WO2021177209A1

WO2021177209A1 - Laminin cross-linked product, culture medium including laminin cross-linked product, three-dimensional culturing kit, culture substrate, composition, structure, and culturing method

Info

Publication number: WO2021177209A1
Application number: PCT/JP2021/007634
Authority: WO
Inventors: 裕介村澤; 一乘水野; 和将藤田; 雄基熊澤; 志乃海八木; 暁艶陽
Original assignee: 株式会社ニッピ
Priority date: 2020-03-02
Filing date: 2021-03-01
Publication date: 2021-09-10
Also published as: JPWO2021177209A1

Abstract

Provided are: a laminin cross-linked product that is formed by bonding collagen or a glycosaminoglycan that has a molecular weight of 1×10⁴–5×10⁶ to laminin or a laminin fragment by means of a cross-linking agent; and a three-dimensional culture substrate and the like that include the laminin cross-linked product. A mixture of the laminin cross-linked product and collagen gels when heated to 37°C and, because the laminin cross-linked product is uniformly dispersed throughout the gel, can be used as a three-dimensional culture substrate for pluripotent stem cells, cancer cell aggregates, cells that are in physiological contact with a basement membrane, and the like.

Description

Laminin cross-linked product, culture medium containing laminin cross-linked product, three-dimensional culture kit, culture substrate, composition, structure and culture method

The present disclosure relates to a laminin crosslinked product, a culture medium containing a laminin crosslinked product, a three-dimensional culture kit, a culture substrate, a composition, a structure, a culture method, and the like.

There is a three-dimensional culture in which cells are cultured with a thickness in the vertical direction, as opposed to a 2D culture in which cells are cultured in a single layer in a plane, and it is said to be an experimental model that more closely imitates the in-vivo environment. For example, in a method of differentiating human pluripotent stem cells into mature hepatocytes or the like, there is a method of culturing human pluripotent stem cells in 2D and then culturing them in 3D scaffold for maturation (Patent Document 1). .. A new 3D culture system was developed in view of the fact that 2D culture of pluripotent stem cells often fails to reproduce the characteristics of mature cell types. In Patent Document 1, as a 3D scaffold, a porous alginate sponge, a biodegradable poly (urethane urea) polymer, an emulsion template polystyrene, a synthetic nanofiber composite material, and a porous sponge made of poly (L-lactic acid) are used. Is illustrated.

In addition, a collagen-binding molecule is bound to at least one of the N-terminal of the α chain, the N-terminal of the β chain, and the N-terminal of the γ chain of the laminin fragment forming a laminin or a heterotrimer. There is a modified laminin characterized by this, and it is used as a culture medium for pluripotent stem cells and the like (Patent Document 2). As an extracellular matrix material that replaces Matrigel (registered trademark), it is an object of the present invention to provide an extracellular matrix material that is useful for constructing a three-dimensional tissue structure for regenerative medicine that is safe for humans. As the collagen-binding molecule, a fragment containing collagenase or a collagen-binding active site thereof is used, and in the examples, a modified human laminin to which the collagen-binding molecule is bound is produced by gene recombination.

Further, there is also a medium composition containing a structure capable of suspending and culturing cells or tissues, which contains either collagen, hyaluronic acid or proteoglycan as an extracellular matrix (Patent Document 3). It provides a medium composition for culturing animal and plant cells and tissues in a three-dimensional or floating state, and according to the above medium composition, the cells and tissues are maintained in a floating state without increasing the viscosity in a liquid medium. It is said that it can grow, differentiate or maintain as it is.

Special Table 2013-532966 International Publication No. 2014/103534 Japanese Unexamined Patent Publication No. 2019-150064

The present disclosure describes laminin cross-linked products suitable for culturing epithelial cells, endothelial cells and other somatic cells, pluripotent stem cells and tissues containing them, culture media and structures suitable for three-dimensional culture containing laminin cross-linked products, and the like. An object of the present invention is to provide related substances, a culture method, and the like.

The present inventors have stated that hyaluronic acid and collagen, which are extracellular matrix (ECM) components, can bind to laminin and its E8 fragment via a cross-linking agent to form a laminin cross-linked product. We have found that the culture medium containing the substance and collagen gels at the culture temperature to become a culture medium having superior cell differentiation ability as compared with the conventional three-dimensional culture medium, and particularly excellent in culturing pluripotent stem cells and the like. Was completed.

That is, the present disclosure describes a ^{laminin crosslinked product in which collagen and / or glycosaminoglycans having a molecular weight of 1 × 10 4} to 5 × 10 ⁶ are bound to laminin or a laminin fragment having a partial structure of the laminin by a cross-linking agent. It is to provide.

The present disclosure also provides the laminin crosslinked product, wherein the laminin fragment is a laminin fragment having an integrin-binding activity of the laminin.

The present disclosure also provides the laminin crosslinked product in which the glycosaminoglycan is hyaluronic acid or chondroitin.

Further, in the present disclosure, the laminin is any one or more of laminin 511, laminin 332, laminin 111, laminin 121, laminin 211, laminin 221 and laminin 311, laminin 321 and laminin 411, laminin 421, or laminin 521. It provides the laminin crosslinked product.

The present disclosure also provides the laminin crosslinked product, wherein the laminin fragment is a laminin E8 fragment.

The present disclosure also provides a culture medium containing the laminin crosslinked product and a temperature-responsive substrate that remains in a solution state at 10 ° C. or lower and becomes a gel at 20 to 45 ° C.

The present disclosure also provides the culture medium, which is characterized in that the temperature-responsive substrate is collagen.

The present disclosure also provides a three-dimensional culture kit containing the culture medium and a pH adjuster.

Further, the present disclosure further provides the above-mentioned three-dimensional culture kit containing a culture solution.

The present disclosure also provides a culture medium, characterized in that the laminin crosslinked product is coated with collagen and / or gelatin.

The present disclosure also provides a composition containing the laminin crosslinked product and collagen and / or gelatin.

The present disclosure also provides a structure composed of the laminin crosslinked product and collagen and / or gelatin.

The present disclosure also provides a method for culturing cells or tissues, which comprises culturing in the presence of the laminin crosslinked product.

The present disclosure also provides a three-dimensional culture method for cells or tissues, which comprises burying cells or tissues in the culture medium and culturing the cells or tissues.

The present disclosure also provides the culture method in which the cells are pluripotent stem cells or somatic stem cells.

According to the present disclosure, a laminin crosslinked product obtained by cross-linking laminin to collagen or glycosaminoglycan, a culture medium containing the laminin cross-linked product, or the like is provided.

It is a figure which shows the result of Example 2. FIG. A pH 7.5 collagen- (hyaluronic acid-laminin crosslinked product) mixture containing a hyaluronic acid-laminin crosslinked product and collagen is liquid at a temperature of 4 to 10 ° C., but maintains a gel state at a temperature of 20 to 35 ° C. It is a figure which shows that. The figure which shows the result of the bright-field image of the tissue 7 days after tissue culture of the mouse developmental kidney tissue of embryonic day 11.5 days, ureter formation (DBA staining), and nephron progenitor cell (WT-1 staining) in Example 3. Is. In Example 3 using the collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared in Example 2, ureteral buds diverged in the gel in the developing kidney tissue as compared with the case of using the Matrigel of Comparative Example 1. It was confirmed that the formation of ureters was excellent, and that the formation of nephron precursor cells, which are the precursor tissues of glomeruli, was excellent at the tips of ureteral buds. It is a figure which shows the result of Example 6, and is the figure which shows the number of ureteral bud branching and the ureteral bud branching area at the time of culturing the renal tissue in the mixture gel with different final concentrations of laminin 511E8. It is a figure which shows the result of Example 7. It is a figure which shows that the vascular structure and the glomerular structure are formed when the cells derived from the renal tissue of a mouse embryo are cultured with a collagen- (hyaluronic acid-laminin crosslinked product) mixture. It is a figure which shows the result of Example 8, FIG. 5A is a stained image using a type I collagen antibody, FIG. 5B is a HABP, and FIG. 5C is a stained image using a laminin 511E8 antibody. Further, FIGS. 5D and 5E are images obtained by freeze-drying the mixture gel and collagen gel, depositing platinum on them, and capturing the images with a scanning electron microscope. It is a figure which shows the result of Example 9, and is the figure which shows the result of having evaluated the gelation ability of a collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared using various collagens. It is a figure which shows the result of Example 10, and is the result of embedding culture of collagen- (hyaluronic acid-laminin crosslinked product) mixture, collagen- (hyaluronic acid-laminin uncrosslinked product) mixture, and cells derived from the developing kidney by Matrigel. It is a figure which shows. It is a figure which shows the result of Example 11, and is the figure which shows the result of embedding and culturing the cells derived from the developing kidney with collagen- (hyaluronic acid-laminin crosslinked product) mixture and Matrigel for 1 week. It is a figure which shows the result of Example 11, and is the figure which shows the result of embedding and culturing the cell derived from the developing kidney with a collagen- (hyaluronic acid-laminin crosslinked product) mixture and Matrigel. It is a figure which shows the result of Example 12, and the molecular weight of hyaluronic acid is changed to prepare the collagen- (hyaluronic acid-laminin cross-linked product) mixture, and this mixture is used for the cell derived from the developmental kidney cell for 7 days. It is a figure which showed that the difference in the molecular weight of hyaluronic acid causes a difference in renal tissue organization. It is a figure which shows the result of Example 13. It is a figure which shows the result of culturing the cell of the developmental kidney origin on the gel on the upper surface of the type I + type IV mixture gel and the type I mixture gel. It is a figure which shows the result of Example 14. The results of embedding and culturing cells derived from developing kidney using a type I mixture, a type I + type III mixture, and a type I + V type mixture are shown. It is a figure which shows the result of Example 14, and shows the result of embedding culture of the cell derived from the developmental kidney using the type I + type IV mixture. It is a figure which shows the result of Example 15. The results of embedding and culturing cells derived from the developing liver and cells derived from the intestinal tract using a collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared by using laminin 332E8 and laminin 111E8 instead of laminin 511E8 are shown. .. It is a figure which shows the result of Example 16. The results of embedding and culturing cells derived from the developing kidney using a collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared using DSG instead of genipin are shown.

The first of the present disclosure ^{is laminin cross-linking in which collagen and / or glycosaminoglycans having a molecular weight of 1 × 10 4} to 5 × 10 ⁶ are bound to laminin or a laminin fragment having a partial structure of the laminin by a cross-linking agent. It is a thing.

Glycosaminoglycans are long-chain, normally unbranched polysaccharides that are ubiquitous in all tissues, especially connective tissues in animals. In their natural form, hyaluronic acid, chondroitin, heparin, keratan sulfate, etc. There is. As the glycosaminoglycan used in the present disclosure, hyaluronic acid and chondroitin, which are ECM components, can be preferably used. The molecular weight of the glycosaminoglycan used is 1 × 10 ⁴ to 5 × 10 ⁶ , preferably 2 × 10 ⁴ to 4 × 10 ⁶ , and more preferably 3 × 10 ⁴ to 4 × 10 ⁶ . Within this range, the gel can be maintained after being mixed with collagen and heated to a temperature of 20 to 45 ° C.

Collagen is one of the proteins that mainly make up the dermis, ligaments, tendons, bones, cartilage, etc. of vertebrates, and is contained in crustaceans, mollusks, etc. The collagen used in the present disclosure may be derived from any animal, and the animal species is not limited. In general, enzyme-treated solubilized collagen in which collagen tissue contained in raw materials such as animal skin and bone is solubilized by adding protease such as pepsin, alkali-treated solubilized collagen solubilized by adding alkali, and acid are added. Solubleized acid-treated solubilized collagen can be used. In order to extract collagen molecules from these collagen solubilized solutions, a salting out method or an isoelectric point precipitation method is generally adopted. Collagen includes fibrous collagen that forms fibers and non-fibrotic collagen that does not form fibers, and is named by Roman numerals such as type I, type II, type III, IV, and V. The collagen used in the present disclosure is not particularly limited as long as it has a triple helix structure at least in part, is a solution at a temperature of 10 ° C. or lower, and can be gelled at a temperature of 20 to 45 ° C. Further, asparagine residue, glutamine residue and the like may be deamidated and changed to aspartic acid residue and glutamic acid residue at the time of alkali solubilization of collagen. The animal species from which it is derived does not matter. Further, the extract is not limited to an extract from animal tissue, and may be purified from collagen high-expressing cells using a known technique or produced as a recombinant protein. For example, CHO cells or tobacco cells may be synthesized by genetic recombination technology.

Laminin is an ECM component that interacts with the cell surface receptor "integrin" and transmits signals necessary for maintaining cell function and controlling proliferation and differentiation induction. Its structure is a cross-shaped molecule in which α, β, and γ chains are associated in a coiled coil region, and there are several types in each chain. Therefore, 5 types of α chains (LAMA1 to 5), 4 types of β chains (LAMB1 to 4), and 3 types of γ chains (LAMC1 to 3) are combined and called, and laminin 511 means laminin α5β1γ1. Each laminin has its own unique effect. As the laminin used in the present disclosure, it is preferable to use laminin 511, laminin 332, laminin 111, laminin 121, laminin 211, laminin 221 or laminin 311, laminin 321 or laminin 411, laminin 421 or laminin 521. The origin of laminin is not particularly limited, and laminin derived from various organisms can be used. It is preferably a mammalian-derived laminin. Examples of mammals include, but are not limited to, humans, mice, rats, cows, pigs and the like. Further, it may be produced by the gene recombination technique by appropriately using a known gene recombination technique. Nucleotide sequence information of genes encoding α-chain, β-chain, and γ-chain constituting laminin of major mammals and amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.).

Further, it may be a laminin fragment having a partial structure of the laminin. As such a laminin fragment, there is a laminin fragment having an integrin-binding activity. A laminin fragment having integrin-binding activity can bind to integrin and exert the same effect as full-length laminin. For example, the integrin binding site of laminin 511 consists of three spherical domains (LG1-3) on the α-chain C-terminal side of the laminin molecule and a γ-chain C-terminal region (γ-tail). The three spherical domains associate in a trefoil shape and bind to integrin on the bottom surface side thereof, and the γ-tail is guided to a predetermined position of the ligand recognition spot of integrin to form a stable laminin-integrin complex. As such an integrin binding site, there is a laminin E8 fragment. Generally, the "laminin E8 fragment" is a fragment forming a heterotrimer obtained by digesting mouse laminin 111 with elastase and identified as a fragment having strong cell adhesion activity (Edgar D et). al., J.Cell Biol., 105: 589-598, 1987). However, the "laminin E8 fragment" in the present disclosure broadly includes a fragment of laminin having a site capable of binding to integrin, and may be derived from any animal species or laminin species. Furthermore, the "laminin E8 fragment" does not need to be a digested product of laminin elastase, but may be produced by genetic recombination or the like. As described above, the integrin binding site of laminin 511 consists of three spherical domains (LG1-3) on the α-chain C-terminal side of the laminin molecule and the γ-chain C-terminal region (γ-tail), and N of the α-chain. Since the terminal side forms a heterotrimer linked to the C-terminal side of the β chain and the γ chain by an SS bond, such a heterotrimer can be used as a laminin E8 fragment. The laminin fragment having an integrin-binding activity is not limited to a laminin fragment having a heterotrimer on condition that it has an integrin-binding activity. The fact that the laminin fragment has integrin-binding activity can be confirmed by a solid-phase binding assay or the like, and the fact that the laminin fragment forms a heterotrimer indicates that the laminin fragment forms SDS-PAGE. It can be confirmed by detecting the number of bands and the like. In the present disclosure, commercially available laminin fragments having integrin-binding activity can also be used. Examples of such a laminin fragment include iMatrix-511, iMatrix-411, and iMatrix-221 manufactured by Nippi Co., Ltd. A laminin crosslinked product composed of laminin or a laminin fragment having integrin-binding activity is excellent in integrin-binding ability. Therefore, this laminin crosslinked product can be used in place of or in addition to collagen, glycosaminoglycan, and laminin in the conventional culture medium.

On the other hand, the laminin fragment used in the present disclosure is not limited to those having integrin-binding activity. Laminin is known to have various functions other than integrin binding. For example, it has been reported that the LG domain of the laminin α chain has binding sites such as lutelan and syndecane and is involved in the regulation of cell adhesion. Such a laminin fragment may be used as long as it has a partial structure of laminin. By cross-linking such a laminin fragment with collagen or glycosaminoglycan, the activity of laminin can be utilized.

The amount of laminin and laminin fragment used is 0.01 to 1000 parts by weight, more preferably 0.05 to 500 parts by weight, particularly preferably 0.05 to 500 parts by weight, based on 100 parts by weight of the total amount of collagen and glycosaminoglycan constituting the laminin crosslinked product. It is preferably 1 to 10 parts by weight. This is because cell culture using a laminin crosslinked product prepared in this range is excellent in tissue formation that mimics the in vivo of monodisperse cells derived from a living body.

As the cross-linking agent used in the present disclosure, those capable of cross-linking glycosaminoglycan with laminin or the laminin fragment, or those capable of cross-linking collagen with laminin or the laminin fragment can be widely used. Glycosaminoglycans, collagen, and laminin contain hydroxyl groups, carboxyl groups, amino groups, amide bonds, and the like, and can be crosslinked by a cross-linking agent that binds these. Examples of such cross-linking agents include N- (6-Maleimidocaproyloxy) sullfosuccinimide, sodium salt, N-[(4-Maleimidomethyl) cyclohexyl carbonyloxy] sulfosuccinimide, sodium salt, N- (4-Maleimidobutyryloxy) sulfosuccinimide, sodium salt, N- { 6-[3- (2-Pyridyldithio) propionamido] hexanoyloxy} sulfosuccinimide, sodium salt, N- (8-Maleimidocapryloxy) sulfosuccinimide, sodium salt, Methyl (1S, 2R, 6S) -2-Hydroxy-9-(hydroxymethyl)- There are 3-oxabicyclo [4.3.0] nona-4,8-diene-5-carboxylate (genipin). Genipin is preferred because the reaction proceeds near neutrality. The blending amount of the cross-linking agent can be appropriately selected depending on the cross-linking agent used. Generally, it is 0.1 to 1000 parts by mass, more preferably 0.5 to 500 parts by mass, and particularly preferably 1 to 300 parts by mass with respect to 100 parts by mass of the total mass of laminin and the laminin fragment.

To prepare a laminin cross-linked product, glycosaminoglycan, collagen, laminin or the laminin fragment are each dissolved in a solvent, and for example, a lycosaminoglycan lysate and a laminin lysate are mixed, and a cross-linking agent is added thereto. Add and react. Examples of the solvent include water, PBS, physiological saline, trishydroxymethylaminomethane, or physiological saline having a neutral pH of 4- (2-hydroxyethyl) -1-piperazine ethanesulphonic acid. Depending on the cross-linking agent used, the reaction is at a temperature of 4 ° C. to 24 ° C. for 0.5 to 30 hours, more preferably 1 to 20 hours.

When preparing a laminin crosslinked product, a quenching agent that binds to the reactive group of the crosslinking agent can be included. The addition of the quenching agent can reduce the influence of the unreacted cross-linking agent without affecting the gel strength using the laminin cross-linked product. The quenching agent can be appropriately selected depending on the cross-linking agent used. For example, when the cross-linking agent is genipin, glycine, trishydroxymethylaminomethane (tris (hydroxymethyl) aminomethane, THAM), or the like can be preferably used as the quenching agent. When a quenching agent is used, for example, the quenching agent may be added and mixed at the time of mixing the glycosaminoglycan solution and the laminin solution, or the quenching agent may be mixed after the cross-linking reaction. When a quenching agent is used, the blending amount is, for example, 1 to 10000 mol times, preferably 10 to 5000 mol times, and more preferably 100 to 2000 mol times with respect to genipin.

After the cross-linking reaction, the obtained laminin cross-linked product can be purified by dialysis, chromatography, or the like. The purified laminin crosslinked product may be dried and crushed into a powder, or may be used as it is dissolved or dispersed in a solution. At the time of drying, depending on the cross-linking agent used, heat treatment may be appropriately performed as long as laminin, collagen and glycosaminoglycan are not deteriorated.

The cells that can be cultured with the laminin crosslinked product of the present disclosure are mammalian cells such as humans, mice, rats, cows, and pigs, and epithelial cells, endothelial cells, myocardial cells, smooth muscle cells, skeletal muscle cells, and muscle satellites. Cells that are in contact with the basal membrane in vivo, such as cells, nerve schwan cells, fat cells, and pluripotent stem cells; somatic stem cells such as nerve stem cells, mesenchymal stem cells, hematopoietic stem cells, heart stem cells, liver stem cells, and small intestinal stem cells. ; ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), mGS cells (pluripotent reproductive stem cells), pluripotent stem cells such as fusion cells of ES cells and somatic cells, tissues containing these cells Suitable for culturing. The cells may be cancer cells, fusion cells, or the like, in addition to normal cells.

The second aspect of the present disclosure is to maintain a solution state with the laminin crosslinked product at 10 ° C. or lower, more preferably 1 to 10 ° C., particularly preferably 1 to 7 ° C., and 20 to 45 ° C., preferably 25 to 40 ° C. , More preferably a culture medium containing a temperature-responsive substrate that gels at 30-40 ° C.
Examples of the temperature-responsive substrate include poly (N-alkylacrylamide), poly (N-vinylalkylamide), polyvinylalkyl ether, polyethylene glycol / polypropylene glycol block copolymer, poly (N-isopropylacrylamide), collagen and the like. be. In the present disclosure, collagen or poly (N-isopropylacrylamide) having a lower limit critical solution temperature near the body temperature can be preferably used. For example, when an acidic collagen solution is neutralized and then heated, a plurality of collagen molecules are displaced by 65 nm to form fibrosis and form a collagen gel. Taking advantage of gel formation by heating, by mixing a liquid collagen solution with cells and medium and then adding it to various culture equipment, tertiary in the collagen gel and on the collagen gel according to the shape of the culture equipment. Original culture becomes possible.

In the culture medium of the present disclosure, when a crosslinked product of glycosaminoglycan and laminin or the laminin fragment (hereinafter, also referred to as a G-laminin crosslinked product for convenience) is used as the laminin crosslinked product, G for collagen is used. -The blending ratio of the laminin crosslinked product is not particularly limited. It is 0.01 to 500 parts by weight, more preferably 0.1 to 100 parts by weight, and particularly preferably 10 to 50 parts by weight with respect to 100 parts by weight of collagen. Within this range, the solution of the mixture of G-laminin crosslinked product and collagen can maintain a gel state under neutral and heating conditions at a temperature of 20 to 45 ° C. Since the gel strength differs depending on the collagen concentration, a collagen concentration suitable for cell culture can be selected within the above range. Other components that can be used for cell culture, such as gelatin, fibronectin, vitronectin, proteoglycan, tenascin, elastin, heparin, laminin, fibrinogen (fibrin), alginate, thrombin, xanthan gum, etc., are added to this culture medium. May be good.

On the other hand, when a crosslinked product of collagen and laminin or the laminin fragment (hereinafter, also referred to as a Col-laminin crosslinked product for convenience) is used as the laminin crosslinked product, the blending ratio of the Col-laminin crosslinked product to collagen is particularly high. There is no limit. In any range, the Col-laminin crosslinked product also gels by neutralization and heating, so that the blending amount of the laminin crosslinked product and collagen suitable for cell culture can be selected in the above range. To this culture medium, all or part of other components that can be used for cell culture, such as hyaluronic acid, gelatin, fibronectin, vitronectin, proteoglycan, tenascin, elastin, laminin, fibrinogen (fibrin), etc., are added. You may.

The culture medium of the present disclosure contains G-laminin cross-linked product and / or Col-laminin cross-linked product and collagen. Such a culture medium can be prepared, for example, by dissolving or dispersing laminin crosslinked product and collagen in a solvent, respectively, and mixing them so that the blending ratio of collagen and laminin crosslinked product is within the above range. .. Examples of such a solvent include water, acetic acid, hydrochloric acid, PBS, physiological saline, trishydroxymethylaminomethane or physiological saline having a neutral pH of 4- (2-hydroxyethyl) -1-piperazine ethanesulphonic acid. There is. In particular, when collagen is dispersed in water but not dissolved, acetic acid or the like may be added to adjust the pH to 2 to 5. The concentration of the laminin crosslinked product when dissolved in the solvent is 0.000000001 to 0.003 g / mL, preferably 0.000001 to 0.003 g / mL, and more preferably 0.000004 to 0.001 g / mL. The culture medium thus obtained is liquid at a temperature of 1 to 10 ° C. The collagen concentration contained in the culture medium is 0.00005 to 0.005 g / mL, preferably 0.0001 to 0.003 g / mL, and more preferably 0.0004 to 0.001 g / mL.

The culture medium is a mixture of G-laminin crosslinked product and / or Col-laminin crosslinked product and collagen, and a pH adjuster such as Tris-HCl, sodium hydroxide, or potassium hydroxide is further added thereto. The pH can be adjusted to 6.5 to 8.5 and heated to a temperature of 20 to 45 ° C. By neutralization and heating, collagen molecules associate in the mixture to become fibrous and gel-like. The laminin crosslinked product contained in the mixture uniformly disperses laminin and the laminin fragment in the gel. Therefore, three-dimensional culture can be performed by seeding, embedding, and suspending cells and tissues in this gel. In particular, pluripotent stem cells, somatic stem cells, or tissues containing these cells can be stably cultured in three dimensions for a long period of time. The concentration of laminin contained in the culture medium or the laminin fragment having a partial structure of the laminin can be appropriately selected depending on the cells and tissues used. For example, when performing three-dimensional culture of developing kidney, as shown in the examples described later, if the content is 4 μg / mL or more in the medium, the number of ureteral buds and the ureteral bud branching area should be significantly increased as compared with Matrigel. Can be done. It is preferably 1.5 to 100 μg / mL.

In the culture medium of the present disclosure, the collagen concentration of the mixed solution of collagen and the laminin crosslinked product is preferably 0.00005 to 0.005 g / mL regardless of the type of the laminin crosslinked product. This is because when neutralized and heated thereafter, collagen is fibrotic to form a gel, and the gel state can be maintained.

In the culture medium of the present disclosure, as shown in Examples described later, a gel is prepared and cells obtained from renal tissue are dispersed in the gel, or an aggregate of cells is embedded and cultured, or cells are placed on the gel. It was found that when the cells were dispersed and cultured on the gel, the cells formed a cell aggregate and differentiated to form a vascular structure and a glomerular structure three-dimensionally in the gel by any of the culture methods. Therefore, it is effective for the formation of organoids such as renal organoids.

The third of the present disclosure is a three-dimensional culture kit containing the culture medium and a pH adjuster. The culture medium in the kit of the present disclosure may be one in which the laminin crosslinked product and collagen are stored in separate containers, or a mixture of the laminin crosslinked product and collagen and stored in one container. good. Further, the laminin crosslinked product and collagen may be dried products, respectively, and may be in the form of a solution or a dispersed liquid in which the laminin crosslinked product and collagen are dissolved or dispersed in a solvent.

As the pH adjuster included in the 3D culture kit, a pH adjuster that can be added to a mixture containing a laminin crosslinked product and collagen to adjust the pH to 6.5 to 8.5 can be widely used. For example, a solution in which Tris-HCl, sodium hydroxide, and potassium hydroxide are dissolved in water or the solvent can be exemplified.

As a culture medium, prepare a solution containing collagen and a laminin crosslinked product, and add a pH adjuster to the solution to adjust the pH of the solution to pH 6.5 to 8.5. Since this solution becomes a gel at a temperature of 20 to 45 ° C., it can be used for three-dimensional culture.

The three-dimensional culture kit of the present disclosure may further contain these solvents when the laminin crosslinked product and / or collagen is provided as a dried product. As such a solvent, physiological saline having a neutral pH of water, acetic acid, hydrochloric acid, PBS, physiological saline, trishydroxymethylaminomethane or 4- (2-hydroxyethyl) -1-piperazine ethanesulphonic acid. There is water and so on.

Further, the three-dimensional culture kit of the present disclosure may include a culture solution for cell culture. Examples of such a culture solution include DMEM, EMEM, IMDM, F12K, RPMI, etc., which can be appropriately selected according to the cells and tissues to be cultured.

The fourth of the present disclosure is a culture substrate, characterized in that the laminin crosslinked product is coated together with collagen and / or gelatin. Since the laminin crosslinked product and collagen and / or gelatin are coated on the culture vessel, the culture operation can be easily performed. When temperature-responsive collagen is used in the culture vessel, three-dimensional culture is possible, but the culture substrate of the present disclosure is not limited to this, and even if it is intended for two-dimensional culture. good.

As the culture container, a glass or plastic petri dish, a flask, a multi-well plate, a culture slide, a microcarrier, a polymer film such as a polyvinylidene fluoride film, a test tube or other culture container can be used.

The collagen and / or gelatin used for coating the culture substrate is not particularly limited, and known collagen and / or gelatin used for cell culture applications can be preferably used. When used for cell culture for regenerative medicine, it is preferable to use collagen and / or gelatin whose safety for medical use has been confirmed, and it is preferable to use collagen and / or gelatin derived from humans. Collagen and / or gelatin whose safety has been confirmed for medical use includes atelocollagen (Koken), porcine skin collagen solution (Nipponham), Nippi high-grade gelatin (Nippi Co., Ltd.), and Medigelatin (Nippi Co., Ltd.). ) And so on. When such collagen is neutral and has a temperature responsiveness that makes it gel-like by heating, it is heated to a temperature of 20 to 45 ° C. to gel it, and cells are embedded or suspended in the gel and cultured. Three-dimensional culture can be performed in liquid.

The method for preparing the culture substrate of the present disclosure can be prepared, for example, by applying a mixed solution of a laminin crosslinked product and collagen and / or gelatin to a culture vessel. Such coating can be performed by a conventionally known method. The coating may be a step of placing the mixed solution on the culture vessel. On the other hand, when collagen has temperature responsiveness, the mixed solution may be adjusted to pH 6.5 to 8.5 and coated at a low temperature of 1 to 10 ° C. Since the culture substrate thus prepared gels when heated to a temperature of 20 to 45 ° C., it can be suitably used for three-dimensional culture.

On the other hand, the laminin crosslinked product and collagen and / or gelatin may be separately applied to serve as a culture medium. For example, a collagen or gelatin solution is applied to the culture vessel in advance, and then a solution of the laminin crosslinked product is applied to the formed film. The coating order may be reversed. When collagen has temperature responsiveness, a collagen solution having a pH of 6.5 to 8.5 is applied to the culture vessel in advance, and the mixture is heated to a temperature of 20 to 45 ° C. to gel. Next, a solution of the laminin crosslinked product may be applied onto the gel-like product. When the laminin crosslinked product is a Col-laminin crosslinked product, the pH of the Col-laminin crosslinked product may be adjusted to 6.5 to 8.5 after being dissolved in a solvent, and the coating may be applied at a low temperature of 1 to 10 ° C. Since the culture vessel prepared in this way gels when heated to a temperature of 20 to 45 ° C., it can be used for three-dimensional culture.

Suitable cells for culturing with the three-dimensional culture kit are mammalian cells such as humans, mice, rats, cows, and pigs, and epithelial cells, endothelial cells, myocardial cells, smooth muscle cells, skeletal muscle cells, and muscles. Cells that are in contact with the basal membrane in vivo, such as satellite cells, nerve schwan cells, fat cells, and pluripotent stem cells; somatics such as nerve stem cells, mesenchymal stem cells, hematopoietic stem cells, heart stem cells, liver stem cells, and small intestinal stem cells. Stem cells: ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), mGS cells (pluripotent reproductive stem cells), pluripotent stem cells such as fusion cells of ES cells and somatic cells, including these cells Suitable for culturing tissues. The cells may be cancer cells, fusion cells, or the like, in addition to normal cells. The three-dimensional culture kit is not limited to the above cells, and can be suitably used for culturing tissues containing these cells.

Fifth of the present disclosure is a composition containing the laminin crosslinked product and collagen and / or gelatin. The shape of this composition may be powder, granules, other granules, liquid dissolved or dispersed in a solvent, or semi-solid gel.
In the case of granularity, the laminin crosslinked product is previously dried and pulverized to obtain a granular laminin crosslinked product, which is mixed with powder or other granular collagen and / or gelatin, respectively, and appropriately pulverized, granulated, etc. to granulate. Can be molded into. These can be dissolved or dispersed in a solvent or the like at the time of use and used for cell culture or the like. On the other hand, the laminin crosslinked product, collagen and / or gelatin can be dissolved or dispersed in a solvent, respectively, and mixed to obtain a liquid composition. Further, a laminin crosslinked product or a gel-like product mixed with collagen can be prepared.

The blending ratio of the laminin crosslinked product, collagen and / or gelatin in this composition is not particularly limited. The laminin crosslinked product contains a laminin fragment and can be used in place of a conventionally known laminin fragment, collagen can be used as a culture medium, and gelatin can be used as a nutritional component during cell culture. can. In the composition of the present disclosure, it can be blended within a range in which a desired effect is exhibited, depending on the intended use.

When the composition of the present disclosure is granular in shape, for example, it can be used as one component to be added to the culture medium at the time of cell culture.
When the composition of the present disclosure is liquid, it can be applied to a culture vessel and used as a culture substrate. Examples of such a solvent include water, acetic acid, hydrochloric acid, PBS, physiological saline, trishydroxymethylaminomethane or physiological saline having a neutral pH of 4- (2-hydroxyethyl) -1-piperazine ethanesulphonic acid. There is.
When the composition of the present disclosure is in the form of a gel, it can be used for cell or tissue culture in the form of a gel. However, the use of the composition of the present disclosure is not limited to cell and tissue culture, and can also be used for hemostasis, wound treatment members, and the like.

The compositions of the present disclosure further include natural polymers such as fibrin, hyaluronic acid, alginic acid, starch, chitin, pectic acid, co-organizing peptides with self-assembling ability, polylactic acid, polyglycolic acid, lactic acid and glycol. Copolymer with acid, poly-ε-caprolactone, copolymer of ε-caprolactone with lactic acid or glycolic acid, polycitrate, polyapple acid, poly-α-cyanoacrylate, poly-β-hydroxybutyric acid, polytrimethylene Synthetic polymers such as oxalate, polytetramethylene oxalate, polypropylene carbonate, poly-γ-benzyl-L-glutamate, poly-γ-methyl-L-glutamate, poly-L-alanine, hydroxyapatite, tricalcium phosphate Inorganic materials such as, etc. may be blended.

The sixth of the present disclosure is a structure composed of the laminin crosslinked product and collagen and / or gelatin. In the present disclosure, the "structure" means a solid material having a predetermined shape. Such predetermined shapes include three-dimensional structures such as sponge-like, film-like, mesh-like, non-woven fabric-like, and knitted cloth-like. For example, a laminin crosslinked product and collagen and / or gelatin can be dissolved in a solvent to prepare a liquid, which can be freeze-dried to prepare a sponge. When collagen has temperature responsiveness, a mixed solution of a laminin crosslinked product and collagen is heated to a pH of 6.5 to 8.5 and a temperature of 20 to 45 ° C. to prepare a gel, and the gel is prepared. The product can be lyophilized and prepared into a sponge shape. Further, the laminin crosslinked product and collagen and / or gelatin are mixed and coated on a flat surface and dried to prepare a film or sheet, or the laminin crosslinked product and collagen and / or gelatin are separated from each other. It may be prepared in the form of a two-layer film or sheet by dissolving it in a solvent, coating it on a flat surface, and drying it. Further, a laminin crosslinked product and a product dissolved in a solvent separately from collagen and / or gelatin can be discharged into a fibrous form in ethanol or other solvent to prepare a mesh, non-woven fabric, or knitted cloth.

When the structure of the present disclosure is spongy, a sponge containing a laminin crosslinked product and collagen and / or gelatin is provided. Such structures can be used as an alternative to areas where collagen or collagen sponge has traditionally been used in bone grafts and the like. Since the laminin crosslinked product is uniformly dispersed in the structure of the present disclosure, it can be particularly preferably used as a scaffold for pluripotent stem cells, somatic stem cells, and tissues containing them in three-dimensional culture. Furthermore, since the structure of the present disclosure functions as a scaffold for cell culture when embedded in a living body, it can also be used as a tissue regeneration induction device.

The seventh of the present disclosure is a method for culturing cells or tissues, which comprises culturing in the presence of the laminin crosslinked product. The laminin cross-linked product is a product obtained by cross-linking laminin or the laminin fragment to glycosaminoglycan or collagen with a cross-linking agent. Since glycosaminoglycan and collagen are used as a culture medium, they can be used not only for cell culture but also for tissue culture as long as they are three-dimensional culture. Further, the culture is not limited to the three-dimensional culture, and may be a two-dimensional culture. For example, a culture vessel coated with a laminin crosslinked product can be prepared, mammalian cells can be seeded, and the cells can be cultured in a suitable culture medium. The mammal is not particularly limited, and examples thereof include humans, mice, rats, cows, and pigs. Of these, humans are preferable.

The culturing method broadly includes culturing using a medium substrate containing at least a laminin crosslinked product, and may be culturing on a gel, embedding culturing, solution culturing, or the like. In addition, when culturing, if a nutrient medium or basal medium suitable for the purpose of culturing is used, tissues can be formed from cultured cells as shown in Examples described later. As such a nutrient medium or a basal medium, a composition known in the art can be used.

The culturing method of the present disclosure is also suitable for culturing cells derived from mammals such as humans, mice, rats, cows and pigs. Such cells include normal cells derived from various organs, osteosarcoma, leukemia cells and other tumorigenic cells, recombinant cells, stem cells and the like. Furthermore, it can also be used to culture cells in contact with the basement membrane in vivo, such as epithelial cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, muscle satellite cells, nerve Schwann cells, adipocytes, and pluripotent stem cells. Suitable. In particular, it can be suitably used for culturing stem cells. Stem cells mean cells having self-renewal ability and pluripotency, and include somatic stem cells, pluripotent stem cells, and the like. Examples of somatic stem cells include neural stem cells, mesenchymal stem cells, hematopoietic stem cells, heart stem cells, liver stem cells, and small intestinal stem cells. Examples of pluripotent stem cells include ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), mGS cells (pluripotent reproductive stem cells), and fusion cells of ES cells and somatic cells. That is, the culturing method of the present disclosure is preferably used for culturing human stem cells. According to the culture method of the present disclosure, stem cells can be cultured three-dimensionally in the presence of laminin crosslinked products, thereby inducing differentiation of stem cells and efficiently constructing a three-dimensional tissue structure for regenerative medicine. can.

A three-dimensional culture substrate may be prepared using a laminin crosslinked product, and the cells or tissues may be embedded and cultured.

Next, the present disclosure will be specifically described with reference to examples, but these examples do not limit the present disclosure in any way.

(Example 1)
All the following operations were performed in ice at 4 ° C. To 1 ml of a 10 mg / ml hyaluronic acid aqueous solution (molecular weight 2 × 10 ⁶ ), 0.5 mL of 500 μg / mL laminin 511E8 (iMatrix-511 manufactured by Nippi Co., Ltd .; E8 fragment of human laminin 511) was added, and 2M Tris- 200 ul of HCl was added, 25 μl of 10 mM (millimol / L) genipin (manufactured by Fuji Film WAKO) was added thereto, and sterilized water was added to make a volumetric flask to obtain a mixed solution having a total volume of 5 mL. Then, a cross-linking reaction was carried out for 12 hours to obtain a hyaluronic acid-laminin cross-linked product (final concentration: laminin 511E8 50 μg / ml, hyaluronic acid 2 mg / ml, genipin 0.05 mM).

(Example 2)
All the following operations were performed on an ice container at 4 ° C. Pepsin treatment solubilized molecular weight 3 × 10 ⁵ of type I collagen (hereinafter referred to as PSC.) The collagen solution 1mL dispersed in 0.05M acetic acid such that the 3 mg / ml, in Tris-HCl buffer 2M The pH was adjusted to 7.5 and purified water was added to prepare a 1 mg / ml neutral collagen solution. To 5 ml of this neutral collagen solution, 1 ml of 10-fold concentrated DMEM basal medium and 1 mL of the hyaluronic acid-laminin crosslinked product obtained in Example 1 are added, and the final volume is 10 ml with purified water. -(Hyaluronic acid-laminin crosslinked product) mixture was obtained. 1 mL of this collagen- (hyaluronic acid-laminin crosslinked product) mixture was placed in a centrifuge tube with a conical bottom, allowed to stand at temperatures of 4 ° C, 10 ° C, 20 ° C, 35 ° C for 2 hours, and then inverted to the top. I observed it face to face. Further, as a control, the hyaluronic acid-laminin crosslinked product prepared in Example 1 was used, and after allowing it to stand at 35 ° C. for 2 hours in the same manner, it was inverted and observed with the bottom surface facing up. The results are shown in FIG. Reference numeral 1 is a control (35 ° C.), and reference numerals 2 to 5 are the results of a collagen- (hyaluronic acid-laminin crosslinked product) mixture at temperatures of 4 ° C., 10 ° C., 20 ° C., and 35 ° C., respectively. The collagen- (hyaluronic acid-laminin crosslinked product) mixture fell from the conical part at a temperature of 4 to 10 ° C., but remained gel-like at a temperature of 20 to 35 ° C. without falling from the conical part. The collagen-free hyaluronic acid-laminin crosslinked product fell from the conical portion even at a temperature of 35 ° C.

(Example 3)
200 μL (4 ° C.) of the collagen- (hyaluronic acid-laminin crosslinked product) mixture obtained in Example 2 was dropped onto a sterile circular cover glass having a diameter of 1 cm and placed in a petri dish having a diameter of 3.5 cm. When this was heated to a temperature of 37 ° C., it gelled after 1 hour. Further, 200 μL of Matrigel (manufactured by Corning Inc.) was used as a control, and gelation was carried out in the same manner.
Then, a fetus was removed from a female mouse on the 11.5th day of gestation to obtain renal tissue. This renal tissue was promptly embedded in the gel, 1 mL of the nutrient medium for renal tissue culture shown in Table 1 was added, and embedding culture was performed at a temperature of 37 ° C. for 5 days. After 5 days, the nutrient medium was removed, 1 mL of the basal medium for renal tissue culture shown in Table 2 was added, and the medium was cultured at a temperature of 37 ° C. for 7 days. In addition, regardless of which gel was used, the medium was exchanged once every two days. Tissues 7 days after culturing in the basal medium were stained with DBA (Dolichos Bifulorus Agglutinin; ureteral bud-specific stain) and WT-1 (nephron precursor marker) and observed with a phase-contrast microscope. The results are shown in FIG. As shown in FIG. 2, the renal tissue after 11.5 days of embryonic development was subjected to embedding culture in a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel, and the number of branches and elongation of ureteral buds (DBA staining) were higher than that of Matrigel. It was vigorous, and the formation of nephron precursor (WT-1 staining), which is a precursor tissue of glomeruli, was confirmed especially at the tip of the ureteral bud. 2b and 2f are images fluorescently observed with a Keyence microscope after DBA staining, and FIGS. 2c, 2g, 2d and 2h are images of frozen sections co-stained with DBA and WT1 and then observed with a confocal microscope. It is an image that was made.

(Example 4)
20 μg / ml, 60 μg / ml, and 180 μg / ml hyaluronic acid-laminin crosslinked products were prepared in the same manner as in Example 1 except that the amount of laminin 511E8 added was changed. Depending on the content of laminin 511E8, they are referred to as hyaluronic acid-laminin crosslinked product (20 μg), hyaluronic acid-laminin crosslinked product (60 μg), and hyaluronic acid-laminin crosslinked product (180 μg), respectively.

(Example 5)
A collagen- (hyaluronic acid-laminin crosslinked product) mixture was prepared in the same manner as in Example 2 except that the hyaluronic acid-laminin crosslinked product prepared in Example 4 was used. These were combined with a collagen- (hyaluronic acid-laminin crosslinked product 20 μg) mixture, a collagen- (hyaluronic acid-laminin crosslinked product 60 μg) mixture, and a collagen- (hyaluronic acid-laminin crosslinked product 180 μg) mixture, respectively, according to the laminin 511E8 content. Refer to. The final concentrations of laminin 511E8 contained in each collagen- (hyaluronic acid-laminin crosslinked product) mixture are 4 μg / ml, 12 μg / ml, and 36 μg / ml.

(Example 6)
200 μL (4 ° C.) of each collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared in Example 5 was dropped onto a sterile circular cover glass placed on a petri dish with a diameter of 3.5 cm and heated to a temperature of 37 ° C. It was gelled to give a mixture gel.
In addition, as a control, Matrigel (manufactured by Corning), a collagen solution adjusted to pH 7.5 with Tris-HCl buffer (manufactured by Nippi Co., Ltd., PSC, 0.5 mg / mL), and this collagen solution (0.5 mg / mL). and 2 mg / mL of molecular weight 2 × 10 ⁵ of hyaluronic acid solution 9: 1 mixture, to obtain a similar manipulation to gel.
The renal tissue obtained in the same manner as in Example 3 after 11.5 days of pregnancy was embedded in each gel and cultured, and the tissue on the 3rd day of culture was fixed with a whole mount, stained with DBA and WT-1, and then stained. Images were acquired with an all-in-one fluorescence microscope (BZ-X800, manufactured by KEYENCE). The images were analyzed on the same machine, and the number of ureteral bud branching (branching tip numbers) and the ureteral bud branching area (extended brunching area) were measured based on DBA staining. The results are shown in FIG. FIG. 3A shows the result of the number of ureteral bud branches, and FIG. 3B shows the result of the ureteral bud branch area. In FIG. 3, sample reference numeral 1 is Matrigel, reference numeral 2 is collagen solution (0.5 mg / mL), reference numeral 3 is collagen- (hyaluronic acid-laminin crosslinked product 0 μg), and reference numeral 4 is collagen- (hyaluronic acid-laminin). 20 μg of crosslinked product), reference numeral 5 indicates a collagen- (hyaluronic acid-laminin crosslinked product 60 μg), and reference numeral 6 indicates a collagen- (hyaluronic acid-laminin crosslinked product 180 μg) mixture. When t-test was performed on Matrigel, a statistically significant difference (p <0.05) was detected in all of the above collagen- (hyaluronic acid-laminin crosslinked products) mixtures.

(Example 7)
The renal tissue obtained in Example 3 was dispersed by Accutase. This dispersion was passed through a 40 μm filter to form single cells, and then centrifuged in a 1.5 mL Eppendorf tube at 400 × g for 2 minutes. Then, tap tube was centrifuged for 2 minutes at by mixing with inversion again 400 × g, and cell pellets were obtained (5 × 10 ⁵ cells).
An embedding culture was carried out for 7 days in the same manner as in Example 3 except that the cell pellet was embedded in the gel instead of the renal tissue. The medium was changed once every two days.
The tissue formed after 7 days of culturing was observed. The results are shown in FIG. The whole image of the tissue was observed in a bright field with a phase-contrast microscope (Fig. 4A). In addition, the tissue was immobilized with 4% PFA, and frozen sections were used for observation. Fluorescent staining was performed by PECAM-1 antibody staining (Fig. 4D) as a vascular marker, synaptopodin antibody staining (Fig. 4E) as a marker of glomerular epithelial cells, and cell nucleus staining (DAPI) (Fig. 4C). 4C, 4D, and 4E are images obtained by laser scanning the tissue. In addition, HE staining was performed separately. The results are shown in FIG. 4B. The white part of the bright-field image of FIG. 4A was a gel, and an aggregate of black cells was confirmed inside the gel. As shown in FIG. 4B, it was confirmed that vascular-like tissues (indicated by thin arrows) and glomerular-like structures (indicated by arrow heads) were present in the cell aggregate. FIG. 4C shows the cell nuclei of many cells contained in the cell aggregate, FIG. 4D shows the annular vascular structure (PECAM-1 staining), and FIG. 4E shows the glomerular structure (synaptopodyn staining). Aggregates of developing renal cell-derived cells collected from renal tissue 11.5 days after embryonic formation form vascular and glomerular structures by embedding culture in a collagen- (hyaluronic acid-laminin cross-linked product) mixture gel. It turned out to be.

(Example 8)
Preparation of hyaluronic acid-laminin crosslinked product:
Sterilized water 2.5 mL, 10 x PBS 0.5 mL, 10 mg / ml hyaluronic acid aqueous solution (molecular weight 1.2 x 10 ⁶ to 2.2 x 10 ⁶ ) 1 ml, 500 μg / mL laminin 511E8 (manufactured by Nippi Co., Ltd.) , IMatrix-511; E8 fragment of human laminin 511) mixed with 0.5 mL, 1.5 M glycine solution (in PBS) 0.125 ml, 10 mM genipin (manufactured by Fuji Film WAKO) solution 0.25 mL, and cross-linked reaction for 12 hours. Was done. 0.125 ml of 1.5 M glycine solution (in PBS) was added to the reaction product, and quenching was performed at 4 ° C. for 6 hours to obtain a hyaluronic acid-laminin crosslinked product.

Preparation of collagen- (hyaluronic acid-laminin crosslinked product) mixture:
Molecular weight 3 × 10 ⁵ of type I collagen (manufactured by Nippi trade name PSMC: pepsin-solubilized low endotoxin porcine type I collagen) of 2.5 mg / ml collagen solution in (PSMC in 0.05 M acetic acid) 4 mL, hyaluronic Add 1 ml of acid-laminin crosslinked product, 4 ml of 2.5 x DMEM (manufactured by Nippon Fisheries Co., Ltd., # 05919, L-glutamine, sodium hydrogencarbonate-free), and _{1 ml of 2.5% NaHCO 3} solution and mix to make collagen-. A mixture (hyaluronic acid-laminine crosslinked product) was obtained. This collagen- (hyaluronic acid-laminin crosslinked product) mixture was heated to a temperature of 37 ° C. and gelled.

The mixture gel was frozen to obtain frozen sections. Type I collagen antibody (without cross-reactivity with pig-derived type I collagen), hyaluronic acid-binding protein (HABP), and laminin 511E8 antibody (anti-HIS antibody) were used for this frozen section to form type I collagen, hyaluronic acid, Laminin 511E8 was stained and observed with a confocal microscope. The results are shown in FIG. FIG. 5A is a stained image using a type I collagen antibody, FIG. 5B is a HABP, and FIG. 5C is a stained image using a laminin 511E8 antibody. Each figure is a vertical cross section of the frozen gel, and the upper end faces the lower end of the gel surface to show the inside of the gel. As shown in FIGS. 5B and 5C, the inside of the gel is composed of a hyaluronic acid-laminin crosslinked product in which hyaluronic acid and laminin 511E8 are bound, and as shown in FIG. 5A, white fibers in which collagen molecules are associated with the gel surface. Was confirmed to exist. The uniformly mixed collagen and hyaluronic acid-laminin crosslinked product formed a heterogeneous product between the collagen fiber portion and the hyaluronic acid-laminin crosslinked product portion as the gelation occurred. This non-uniformity is similar to the localized separation between an interstitial portion having a collagen fiber portion as a microenvironment such as skin tissue and a basement membrane portion having a basement membrane structure such as laminin as a microenvironment. It was speculated that the collagen- (hyaluronic acid-laminin crosslinked product) mixture could utilize this localized separation to be used for cell organization in cell culture.

In addition, this mixture gel was fixed with 2% glutaraldehyde / PBS, dehydrated with ethanol, freeze-dried, platinum-deposited, and a scanning electron microscope image was taken. The results are shown in FIG. 5D. As a control, a scanning electron microscope image obtained by treating a mixture prepared in the same manner as the collagen- (hyaluronic acid-laminin crosslinked product) mixture in the same manner as above except that the hyaluronic acid-laminin crosslinked product was replaced with PBS is shown in FIG. 5E. Shown in. As shown in FIG. 5E, the control collagen had many linear fibers, but in the mixture gel shown in FIG. 5D, many fibers showing a bent structure were observed. Also, in the mixture gel of FIG. 5D, a denser network was observed as compared with the control of FIG. 5E.

(Example 9)
A hyaluronic acid-laminin crosslinked product was prepared by the method described in Example 8. Instead of the PSMC used in Example 8, a collagen solution obtained by blending collagen <1> and collagen <2> shown in Table 3 below at the ratios shown in Table 3 was added to the hyaluronic acid-laminin crosslinked product. A collagen- (hyaluronic acid-laminin crosslinked product) mixture was obtained. In the table, type I (PSC) means pepsin-solubilized type I collagen, type I (ASC) means acid-solubilized type I collagen, and type I (PSMC) means pepsin-solubilized low endotoxin type I collagen. In addition, as type III, Nippi Co., Ltd., trade name: bovine dermis-derived type III collagen (pepcin solubilized), and as type IV collagen, Nitta Gelatin, trade name: cellmatrix-type IV (pepcin extraction derived from bovine lens capsule). , Pepsin solubilized extract and purified product from porcine cornea was used as V-type collagen, and various collagen- (hyaluronic acid-laminin crosslinked products) mixtures were prepared.
300 μL of each of these mixtures is dispensed into 8-wells and reacted at 37 ° C. for 2 hours or more, and then zirconia beads (manufactured by Nikkato Corporation, diameter 1.5 mm) are placed on a gel and settled. It was determined whether or not. The results are shown in FIG. In the figure, the arrow heads indicate zirconia beads. As shown in FIG. 6, it was found that the zirconia beads did not settle in any of the mixtures and maintained the gelling ability.

(Example 10)
A hyaluronic acid-laminin uncrosslinked product was prepared in the same manner as in Example 8 except that the same amount of purified water was used instead of the cross-linking agent genipin, and collagen- (hyaluronic acid-laminin uncrosslinked product). A mixture was obtained. This collagen- (hyaluronic acid-laminin non-crosslinked product) mixture, the collagen- (hyaluronic acid-laminin cross-linked product) mixture prepared in Example 8, and Matrigel (manufactured by Corning) as a control were used.
100 μl of collagen- (hyaluronic acid-laminin uncrosslinked) mixture on 14 mm diameter glass in a glass bottom dish (Matsunami Glass Ind. # D11131H) with a diameter of 35 mm, collagen- (hyaluronic acid-laminin) prepared in Example 8. Crosslinked) The mixture or matrigel was spread over the surface of the dish and allowed to stand at 37 ° C. for 1 hour to prepare a gel bed.
Next, a fetus was removed from a female mouse on the 13th day of gestation to obtain a developing kidney. This was subjected to Accutase treatment to separate into cells, and then the dispersion was passed through a 40 μm filter to form single cells, which were then centrifuged at 1,200 rpm for 5 minutes to reaggregate the cells. Centrifuge this, suck the supernatant with a pipette, discard it, add the nutrient medium for renal cells shown in Table 4 to the pellet to 5 × 10 ⁶ cells / mL and suspend it, and obtain the cell suspension. Was dispensed into 10 centrifuge tubes and centrifuged at 1,200 rpm for 5 minutes to reaggregate the cells. Centrifugal tubes containing cells from which the supernatant was discarded were stored in ice.
To this centrifuge tube, each mixture or matrigel before gelation ^{was added to 5 × 10 5} cells / 200 μL drop, suspended well to form a cell resuspension, and 200 μl of each drop was added dropwise to each gel bed. , 37 ° C., allowed to stand in a _{CO 2 incubator for 1 hour.} After 1 hour, after each mixture and the like gelled, 1.5 ml of the nutrient medium for renal cells shown in Table 4 preheated at 37 ° C. was added dropwise from the outer periphery of the gel, and the cells were gelled in _{a CO 2 incubator at 37 ° C.} It was dispersed in the medium and embedded culture was performed. The medium was changed once every two days. Immediately after culturing, observation was performed with a phase-contrast microscope on the first day of culturing and the seventh day of culturing. In addition, on the 7th day of culture, formalin was fixed, stained with DBA (ureteral bud-specific stain), and fluorescently observed with a confocal microscope to evaluate ureteral formation. The results are shown in FIG.
Immediately after culturing, the cells were monodispersed in all the gels (FIGS. 7a, 7e, 7i). In the embedded culture of dispersed cells using a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel, a tendency for the cells to aggregate from 1 day after the culture was observed (Fig. 7b), and urinary tract branching was observed on the 7th day of the culture. Tissue was observed (Fig. 7c). When the 7th day of culture was stained with DBA, which is a ureter-specific marker, the branched structure of the ureter could be confirmed (Fig. 7d, white part).
On the other hand, in the embedding culture using the collagen- (hyaluronic acid-laminin uncrosslinked product) mixture gel, elongated cells were observed 7 days after the culture, but the ureteral structure could not be observed (Fig. 7g, Fig. 7h). ).
In the control Matrigel, cell aggregation was observed 7 days after culturing, but no elongated cells were observed and no ureter was formed (FIGS. 7k, 7l). It was a phenomenon specific to the collagen- (hyaluronic acid-laminin cross-linked product) mixture gel that the dispersed cells expanded after agglutination and led to the organization of the ureter. By blending the hyaluronic acid-laminin crosslinked product, a phenomenon that cannot be realized by the hyaluronic acid-laminin uncrosslinked product or the Matrigel culture was obtained.

(Example 11)
A collagen- (hyaluronic acid-laminin crosslinked product) mixture prepared in the same manner as in Example 8 and Matrigel as a control were used, and the mixture was heated to a temperature of 37 ° C. to gel. Next, the embryos were removed from female mice on the 13th day of pregnancy to obtain developing kidney-derived cells by operating in the same manner as in Example 10 except that DMEM / F12 containing 10% serum was used instead of the nutrient medium for renal cells. Then, the implant culture in which the cells were dispersed in the gel was performed. Cell concentration in the gel is 2.5 × ¹⁰ 6 cells / ml.
By culturing in a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel for 1 week, cell aggregates were confirmed in the gel by a phase-contrast microscope (Fig. 8a: low-magnification (20-fold) phase-contrast observation image). .. A partially enlarged view of FIG. 8a is FIG. 8b. In FIG. 8b, a black cell aggregate and a mesh-like fiber connecting the cell aggregate and the cell aggregate were observed. In addition, FIG. 8c is an image obtained by fluorescently staining the cell aggregate using DBA (ureter marker), which is a specific marker of the developing ureter, and imaging with a confocal microscope (Olympus FV1000), and the white part is an image. It is a DBA staining part.
In addition, the cell aggregates in this gel were fixed and stained with DBA and nephrin (glomerular marker). The merged image is shown in FIG. 8d, the DBA stained image is shown in FIG. 8e, and the nephrin stained image is shown in FIG. 8f. As shown in FIG. 8f, the inside of the cell assembly is stained with nephrin, and as shown in FIG. 8e, the outside of the cell assembly is stained with DBA, and this cell assembly is a precursor tissue of the glomerulus and the urinary tract. It turned out to be a fusion. By dispersing cells derived from the developing kidney in a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel and performing embedding culture for 1 week, a cell aggregate in which glomeruli and ureters are formed is formed. It turned out to be possible.

In the above culture, the embedded culture on the 3rd and 5th days of the culture was fixed, and the type I collagen antibody (without cross-reactivity with pig-derived type I collagen) and the type IV collagen antibody (with bovine-derived type IV collagen) were used. (No crossing), PECAM-1 (vascular marker), Nephrin antibody (nephron epithelial marker), DAPI staining (cell nucleus), and fluorescence observation with a confocal microscope. The results are shown in FIG. The arrows in FIGS. 9a, 9b, 9c, and 9d are areas where DAPI-stained cell nuclei are clustered. By embedding culture, cell aggregates were observed on the 3rd day of culture in both collagen- (hyaluronic acid-laminin crosslinked product) mixture gel and matrigel. Here, type I collagen and type IV collagen were observed inside the cell aggregate on the third day of culture with a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel (Fig. 9e, Fig. 9i), but Matrigel. Type I collagen could not be confirmed in (Fig. 9f, Fig. 9j). In addition, in the collagen- (hyaluronic acid-laminin crosslinked product) mixture gel, staining with PECAM-1 and Nephrin was confirmed inside the cell aggregate on the 5th day of embedding culture (Fig. 9g, Fig. 9k). No staining with PECAM-1 was observed in Matrigel (Fig. 9h, Fig. 9l). When dispersed cells are embedded and cultured using a collagen- (hyaluronic acid-laminin crosslinked product) mixture gel, the cells aggregate to form agglomerates, and at that time, I, which is an stromal component inside the cell aggregate. It was speculated that the type collagen was encapsulated and organized while separating from the basal membrane type IV collagen, and as a result, the tissue had blood vessels and epithelium.

(Example 12)
Hyaluronic acid with a molecular weight of 5 × 10 ⁴ to 1.1 × 10 ⁵ and 2 × 10 ⁵ to 6 × 10 ⁵ were used instead of hyaluronic acid with a molecular weight of 1.2 × 10 ⁶ to 2.2 × 10 ^6. Prepared a hyaluronic acid-laminin crosslinked product and obtained a collagen- (hyaluronic acid-laminin crosslinked product) mixture in the same manner as in Example 8. A total of 3 types of collagen- (hyaluronic acid-laminin crosslinked product) mixture obtained by using molecular weights of 1.2 × 10 ⁶ to 2.2 × 10 ^{6 were used for these and operated in the same manner as in Example 10.} The dispersed cells derived from developing renal cells were embedded and cultured. On the 7th day of culture, formalin was fixed, stained with DBA (ureteral bud-specific stain), and fluorescently observed with a confocal microscope to evaluate ureteral formation. The results are shown in FIG. It was observed that developing renal cells formed a ureteral branch structure inside each gel. The formation of ureteral branch structure increased with the increase in the molecular weight of hyaluronic acid.

(Example 13)
Instead of the total amount of type I collagen, type I collagen mixed with type IV collagen (manufactured by Nitta Gelatin Co., Ltd., trade name cellmatic-type IV (extracted pepsin derived from bovine lens capsule)) is used at a ratio of 1: 0.6. A collagen- (hyaluronic acid-laminin crosslinked product) mixture was obtained by the same procedure as in Example 8. This is a type I + type IV mixture. Further, a collagen- (hyaluronic acid-laminin crosslinked product) mixture using only type I collagen prepared in Example 8 is used as a type I mixture. Type I + IV type mixture was stored on ice on a glass diameter 14mm in the same glass bottom dish as used in Example 10, I-type mixture, or Matrigel was 100μm dropwise as a control, 37 ° C., in a C0 ₂ incubator The mixture was allowed to stand for 1 hour to gel.

Instead of the collagen- (hyaluronic acid-laminin cross-linked product) mixture, the type I + type IV mixture, type I mixture or matrigel prepared above was used and operated in the same manner as in Example 10 to obtain cells derived from developing kidney cells. cell resuspension ⁽¹⁰ 5 cells / ^100μl) was obtained. The cell re-suspension 100μl were pipetted on the upper surface of the gel, 37 ° C., for 2 hours left in the C0 ₂ incubator. Thereafter, the DMEM / F12 medium + 10% FBS were prewarmed at 37 ° C. and 1.5ml dropwise from the outer periphery of the gel, 37 ° C., the cells were dispersed was subjected to gel the culture in the C0 ₂ incubator. In addition, the culture was carried out for 3 days by using DMEM / F12 medium + 10% FBS instead of the above-mentioned nutrient medium for renal cells and exchanging the medium once every 2 days.
The cells were fixed on the 3rd day of culture, stained with DAPI, type IV collagen antibody (without cross-reactivity with bovine type IV collagen), and stained with phalloidin to stain the cytoskeleton, and observed with a confocal microscope. The results are shown in FIG.
In the control Matrigel, the cells dispersed by culturing on the gel for 3 days formed a cell aggregate, and type IV collagen was formed outside the cell aggregate (Fig. 11i). In the type I mixture and the type I + type IV mixture, the cells dispersed by culturing on the gel for 3 days formed a cell aggregate, but unlike Matrigel, type IV collagen was formed inside the cell aggregate. (Fig. 11c, Fig. 11f). In addition, in the type I mixture gel culture, cytoskeleton formation aggregated at the cell margin resulting from the interaction between cells associated with cell assembly was observed (Fig. 11b), and the type I + IV mixture was cultured on the gel. Then, the cells were elongated while forming a strong cytoskeleton (stress fiber formation) (Fig. 11e). In the cell culture of the control Matrigel, the cytoskeleton was observed inside the cells, but unlike the type I + type IV mixture, the stress fiber formation was not aggregated, and the cells were totally diffused inside the cells. (Fig. 11h). It was considered that the localization of type IV collagen, cell behavior, and cell organization are linked to induce different cell organization for each substrate.

(Example 14)
Instead of type IV collagen, type III collagen (manufactured by Nippi Co., Ltd., trade name: bovine dermis-derived type III collagen (pepsin solubilized)) and type V collagen (pepcin solubilized extract and purification from porcine cornea) were used. The same procedure as in Example 13 was carried out to obtain a collagen- (hyaluronic acid-laminin crosslinked product) mixture. This is used as an I + III type mixture and an I + V type mixture, respectively, and a collagen- (hyaluronic acid-laminin crosslinked product) mixture using only type I collagen prepared in Example 8 is used as a type I mixture.
The procedure was the same as in Example 10 except that the type I mixture, the type I + type III mixture, and the type I + type V mixture were used to obtain a cell resuspension of cells derived from the developing kidney, and the cells in the gel. Was dispersed and embedded culture was performed.
The phase difference observation image on the 7th day of culture is shown in FIG. In the embedding culture in which cells were dispersed with a type I mixture gel, it was observed that the gel contracted after 7 days of culture, a tissue-like structure was formed inside, and a ureteral branch was formed (Fig. 12a, FIG. Black mesh structure). In the type I + type III mixture gel and the type I + V type mixture gel, denser ureteral branching was observed in the gel (FIGS. 12b, 12c, black mesh). In addition, the culture of the type I + type V mixture gel was fixed-stained with holes, stained with Dorichos Biflorus, which is a ureter-specific marker, and fluorescently observed with a fluorescence microscope (Kieens, BZ-X800). It was observed that the renal cells promoted ureteral branching inside (Fig. 12d, white mesh).

Further, the operation was carried out in the same manner as in Example 10 except that the type I mixture gel and the type I + IV mixture prepared in Example 13 were used, and DMEM / F12 medium + 10% FBS was used instead of the nutrient medium for renal cells. A cell resuspension of cells derived from the developing kidney was obtained, and the cells were dispersed in a type I mixture gel or an I + IV type mixture gel and subjected to embedding culture.
The gel on the 10th day of culture using the type I mixture gel and the type I + IV mixture gel was fixed with formalin, stained with an antibody of nephrin, which is a renal epithelial cell marker, and PECAM-1 which is a vascular marker, and observed with a confocal microscope. .. The results are shown in FIG. In each of the mixture gels, the cells were assembled and organized on the 10th day of the embedding culture in which the cells were dispersed, and ureters and glomeruli were formed (FIGS. 13-b, f). In particular, in the I + IV type mixture gel, cell aggregates stained with the vascular marker PECAM-1 were concentratedly localized at the tip of the nephron structure (Fig. 13-g) stained with the renal epithelial cell marker nephrin (Fig. 13-g). FIG. 13-h).

(Example 15)
Laminin 332E8 (Nippi Co., Ltd., E8 fragment of Hitoraminin 322), Laminin 111E8 (Nippi Co., Ltd., E8 fragment of Hitoraminin 111) instead of Laminin 511E8 (Nippi Co., Ltd., iMatrix-511; E8 fragment of Hitoraminin 511) A collagen- (hyaluronic acid-laminin crosslinked product) mixture was prepared in the same manner as in Example 8 except that the above was used. These are referred to as 511 mixture, 332 mixture and 111 mixture, respectively. In addition, Matrigel (manufactured by Corning Inc.) was used as a control, and gelation was carried out in the same manner as in Example 8.
Then, the fetus was removed from the female mouse on the 13th day of gestation to obtain the developing liver tissue and the developing intestinal tissue. The developing liver tissue and the developing intestinal tissue were each dispersed by Accutase, and the dispersion was passed through a 40 μm filter to form a single cell. Instead of the above-mentioned nutrient medium for renal cells, the nutrient medium for hepatic cells shown in Table 5 below is used for culturing cells derived from developing hepatic tissue, and the cells derived from developing intestinal tissue are used for intestinal cells shown in Table 6. The cells were dispersed in the gel and embedded culture was carried out in the same manner as in Example 10 except that the nutrient medium was used.

On the 7th day of culture, the gel was fixed, stained with antibodies of CPSI, which is a mature liver marker, and CDX2, which is a mature intestinal marker, and fluorescence observation was performed. The results are shown in FIG.
In the embedding culture of developing hepatocytes, hepatocyte cell aggregates were formed in all of the 511 mixture, 332 mixture, 111 mixture, and Matrigel (arrows in FIGS. 14a, 14b, 14c, and 14d). In addition, cell aggregates stained with mature liver markers were observed in the 511 mixture, 332 mixture, and Matrigel (white in FIGS. 12e, 12f, and 12h). On the other hand, in the 111 mixture, the liver marker was negative, but organization into a tubular structure was observed (arrowhead in FIG. 14c).
In addition, in the embedding culture of developing intestinal cells, cell aggregates were observed on each substrate (arrows in FIGS. 14i, 14j, 14k and 14l). These cell aggregates were stained with the intestinal marker CDX-2 (white in FIGS. 14m, 14n, 14o and 14p). On the other hand, in the 111 mixture and Matrigel, radial tubes were formed from the cell aggregates, and the connections between the agglomerates were confirmed (arrowheads in FIGS. 12k and 12l), and the tubular structuring was particularly intense in the 111 mixture (FIG. 12k, arrowhead in FIG. 12l). FIG. 14k arrow head). It was suggested that the difference in laminin isoforms could provide a pericellular microenvironment suitable for organizing each cell, and could lead to various cell-specific organization.

(Example 16)
Collagen- (hyaluronic acid-laminine crosslinked product) mixture operated in the same manner as in Example 8 except that disuccinidiyl glutarate (DSG); Thermo Fisher # 20593 was used instead of genipin as a cross-linking agent. Was prepared, heated to 37 ° C., and gelled.
Next, a cell pellet of renal cells was obtained in the same manner as in Example 7 except that a female mouse on the 12th day of pregnancy was used, and embedding culture was performed. FIG. 15 shows a phase difference observation image on the 7th day of culture. As is clear from FIG. 12a, the mixture gel using DSG as a cross-linking agent was also able to grow ureteral branches in the same manner as when genipin was used.

The present disclosure allows for various embodiments and modifications without departing from the broad spirit and scope of the present disclosure. Further, the above-described embodiments and examples are for explaining the present disclosure, and do not limit the scope of the present disclosure. That is, the scope of the present disclosure is indicated by the claims, not by the embodiments and examples. And various modifications made within the scope of the claims and within the equivalent meaning of the disclosure are considered to be within the scope of the present disclosure.

This application is based on Japanese Patent Application No. 2020-035152, which was filed on March 2, 2020. The specification, claims, and the entire drawing of Japanese Patent Application No. 2020-035152 shall be incorporated into this specification as a reference.

Claims

A laminin crosslinked product in which collagen and / or glycosaminoglycans having a molecular weight of 1 × 10 4 to 5 × 10 6 are bound to laminin or a laminin fragment having a partial structure of the laminin by a cross-linking agent.
The laminin crosslinked product according to claim 1, wherein the laminin fragment is a laminin fragment having an integrin-binding activity of the laminin.
The laminin crosslinked product according to claim 1 or 2, wherein the glycosaminoglycan is hyaluronic acid or chondroitin.
Claims 1 to 3 wherein the laminin is any one or more of laminin 511, laminin 332, laminin 111, laminin 121, laminin 211, laminin 221 and laminin 311, laminin 321 and laminin 411, laminin 421, or laminin 521. The laminin crosslinked product described in any of the above.
The laminin crosslinked product according to any one of claims 1 to 4, wherein the laminin fragment is a laminin E8 fragment.
A culture medium containing the laminin crosslinked product according to any one of claims 1 to 5 and a temperature-responsive substrate that remains in a solution state at 10 ° C. or lower and becomes a gel at 20 to 45 ° C.
The culture medium according to claim 6, wherein the temperature-responsive substrate is collagen.
A three-dimensional culture kit containing the culture medium according to claim 6 or 7 and a pH adjuster.
The three-dimensional culture kit according to claim 8, further containing a culture solution.
A culture substrate, wherein the laminin crosslinked product according to any one of claims 1 to 5 is coated with collagen and / or gelatin.
A composition containing the laminin crosslinked product according to any one of claims 1 to 5 and collagen and / or gelatin.
A structure composed of the laminin crosslinked product according to any one of claims 1 to 5 and collagen and / or gelatin.
A method for culturing cells or tissues, which comprises culturing in the presence of the laminin crosslinked product according to any one of claims 1 to 5.
A three-dimensional culture method for cells or tissues, which comprises burying cells or tissues in the culture medium according to claim 6 or 7 and culturing the cells or tissues.
The culture method according to claim 13 or 14, wherein the cells are pluripotent stem cells or somatic stem cells.