206] Regarding the four kinds of hydrogels produced by using azide-modified gelatin and DBCO-PC-4armPEG, of which a composition is shown in Table 7, which are PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), and PD-gelatin (100); the four kinds of hydrogels produced by adding Matrigel, of which a composition is shown in Table 7, which are PD-gelatin (25)_M+, PD-gelatin (50)_M+, PD-gelatin (75)_M+, and PD-gelatin (100)_M+; and the photodegradable hydrogel produced by using gelatin and the active ester-type crosslinker at the same concentration of these hydrogels, the results of investigating the cell survival rate when DU145 cells and HeLa cells were embedded in the photodegradable gel in the manner described above are shown in FIG. 7.

[0207] In a case of using the click-type crosslinker and DBCO-PC-4armPEG (FIG. 7A and FIG. 7B), it was checked that the embedded cells showed a high survival rate in a wide concentration range of the crosslinker. On the other hand, in a case of the active ester-type crosslinker (FIG. 7C), it was checked that the survival rate of the cell drastically decreased from the point where the crosslinker concentration exceeds 1.8 mM.

[0208] From these results, it was checked that the click-type photocleavable crosslinker of the present embodiment is an excellent crosslinker with lower cytotoxicity compared to the active ester type photocleavable crosslinker shown in the literature of the related art.

Example 8

Cell Culture Test

[0209] The culture test of cells embedded in the hydrogel was carried out by the following procedure.

[0210] As cells, HeLa cells provided by RIKEN BioResource Center were used to be cultured at 37 C. under a 5% CO.sub.2 environment using Dulbecco's modification of Eagle's medium (DMEM, Life technologies) in which 10% serum was added as a culture medium.

[0211] A photodegradable hydrogel containing the cells was prepared according to the protocol of Example 7 except that HeLa cells were mixed with an azide gelatin solution at a concentration of 3.310.sup.3 cells/mL. Cell observation was carried out after 24, 48, and 72 hours using an inverted microscope (IX-71, Olympus).

[0212] Regarding eight kinds of hydrogels of which a composition is shown in Table 7, which are PD-gelatin (25), PD-gelatin (50), PD-gelatin (75), PD-gelatin (100), PD-gelatin (25)_M+, PD-gelatin (50)_M+, PD-gelatin (75)_M+, and PD-gelatin (100) M+, the state of cell growth and form change when HeLa cells are embedded to be cultured for 3 days according to the procedure described in Example 7 are shown in FIG. 8A and FIG. 8B. It was checked that the cells showed more remarkable growth in the photodegradable hydrogel (FIG. 8B) to which Matrigel was added compared to a system in which Matrigel is not added (FIG. 8A).

[0213] From the above results, it was checked that by using the click-type crosslinker, it is possible to form the photodegradable hydrogel even in a system containing an additive factor called Matrigel. It was also checked that the cell growth in the photodegradable hydrogel was remarkable in the presence of Matrigel. It was checked that the click-type photocleavable crosslinker of the present embodiment had better properties than the active ester type photocleavable crosslinker shown in the literature of the related art.

INDUSTRIAL APPLICABILITY

[0214] Cell transplantation using cell sheets and cell suspensions is currently the mainstream in regenerative medicine, but in the future, it is considered that regenerative medicine using Engineered Tissue in which tissues having complex structures such as blood vessels and nerves, larger tissues such as liver tissues and kidney tissues, and organs are extracorporeally and artificially processed to be transplanted will become important. In order to extracorporeally and artificially form the large tissue, it is required to form, in the tissue, a network of a blood vessel-like passage for exchanging oxygen, nutrients, and waste products, and a three-dimensional structure thereof inevitably becomes complicated.

[0215] A gel that degrades by light can form a complex structure by a combination with micropatterned light irradiation and scan irradiation. The photodegradable gel disclosed in the present invention can be used for processing a tissue having a complex three-dimensional structure, since the gel has low cytotoxicity, the cells can be embedded therein, and the gel degrades by light. The processed tissue can be applied to regenerative medicine.

[0216] The photodegradable gel of the present invention can also be used for cell separation. For example, the cells are embedded in the photodegradable gel to be cultured, or in a state where the cells are cultured on the photodegradable gel, the vicinity of the cells to be separated is irradiated with light so that the gel dissolves, and therefore, the cells can be separated. The photodegradable gel of the present invention is a useful material for such cell separation, because the gel has no cytotoxicity, and it is possible to provide the photodegradable gel having cell adhesiveness by a simple method in which two solutions are mixed.