JP2019137639A - Collagen structure - Google Patents

Abstract

To provide a novel structure collagen structure for solving problems for example, in prior cell culture in vitro of PHHs using a collagen coat, a liver function and viability of PHHs are deteriorated for a few days, so that application to a middle/long-term drug development related test, namely, there is a problem in which a function of PHHs cannot be maintained for a few days.SOLUTION: The invention found that, a collagen structure comprising a collagen fibril having a dimple on a surface thereof can solve the above-mentioned problem.SELECTED DRAWING: None

Description

  The present invention relates to a novel collagen structure, a collagen carrier containing the collagen structure, and a cell culture method using the carrier.

(collagen)
Collagen is an extracellular matrix (ECM) generally used for culturing human primary cultured hepatocytes (PHHs) and the like.
Conventional two-dimensional culture of hepatocytes is a simple and most common culture method (Non-patent Document 2). Conventional collagen coats (such as culture vessels coated with collagen on the surface) in the culture of PHHs are cultured with a collagen density of the order of microgram / cm ² .

(Hepatocytes)
Human primary cultured hepatocytes are widely used in efficacy and toxicity tests such as hepatic drug metabolism, clearance, drug interaction, transporter activity, hepatotoxicity, and liver regeneration studies of candidate drugs in drug discovery ( Non-patent documents 1 to 3).
Primary cultured hepatocytes cultured on conventional collagen coats are a culture model suitable for up to about 1 day (at 24 hours) because of their rapid loss of function, but are not suitable for medium- to long-term toxicity tests (non-patented) Reference 4).

(Hepatocyte-like cells)
Artificial pluripotent stem (iPS) cells or embryonic stem (ES) cell-derived hepatocyte-like cells {HLC (s)} are promising for use in drug tests and the like because of their proliferation ability and differentiation ability. However, HLCs have an immature phenotype and do not necessarily exhibit functions similar to PHHs (Non-patent Documents 5 and 6).

(Prior Patent Literature)
A collagen-coated cell culture vessel characterized by polymerizing collagen fibers by applying a collagen solution of a predetermined concentration to an arbitrary culture vessel and then allowing it to stand on an inclined surface with a predetermined angle has been reported. (Patent Document 1). However, the collagen-coated cell culture container described in Patent Document 1 is clearly different in structure from the collagen structure of the present invention.
A collagen-coated cell incubator has been reported in which a collagen layer is formed on the surface of a substrate having an amino group, and the amino group and collagen are chemically bonded (Patent Document 2). However, the collagen-coated cell culture container described in Patent Document 2 is clearly different in structure from the collagen structure of the present invention.
A collagen-coated carrier characterized in that at least a part of the surface of a carrier composed of a calcium phosphate compound at least near the surface is coated with the collagen via a protein having a high affinity for collagen has been reported. (Patent Document 3). However, the collagen-coated carrier described in Patent Document 3 is clearly different in structure from the collagen structure of the present invention.

JP2002-142752 JP-A-8-168372 JP 2006-325467 A

Godoy, P. et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch. Toxicol. 87, (2013). Zeilinger, K., Freyer, N., Damm, G., Seehofer, D. & Kn ö spel, F. Cell sources for in vitro human liver cell culture models.Exp. Biol. Med. 241, 1684-1698 (2016 ). Fraczek, J., Bolleyn, J., Vanhaecke, T., Rogiers, V. & Vinken, M. Primary hepatocyte cultures forpharmaco-toxicological studies: At the busy crossroad of variousanti-dedifferentiation strategies. Arch. Toxicol. 87, (2013 ). Beigel, J., Fella, K., Kramer, P.-J., Kroeger, M. & Hewitt, P. Genomics and proteomics analysis of cultured primary rat hepatocytes. Toxicol. In Vitro 22, 171-181 (2008). Godoy, P. et al. Gene networks and transcription factormotifs defining the differentiation of stem cells into hepatocyte-like cells. J. Hepatol. 63, 934-942 (2015). Baxter, M. et al. Phenotypic and functional analyzes showstem cell-derived hepatocyte-like cells better mimic fetal rather than adulthepatocytes. J. Hepatol. 62, 581-589 (2015).

In vitro cell culture of PHHs using a conventional collagen coat reduces the liver function and viability of PHHs within a few days, and therefore is limited to medium- to long-term drug discovery-related tests. That is, there is a problem that the function of PHHs cannot be maintained for several days.
An object of the present invention is to provide a collagen structure having a novel structure that solves the above problems.

  As a result of researches to solve the above problems, the present inventors have found that a collagen structure containing collagen fibrils having dimples on the surface (hereinafter sometimes referred to as “collagen structure of the present invention”) The present inventors have found that the problem can be solved and have completed the present invention.

That is, the present invention is as follows.
1. A collagen structure containing collagen fibrils having dimples on the surface.
2. 2. The collagen structure according to item 1, wherein the dimple on the surface is a hollow space formed by overlapping a plurality of collagen fibrils.
3. 3. The collagen structure according to item 1 or 2, wherein the dimple has 1 to 4,000 per 475 μm ^{2 of} surface.
4). 4. The collagen structure according to any one of items 1 to 3, wherein the number of motifs per surface 475 μm ² is 4,377 or less.
5). 5. The collagen structure according to item 4 above, wherein the average area of the motif is 0.12 μm ² or more.
6). 6. The collagen structure according to any one of 1 to 5 above, wherein the surface has a random orientation.
7). 7. The collagen structure according to any one of items 1 to 6, wherein a space is formed between a plurality of collagen fibrils forming the collagen structure.
8). The collagen structure according to any one of the preceding items 1 to 7, wherein the plurality of collagen fibrils forming the collagen structure are randomly oriented.
9. The collagen according to any one of the preceding items 1 to 8, wherein the collagen fibril is atelocollagen fibril.
10. 10. The collagen structure according to any one of items 1 to 9, wherein a collagen density of the collagen structure is 0.21 mg / cm ² or more.
11. A collagen carrier comprising the collagen structure according to any one of 1 to 10 above.
12 12. The collagen carrier according to 10 or 11 above, which is for cell culture.
13. 13. The collagen carrier according to item 12 above, which has an action of enhancing enzyme activity of cultured cells.
14 14. The collagen carrier according to item 12 or 13, which has an action of enhancing drug responsiveness of cultured cells.
15. 15. The collagen carrier according to any one of 12 to 14 above, which has a dedifferentiation inhibitory action on cultured cells.
16. A method for producing a collagen structure comprising collagen fibrils having dimples on the surface, comprising the following steps:
(1) A step of coating the surface of the object to be coated with a collagen solution having a concentration of 0.1 mg / ml to 100 mg / ml so that the collagen density is 0.21 mg / cm ² or more.
17. The method for producing collagen according to item 16, further comprising the following steps:
(2) A step of crosslinking the collagen structure.
18. 18. The method for producing collagen according to 16 or 17 above, wherein the collagen is atelocollagen.

The collagen structure of the present invention (or the collagen carrier of the present invention) has at least one of the following effects.
(1) Has an effect of forming a hepatocyte shape on cultured cells.
(2) It has a cell-cell interaction enhancing action and a dedifferentiation inhibiting action of cultured cells.
(3) Has an effect of enhancing esterase activity of cultured cells.
(4) It has an action to promote albumin secretion of cultured cells.
(5) It has a CYP3A4 enzyme activity enhancing effect on cultured cells.
(6) It has a drug responsiveness (CYP3A4-inducing enzyme activity by rifampicin) enhancing effect of cultured cells.
(7) It has the effect of promoting or maintaining the expression of liver-specific secreted protein gene in cultured cells. In addition, it has the effect of enhancing or maintaining the production of liver-related protein in cultured cells.
(8) It has the effect of enhancing or maintaining CYP activity of cultured cells.
(9) It has an action of promoting or maintaining nuclear receptor gene expression in cultured cells.
(10) It has an action of maintaining the expression of nuclear receptor genes in cultured cells.
(11) By suppressing the expression of the integrin gene in the cultured cells, it has the effect of suppressing activation of FAK signal and suppressing dedifferentiation and liver function deterioration.
(12) It has the effect of suppressing or maintaining DDR1 gene expression in cultured cells.
(13) It has an action of promoting albumin secretion of human iPS cell-derived hepatocyte-like cells.
(14) It has a CYP3A4 enzyme activity enhancing action of human iPS cell-derived hepatocyte-like cells.
(15) It has a liver-specific secretory protein gene expression promoting action of human iPS cell-derived hepatocyte-like cells.
(16) It has an action of promoting the expression of the drug metabolizing enzyme Phase 2 gene of human iPS cell-derived hepatocyte-like cells.
(17) It has an action of promoting the expression of drug metabolizing enzyme Phase 3 gene in human iPS cell-derived hepatocyte-like cells.
(18) It has an action of promoting nuclear receptor gene expression of human iPS cell-derived hepatocyte-like cells.

Evaluation of structural characteristics of the collagen structure of the present invention. (a)-(d) show the observation of the collagen coat and structure surface by SEM. (e) to (h) show motif analysis by Digital surf (trademark) MountainsMap analysis software. (k) and (l) show observation of the collagen cross section by TEM. (a) A conventional collagen coat having a collagen density of 0.04 mg / cm ² . (b) A conventional collagen coat having a collagen density of 0.2 mg / cm ² . (c) A collagen structure having a collagen density of 1 mg / cm ² . (d) A collagen structure having a collagen density of 2 mg / cm ² . In (c) and (d), arrows indicate representative collagen fibrils. An arrow head indicates a typical dimple. Scale bar: 10 μm. (e) Conventional collagen coat having a collagen density of 0.04 mg / cm ² . (f) A conventional collagen coat having a collagen density of 0.2 mg / cm ² . (g) A collagen structure having a collagen density of 1 mg / cm ² . (h) A collagen structure having a collagen density of 2 mg / cm ² . (i) Number of motifs at collagen density 0.04 mg / cm ² , 0.2 mg / cm ² , 1 mg / cm ² , 2 mg / cm ² . (j) Average area of motifs at collagen densities of 0.04 mg / cm ² , 0.2 mg / cm ² , 1 mg / cm ² and 2 mg / cm ² . (k) A conventional collagen coat having a collagen density of 0.04 mg / cm ² . The two wounds found in collagen are artifacts. (l) A collagen structure having a collagen density of 2 mg / cm ² . Arrows indicate representative collagen fibrils. An arrow head indicates a cross section of a typical dimple (difference in cross-sectional shape of each dimple is caused by a difference in cutting position). Scale bar: 1 μm. All images (a) to (h), (k), and (l) were acquired from near the center of the well. LIVE / DEAD staining. (A, b, c) Conventional 0.04 mg / cm ² collagen. (D, e, f) 0.2 mg / cm ² collagen. (G, h, i) 1 mg / cm ² collagen. (J, k, l) 2 mg / cm ² collagen. Arrowheads show multiple cell island-like morphology suggesting cell-cell interactions. The merge images of phase contrast observation of Hoechst33342 (A: cell nucleus), Calcein AM (B: cytoplasm of live cells) and Ethidium homodimer-1 (C: nucleus of dead cells) were shown. Scale bar: 100 μm. Evaluation of albumin secretion and CYP3A4 enzyme activity. (A) Albumin secretion amount. (B) CYP3A4 basic enzyme activity. (C) CYP3A4 induced enzyme activity (Rifampicin or DMSO 48 hours). N = 4, Mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Statistical analysis used One-way ANOVA tukey test (A, B) and t-test (C). Evaluation of liver-related gene expression (qRT-PCR, 3 days after the start of culture). (A) Liver-specific secreted protein. (B, c, d) Drug metabolism-related genes Phase1, Phase2, Phase3. (E) Nuclear receptor. Relative values with 0.04 mg / cm ² as 1. N = 4, Mean ± SD. Evaluation of liver-related gene expression maintenance (qRT-PCR, 1, 3, 7 days after start of culture). (A) Liver-specific secreted protein. (B, c, d) Drug metabolism-related genes Phase1, Phase2, Phase3. (E) Nuclear receptor. N = 4, Mean ± SD. Expression of 0.04 mg / cm ² (dotted line) and 2 mg / cm ² (solid line) were compared. Evaluation of gene expression of cell adhesion factor (qRT-PCR, 3 days after start of culture). Relative values with 0.04 mg / cm ² as 1. Day 0 is the expression level of hepatocytes before the start of culture. Day 0 is N = 1, others are N = 4, Mean ± SD. Evaluation of liver function of hepatocytes derived from iPS cells (21, 26, 31 days after initiation of induction). (A) Albumin secretion amount. (B) CYP3A enzyme activity. Conv. Shows the conventional method. (C) Liver-related gene evaluation results (qRT-PCR, 31 days after initiation of induction). Corrected with GAPDH. The relative expression level is shown with the expression level of Conv. n = 4. As a conventional method (abbreviated as C), differentiation induction on the Matrigel coat was used as a control.

The present invention relates to a collagen structure including collagen fibrils having dimples on the surface, a collagen carrier including the collagen structure, and a method for producing the collagen structure. The present invention is described in detail below.
The collagen carrier in the present invention means a collagen structure for supporting any substance, cell, or tissue. In the collagen carrier, only the collagen structure or the main component may be substantially composed of the collagen structure, or a certain structure (particularly, the surface of the container) may be coated with the collagen structure.
Examples of the container include a well plate, a dish, and a flask. The material of the container is not particularly limited, and examples thereof include plastic, polyethylene, polycarbonate, polystyrene, and glass.

(Collagen material)
As the collagen used as the material of the collagen structure of the present invention (or the collagen carrier of the present invention), collagen known per se can be used. For example, insoluble collagen collected from living tissue, such as tendon collagen derived from Achilles tendon, collagen derived from skin, soluble collagen, solubilized collagen, such as enzyme-solubilized collagen (atelocollagen), alkali-solubilized collagen, acid-soluble collagen, salt-soluble Collagen or the like can be used, but atelocollagen is particularly desirable.
There is no particular limitation on the animal species from which the collagen is derived, and there is no problem as long as the collagen has a denaturation temperature at which the collagen does not undergo thermal denaturation during culture. Specifically, it can be derived from mammals such as cows and pigs, birds such as chickens, fishes such as tuna and sea bream. Recombinant collagen can also be used.
Furthermore, if necessary, the constituent amino acid side chains of collagen may be chemically modified. Specific examples include collagens that have been acylated, such as acetylated, succinylated, and phthalated, alkylated such as methylated and ethylated, and esterified.

(Atelocollagen material)
Atelocollagen is collagen in which telopeptides are enzymatically depleted and has low immunogenicity. Furthermore, atelocollagen has the same typical triple helix structure as natural collagen, and can form collagen fibrils under physiological conditions. Therefore, it can be applied to both in vivo and in vitro such as regenerative medicine and drug delivery system.

Collagen gels and vitrigels are known as thick collagen carriers made of high-density atelocollagen. Scanning electron microscope (SEM) observations reveal that both collagen gels and vitrigels have a network structure consisting of many collagen fibrils as well as features of striated lines every 70 nm from individual collagen fibrils. Has been reported (see Takezawa, T. et al. Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo. J. Biotechnol. 131, 76-83 (2007)).
The following examples confirm that the structure of the collagen structure of the present invention is significantly different from the structures of the conventional collagen coat (collagen coat with a collagen density of 0.20 mg / cm ² or less), collagen gel and vitrigel.
Specifically, the collagen structure of the present invention includes collagen fibrils and has dimples on the surface, whereas the conventional collagen coat does not include collagen fibrils and does not have dimples on the surface. Gels and vitrigels contain collagen fibrils but do not have dimples on the surface.

(Collagen structure of the present invention)
The “collagen structure including collagen fibrils having dimples on the surface” which is the collagen structure of the present invention has at least a depression shape (for example, a substantially hemispherical depression formed by overlapping a plurality of collagen fibrils. ) Dimples which are spaces.
Furthermore, the collagen structure of the present invention has at least one of the following characteristics, preferably 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all characteristics.
(1) The dimple has a substantially hemispherical shape with a diameter of 100 μm or less, preferably 0.01 μm to 50 μm, more preferably 0.5 μm to 30 μm, and still more preferably 1 μm to 3.5 μm.
(2) 1 to 4,000, preferably 10 to 2,000 dimples per 475 μm ^{2 on} the surface (open surface not in contact with the surface to be coated and / or surface in contact with the surface to be coated) , More preferably 70 to 700.
Note that “per surface 475 μm ² ” means, for example, “per SEM 5,000 × observation photograph”. The number calculation method is to measure the number of dimples described in the photograph.
(3) The number of motifs per surface 475 μm ² is 4,377 or less, preferably 1,000 to 4,000, more preferably 2,000 to 3,000, still more preferably 2,100 to 2,500, and most preferably 2,200 to 2,400.
The “motif” is an area segmented by motif analysis corresponding to ISO 25178. The “number of motifs” is obtained by analyzing a motif of a SEM image of the surface of a collagen structure 475 μm ² using MountainsMap ™ software (Digital Surf) under the conditions of “bottom detection (bottom detection)”.
(4) The average area of the motif is 0.12 μm ² or more, preferably 0.12 μm ^{2 to} 1.00 μm ² , more preferably 0.20 μm ^{2 to} 0.24 μm ² .
(5) The surface structure has random orientation.
(6) The collagen structure has collagen fibrils, and the diameter of the collagen fibrils is 9 μm or less, preferably 1 μm or less, more preferably 0.01 μm to 0.3 μm.
(7) Collagen fibrils are preferably oriented randomly, and a space of 10 μm ² or less is present between the collagen fibrils, more preferably a space of 0.01 μm ² to 1 μm ² .

(Collagen carrier of the present invention)
The collagen carrier of the present invention can be used as a support (extracellular matrix) for culturing cells or the like as described in the following examples. The form and shape of the collagen carrier are not particularly limited, but are preferably collagen structures coated on a container.
In addition, the collagen carrier of the present invention can be used for a carrier of a substance containing genes, proteins, etc., various scaffolds, tissue culture and the like.

  Cells to be cultured with the collagen carrier of the present invention are not particularly limited, but preferably hepatocytes, more preferably human primary culture hepatocytes, human iPS-derived hepatocyte-like cells, ES-derived hepatocyte-like cells, Muse-derived hepatocyte-like cells. Cells, MSC-derived hepatocyte-like cells, human liver tumor-derived cells {eg, HepaRG (trademark), etc.}, human liver cancer-derived cell lines (eg, HepG2, HuH7, etc.), more preferably human primary cultured hepatocytes, human iPS It is a derived hepatocyte-like cell.

(Method for producing collagen structure of the present invention)
The method for producing a collagen structure containing collagen fibrils having dimples on the surface of the present invention includes at least the following steps (A) and (B).
(A) After adding and coating a collagen solution having a concentration of 0.1 mg / ml to 100 mg / ml on the surface of the coat, the collagen density is 0.21 mg / cm ² or more or 0.21 mg / cm ² to 50 mg / cm ² And (B) a step of crosslinking the collagen structure.
Furthermore, it preferably includes at least the following steps (1) to (3).
(1) A step of adding a collagen solution having a concentration of 0.1 mg / ml to 100 mg / ml to the surface to be coated and coating the surface;
(2) a step of drying the collagen solution to have a collagen density of 0.21 mg / cm ² or more or 0.21 mg / cm ² to 50 mg / cm ² , and (3) a step of crosslinking the dried collagen structure (particularly , A step of crosslinking the dried collagen structure by irradiating with ultraviolet rays (UV)).

Step (1) includes coating the surface to be coated with a collagen solution in which collagen and a solvent are mixed. The “collagen solution in which collagen and a solvent are mixed” may be a solution in which collagen and a solvent coexist, and even if the collagen is not dissolved in the solvent, the collagen is dissolved in the solvent. There may be. Step (1) includes the following step (1-1) and / or step (1-2).
(1-1) A step of preparing a collagen solution by adding collagen to a solvent and mixing,
(1-2) A step of coating the surface of the object to be coated with a collagen solution obtained by mixing collagen and a solvent.
Preferably, step (1) includes both step (1-1) and step (1-2).
Although the density | concentration of the collagen solution in a process (1) is not specifically limited, Preferably it is 0.01 mg / ml-100 mg / ml, More preferably, it is 0.1 mg / ml-50 mg / ml, More preferably, it is 1.0 mg / ml-10 mg / ml, particularly preferably 2.5 mg / ml to 7.5 mg / ml.
The collagen density when the collagen solution is coated on the surface of the coating target should be higher than 0.2 mg / cm ² , preferably 0.25 mg / cm ^{2 to} 50.0 mg / cm ² , more preferably 0.5 mg / cm ^{2 to} 20.0. mg / cm ² , more preferably 0.75 mg / cm ^{2 to} 10.0 mg / cm ² , particularly preferably 1.0 mg / cm ^{2 to} 5.0 mg / cm ² .
In the step (1-1), the solvent for preparing the collagen solution is not particularly limited, but a solvent having an acidic pH is preferable, and for example, hydrochloric acid, acetic acid, citric acid, malic acid and the like can be used. Moreover, the collagen solution may contain an additive within a range not impairing the object of the present invention. More specifically, the pH of the collagen solution is from pH 1 to 4.5, preferably from pH 2 to 4.
In the step (1-2), the collagen solution is not particularly limited, but a solvent having an acidic pH is preferable. More specifically, the pH of the collagen solution is from pH 1 to 4.5, preferably from pH 2 to 4.

  The drying time and drying temperature in the step (2) may be such that at least 50, 60, 70 or 80% of the moisture of the collagen is removed. The drying time is not particularly limited, and may be, for example, 3 hours or more, 12 hours or more, 24 hours or more, preferably 1 day to 10 days, more preferably 2 days to 8 days, and particularly preferably 4 days to 6 days. . A drying temperature is not specifically limited, For example, 0 to 100 degreeC, Preferably it is 10 to 50 degreeC, More preferably, it is room temperature.

The method for cross-linking the collagen structure in the step (3) is not particularly limited, and examples thereof include cross-linking by ultraviolet (UV) irradiation, cross-linking by a cross-linking agent, etc., preferably cross-linking by UV irradiation.
In the case of crosslinking by UV irradiation, the UV wavelength is not particularly limited, but is preferably 100 nm to 400 nm, more preferably 200 nm to 300 nm, still more preferably 240 nm to 270 nm, and particularly preferably 254 nm. The UV irradiation amount is not particularly limited, but preferably 0.01 J / cm ^{2 to} 100 J / cm ² , more preferably 0.1 J / cm ² to 10 J / cm ² , and still more preferably 0.1 J / cm ^{2 to} 5 J /. cm ² , particularly preferably 1 J / cm ² . UV irradiation may be divided into a plurality of times. For example, when the UV irradiation amount is 1 J / cm ² , 0.5 J / cm ² may be irradiated twice.
In the case of cross-linking with a cross-linking agent, a homobifunctional reagent having the same reactive group can be used as the cross-linking agent, but preferably a heterobifunctional reagent having separate reactive groups at both ends is desirable. More preferably, the collagen is a crosslinking reagent capable of reacting in a solution state, such as AMAS (N- [α-Maleimidoacetoxy] succinimideester), BMPH (N- [β-Maleimidopropionic acid] hydrazideoTFA), BMPS (N- [β-Maleimidopropyloxy] succinimide ester), EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride), EMCS (N- [ε-Maleimidocaproyloxy] succinimide ester), GMBS (N- [γ-Maleimidobutyryloxy] succinimide ester), MBS (m- Maleimidobenzoyl-N-hydroxysuccinimideester), SBAP (Succinimidyl3- [bromoacetamido] propionate), SIA (N-Succinimidyl iodoacetate), SIAB (N-Succinimidyl [4-iodoacetyl] aminobenzoate), SMCC (Succinimidyl 4- [N-maleimidomethyl] -cyclohexane -1-carb oxylate), SM (PEG) n (Succinimidyl-([N-maleimidopropionamido] -n-ethyleneglycol) ester), Sulfo-EMCS (N- [ε-Maleimidocaproyloxy] sulfosuccinimide ester), Sulfo-GMBS (N- [γ-Maleimidobutyryloxy) ] sulfosuccinimide ester) and the like.

The collagen carrier of the present invention can have dimples on the surface even if the cell culture container is not coated.
The method for producing a collagen carrier of the present invention when the cell culture container or the like is not coated is produced, for example, by a production method including the following steps.
(1) A step of pouring a collagen solution obtained by mixing collagen and a solvent into a mold.
(2) A step of drying collagen (solution).
(3) A step of removing from the mold.
In addition, you may add the process of bridge | crosslinking to the collagen before or after taking out from a type | mold as needed.

The collagen structure or collagen carrier of the present invention has one or more of the following effects.
(1) It has the function of maintaining the liver function of cultured cells and / or human iPS cells.
(2) The function of enhancing, maintaining, enhancing function and / or enhancing maturation of hepatocytes of human iPS cell-derived hepatocyte-like cells.
(3) By using for culturing hepatocytes (especially human primary cultured hepatocytes), the cells and cells proliferated from the cells are rounder, smaller and more in vivo compared to the conventional collagen coat. It can be formed in a form close to hepatocytes. That is, it has a hepatocyte shape forming action of cultured cells.
(4) By using for culturing hepatocytes (especially human primary cultured hepatocytes), the cells and cells proliferated from the cells can be formed into motility island-like morphology. That is, it has a cell-cell interaction enhancing action and a dedifferentiation inhibiting action of cultured cells.
(5) By using for culturing hepatocytes (especially human primary cultured hepatocytes), the esterase activity of the cells and cells proliferated from the cells can be enhanced (at least for 7 days from the start of culture), and the cells can survive. it can. That is, it has an effect of enhancing esterase activity of cultured cells.
(6) By using it for culturing hepatocytes (particularly human primary cultured hepatocytes), the amount of albumin secretion is enhanced as compared with a conventional collagen coat. That is, it has an action of promoting albumin secretion of cultured cells.
(7) By using for culturing hepatocytes (particularly human primary cultured hepatocytes), the basic enzyme activity of CYP3A4 is enhanced as compared with the conventional collagen coat. That is, it has an effect of enhancing CYP3A4 enzyme activity of cultured cells.
(8) When used for culturing hepatocytes (especially human primary cultured hepatocytes), the inducible enzyme activity of CYP3A4 is enhanced as compared with a conventional collagen coat. That is, it has an action of enhancing drug responsiveness (CYP3A4 inducing enzyme activity by rifampicin) of cultured cells.
(9) Compared with the conventional collagen coat, albumin (ALB), α1-antitrypsin (AAT) and liver-specific secreted proteins are used by culturing hepatocytes (particularly human primary cultured hepatocytes). Increase or maintain tryptophan 2,3-dioxygenase (TO) gene expression. That is, it has the effect of promoting or maintaining liver-specific secretory protein gene expression in cultured cells. In addition, it has the effect of enhancing or maintaining the production of liver-related protein in cultured cells.
(10) Increased or maintained gene expression of CYP1A2, CYP2C9, CYP2C19 and CYP2D6, which are drug metabolizing enzymes, compared to conventional collagen coat by using for culturing hepatocytes (especially human primary cultured hepatocytes) To do. That is, it has the effect of promoting or maintaining the expression of the drug metabolizing enzyme Phase1 gene in cultured cells. It also has the effect of enhancing or maintaining CYP activity of cultured cells.
(11) Increase or maintain gene expression of CAR, PXR, and HNF4α, which are nuclear receptors, by using for culturing hepatocytes (particularly human primary cultured hepatocytes), as compared with conventional collagen coats. That is, it has an action of promoting or maintaining nuclear receptor gene expression in cultured cells.
(12) By using it for culturing hepatocytes (particularly human primary cultured hepatocytes), the expression of Ahr gene, which is a nuclear receptor, is maintained as compared with a conventional collagen coat. That is, it has an action of maintaining the expression of nuclear receptor genes in cultured cells.
(13) By using it for culturing hepatocytes (especially human primary cultured hepatocytes), integrin α2, α3, α5, α6, αV, β1, and β3, which are cell adhesion factors, compared to the conventional collagen coat Reduce or maintain gene expression. That is, it has an action of suppressing or maintaining cell adhesion factor gene expression in cultured cells. Moreover, by suppressing the expression of the integrin gene in cultured cells, it has the action of suppressing activation of FAK signal and suppressing dedifferentiation and liver function deterioration.
(14) By using for culturing hepatocytes (particularly human primary cultured hepatocytes), the gene expression of DDR1 is reduced or maintained as compared with a conventional collagen coat. That is, it has the effect of suppressing or maintaining DDR1 gene expression in cultured cells.
(15) The collagen structure of the present invention increases the amount of albumin secretion as compared with a conventional collagen coat when used for induction of hepatocyte-like cell differentiation of human iPS cells. That is, the collagen structure of the present invention has an action of promoting albumin secretion of human iPS cell-derived hepatocyte-like cells.
(16) By using for induction of human iPS cell-derived hepatocyte-like cell differentiation, CYP3A enzyme activity is enhanced as compared with a conventional collagen coat. That is, it has a CYP3A enzyme activity enhancing action of human iPS cell-derived hepatocyte-like cells.
(17) By using for induction of hepatocyte-like cell differentiation of human iPS cells, the expression levels of ALB, AAT, and TO genes, which are liver-specific secreted proteins, are increased as compared with the conventional collagen coat. That is, it has a liver-specific secretory protein gene expression promoting action of human iPS cell-derived hepatocyte-like cells.
(18) By using for induction of hepatocyte-like cell differentiation of human iPS cells, gene expression of UGT1A1, which is a drug metabolizing enzyme Phase 2, is increased as compared with a conventional collagen coat. That is, it has the effect of promoting the expression of the drug metabolizing enzyme Phase 2 gene in human iPS cell-derived hepatocyte-like cells.
(19) By using for induction of hepatocyte-like cell differentiation of human iPS cells, gene expression of MRP2, which is a drug metabolizing enzyme Phase 3, is increased as compared with a conventional collagen coat. That is, it has an action of promoting the expression of the drug metabolizing enzyme Phase 3 gene in human iPS cell-derived hepatocyte-like cells.
(20) By using for induction of hepatocyte-like cell differentiation of human iPS cells, the expression of Ahr gene, which is a nuclear receptor, is increased as compared with a conventional collagen coat. That is, it has a nuclear receptor gene expression promoting action of human iPS cell-derived hepatocyte-like cells.
(21) It has an effect of reducing FAK activation through integrin expression of cultured cells and / or human iPS cells.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

[Production of Collagen Carrier of the Present Invention]
The collagen carrier of the present invention was produced by the following method.

<Material>
96-well plate (Corning), collagen acidic solution {collagen acidic solution I-PC 5 mg / mL 50 mL Atelocollagen Bovine Dermis (IPC-50), Koken}, 1 mM HCl, Petit Rotor (Wakken Yakuhin), UV cloth A linker (CL-1000, UVP) was used.

<Method>
The following was adjusted in a clean room. Collagen was handled on ice.
1. A collagen solution (pH 3) was prepared. The concentration of the collagen solution is 5 mg / mL (IPC-50 stock solution) in the production of the collagen carrier of the present invention, and 0.25 mg / mL by adding 1 mM HCl to IPC-50 in the production of the conventional collagen coat. Those adjusted to ml and 1.25 mg / ml were used.
2. Using a stirrer (petit rotor (Speed 8.5)) in the refrigerator, the collagen solution was mixed by inversion (4 ° C., 15 minutes).
3. The collagen solution is dispensed into a 96-well plate, the collagen density is 1 mg / cm ² and 2 mg / cm ^{2 in} the production of the collagen carrier of the present invention, and the collagen density is 0.04 mg / cm ^{2 in} the production of the conventional collagen coat. And 0.2 mg / cm ² .
4). Air-dried in a clean bench for 5 days. During air drying, the UV light was turned off to prevent crosslinking by the UV light.
5). The state in which the collagen was completely dried was confirmed with the naked eye and stored until UV crosslinking was performed at a later date.
6). A 96 well plate coated with a conventional collagen coat or a collagen carrier of the present invention in a clean room was placed in a UV crosslinker and irradiated with 1 J / cm ² .

[Evaluation of collagen structure characteristics]
The structural characteristics of the collagen carrier of the present invention produced in Example 1 and the conventional collagen coat were analyzed and compared.

<Material>
Osmium coater (Neoc-STB, Meiwaforsys), scanning electron microscope (SU6600, Hitachi), ultramicrotome (Leica EM UC7, Leica), transmission electron microscope (JEM-1400Plus, JEOL), MountainsMap ™ software (Digital) Surf), a conventional collagen coat of Example 1 (collagen density 0.04 mg / cm ² ) or a 96 well plate coated with a collagen carrier of the present invention (2 mg / cm ² ).

<Method>
{Scanning electron microscope (SEM)}
1. The collagen carrier of Example 1 was fixed to 2% glutaraldehyde.
2. Dehydrated with ethanol.
3. Soaked in t-butyl alcohol. Thereafter, it was freeze-dried and sputter coated with osmium using Neoc-STB.
4). Images were acquired using SU6600.
5). The micro roughness (micro roughness) of the acquired SEM image was analyzed and evaluated by motif analysis (bottom detection) corresponding to ISO 25178 using MountainsMap (trademark) software.

{Transmission electron microscope (TEM)}
1. The collagen carrier of Example 1 was fixed to 2% glutaraldehyde.
2. Dehydrated with ethanol and embedded in Epon.
3. It was sliced to a thickness of 70 nm using an ultramicrotome and stained with uranium acetate.
4). Images were acquired using JEM-1400Plus.

<Result>
SEM and TEM images are shown in FIG.
In order to evaluate the structural characteristics of the collagen structure constituting the collagen carrier of the present invention, it was compared with a conventional collagen coat (collagen density 0.04 mg / cm ² ). The surface of the conventional collagen coat has a fine and flat structure (FIG. 1 (a)). The 0.2 mg / cm ² collagen coat was similar to the conventional collagen coat (FIG. 1 (b)). In contrast, the surface of the 1 mg / cm ² and 2 mg / cm ² collagen structures were rough, randomly oriented structures (FIGS. 1 (c) and (d)). Collagen fibrils (arrows in Fig. 1 (c) and (d)) and characteristic dimples with a diameter of about 2 to 3 μm (Fig. 1 (c) only in 1 mg / cm ² and 2 mg / cm ² collagen structures ) And (d) arrowheads) were observed.

In order to analyze the minute roughness of the surface, motif analysis based on ISO 25178 was performed using analysis software (FIGS. 1 (e) to (j)). This motif analysis is a technique for detecting and quantifying isolated surfaces using a segmentation algorithm based on ISO25178.
Per SEM 5,000 times photograph of collagen coat of 0.04 mg / cm ² and 0.2 mg / cm ² and collagen structure of 1 mg / cm ² and 2 mg / cm ² (corresponding to surface 475 μm ² ) The number of motifs was 5,949, 4,378, 2,320, and 2,201, respectively (FIG. 1 (i)). Therefore, the collagen structures of 1 mg / cm ² and 2 mg / cm ² are composed of a smaller number of motifs than the conventional collagen coat (approximately half or less than the conventional collagen coat), and the collagen structure of the present invention It has been shown that the body has a rougher surface than a conventional collagen coat.
The average motif area of the collagen coat of 0.04 mg / cm ² and 0.2 mg / cm ² and the collagen structure of 1 mg / cm ² and 2 mg / cm ² is 0.08 μm ² , 0.11 μm ² , 0.21 μm ² and 0.22 respectively. It was μm ² (FIG. 1 (j)). Therefore, the collagen structures of 1 mg / cm ² and 2 mg / cm ² are composed of a motif larger than the conventional collagen coat (approximately twice or more than the conventional collagen coat), and the collagen of the present invention. The structure was shown to have a rougher surface than a conventional collagen coat.

  Collagen molecules have a rod-like structure with a length of 300 nm and a width of 1.5 nm. The arrangement of collagen molecules was examined using TEM. In the conventional collagen coat, there was no collagen fibrils, and no space was observed in a small amount of collagen (FIG. 1 (k)). On the other hand, the collagen structure of the present invention was composed of collagen fibrils, which were randomly oriented (FIG. 1 (l), arrow). In the collagen structure of the present invention, there was a space between collagen fibrils (FIG. 1 (l)). The dimples were observed only on the surface of the collagen structure of the present invention (FIG. 1 (l), arrowhead) and coincided with the SEM image (FIG. 1 (d)).

From the above, the collagen structure of the present invention has the following differences in structure compared with the conventional collagen coat.
(1) The surface of the conventional collagen coat has a fine and flat structure, whereas the surface of the collagen structure of the present invention has a rough random orientation structure.
(2) The collagen structure of the present invention has collagen fibrils and characteristic dimples having a diameter of about 2 to 3 μm, but none of the surface of the conventional collagen coat. Collagen fibrils were randomly oriented, and there were spaces between the collagen fibrils.
(3) The surface of the collagen structure of the present invention was composed of a smaller number of motifs (2,320 to 2,201) than the conventional collagen coat, and had a rougher surface than the conventional collagen coat.
(4) The surface of the collagen structure of the present invention was composed of a larger motif (0.21 μm ^{2 to} 0.22 μm ² ) than the conventional collagen coat, and had a rougher surface than the conventional collagen coat.

[Culture of primary human hepatocytes (PHHs) using the collagen carrier of the present invention]
Human primary cultured hepatocytes (PHHs) were cultured using the collagen carrier of the present invention, and compared with culture using a conventional collagen coat.

<Cell>
PHHs 1 vial {Adherent human hepatocytes (for enzyme induction test) CryoHuman Hepatocytes Induction Qualified (HUCPI), lot: HUM4218B (Male, Caucasian, 19 years old, 09-May-2017, frozen cells 7.4 × 10 ⁶ cells / vial , Lonza)}.
<Material>
HCMBullet kit {HCM (trademark) hepatocyte culture medium BulletKit (trademark), basic medium and SingleQuots (trademark) kit (cc-3198, Lonza)}, HCM (+ 2% FBS, incubated at 37 ° C.), each collagen of Example 1 96-well plate coated with a density of the collagen carrier of the present invention, phosphate buffered salt (without Dulbecco, calcium, magnesium) (D-PBS) (DS Pharma), Eagle's minimal essential medium (MEM) (M4655-500ML, Sigma), centrifuge {CF8DL, mounted rotor T3S6, Hitachi}, RNeasy micro kit (Qiagen).

<Culture method>
(Preparation of collagen plate)
1. The 96-well plate coated with the collagen carrier of the present invention prepared in Example 1 was washed twice with 0.1 ml of D-PBS, and 0.05 ml / well of HCM (+ 2% FBS) was added. Care was taken not to dry the collagen during washing.
_2. Incubate overnight at 37 ° C, 5% CO ₂ incubator.

(PHHs thawing)
1. PHHs were half-thawed in a 37 ° C. water bath and suspended in 50 ml of HCM (37 ° C.).
2. After centrifugation, the cells were suspended in 10 ml of HCM (+ 2% FBS, 37 ° C.).
3. Trypan blue staining and cell count were performed.
4). The cell suspension was diluted to 100 × 10 ⁴ viable cells / ml.
5. 100 μl / well was seeded in a 96-well plate.
6. Cultured at 37 ° C., 5% CO ₂ for 7 days.
7). To extract RNA at a later date, the remainder of the seeded cells was dissolved in Buffer RLT of the RNeasy micro kit and stored at −80 ° C.

(Medium exchange)
1. The medium was changed on the day after thawing PHHs (one day after the start of culture). The medium was changed at 0.15 ml / well of HCM (without FBS).
2. Thereafter, the medium was changed to HCM (without FBS) 0.15 ml / well every day.

(Culture result)
The total number of cells in 10 ml of the culture solution was 1.2 × 10 ⁷ cells. The survival rate was 87%, and the viable cell concentration was 1.04 × 10 ⁶ viable cells / ml.

[Confirmation of morphology and survival of primary human hepatocytes (PHHs) using the collagen carrier of the present invention]
In this example, the morphology and survival of human primary cultured hepatocytes (PHHs) using the collagen carrier of the present invention were compared with those of a conventional collagen coat.

<Material>
LIVE / DEADViability / Cytotoxicity Kit, for mammalian cells (Thermo, Ethidium homodimer-1 and Calcein AM are included in the kit contents), HCM bullet kit (Lonza), Hoechst33342 (Dojin Chemical), Working solution {HCM 1ml, Ethidium homodimer-1 (EhtD-1) 2 μl, calcein AM 0.5 μl, Hoechst33342 0.5 μl}, and the cultured cells of Example 3 were used.

<Method>
1. The culture medium was removed 1 day, 3 days, and 7 days after the start of culture in Example 3 above, and 50 μl / 96 well of a previously prepared Working solution was added.
2. Incubated at 37 ° C for 5 minutes.
3. The medium was changed to 50 μl of HCM.
4). Pictures were taken with a fluorescence microscope (IX83, Olympus).

<Result>
PHHs grown on a conventional collagen coat showed a typical polygonal shape on the first day of culture (FIG. 2 (a)), but gradually on the third day of culture and on the seventh day of culture. Elongation (Fig. 2 (b), (c)) showed dedifferentiation.
On the other hand, PPH cultured on the collagen carrier of the present invention (1 mg / cm ² and 2 mg / cm ² ) was round with a very compact cytoplasm (FIGS. 2 (g) and (j)). PPH cultured on the collagen carrier of the present invention (1 mg / cm ² and 2 mg / cm ² ) was very small compared to PHHs grown on the conventional collagen coat (FIG. 2 (g) (J)). The circular shape of the cells grown on the collagen carrier of the present invention was maintained until the third day (FIGS. 2 (h) and (k)). That is, PHHs grown on the collagen carrier of the present invention were in a form closer to in vivo hepatocytes compared to PHHs grown on a conventional collagen coat. On day 7 of the cells grown on the collagen carrier of the present invention, motility island-like morphology was observed (FIGS. 2 (i) and (l)). The island-like morphology detected on day 7 showed higher interaction between cells than on day 3, indicating that dedifferentiation was not progressing.
In the cells grown on the collagen carrier of the present invention, many dead cells were temporarily observed on the third day (FIGS. 2 (h) and (k)). However, dead cells were reduced by medium exchange, and most cells survived on the 7th day (FIGS. 2 (i) and (l)). That is, it was confirmed that the dead cells were caused by nutritional deficiency and were not caused by the structure of the collagen structure constituting the collagen carrier of the present invention. During the culture period, the green fluorescence intensity (calcein AM) was low with the conventional collagen coat, but was high with the collagen carrier of the present invention. This means that the cells grown on the collagen carrier of the present invention have enhanced esterase activity compared to cells grown on a conventional collagen coat.
That is, in human primary cultured hepatocytes (PHHs) using the collagen carrier of the present invention, (1) promoting the interaction between cells as compared with culture using a conventional collagen coat, (2) dedifferentiation Confirmed that it was difficult to progress.

[Evaluation of liver function (albumin secretion)]
In order to evaluate the effect of PHHs on liver function in culture using the collagen carrier of the present invention, the amount of albumin secretion, which is a liver function marker, was examined.

<Material>
HumanAlbumin ELISA kit (Bethyl Laboratories) and cultured cells of Example 3 were used.
<Method>
1. In Example 3 above, the whole culture supernatant was collected 24 hours after the medium was changed 1 day after the start of culture and 2 days after the start of culture, and stored at -80 ° C.
2. Later, the culture supernatant stored at −80 ° C. was thawed, and ELISA was performed using the Human Albumin ELISA kit according to the method attached to the product. The amount of albumin per well was calculated with the amount of supernatant being 150 μl / well.

<Result>
The results are shown in FIG. 3A. The amount of albumin secreted by the cells cultured with the collagen carrier of the present invention was higher than the amount of albumin secreted by the cells cultured with the conventional collagen coat. Specifically, the albumin secretion amount of cells cultured with the collagen carrier of the present invention was about 2.5 times to about 5 times higher than the albumin secretion amount of cells cultured with the conventional collagen coat. For example, the amount of albumin secretion (7.0 ± 1.1 mg / mg RNA) of cells cultured on the collagen carrier of the present invention (collagen density 1 mg / cm ² ) after 24 hours of culturing is Increased by 4.7 times compared to the amount of albumin secreted (1.5 ± 0.3 mg / mg RNA).
From the above, it was confirmed that the collagen carrier of the present invention enhances the secretion amount of albumin, which is a liver function marker of cells.

[Evaluation of liver function (CYP3A4 enzyme activity)]
In order to evaluate the effect of PHHs on liver function in culture using the collagen carrier of the present invention, the activity of CYP3A4 enzyme, which is a liver function marker, was examined.

<Material>
Rifampicin (Sigma), DMSO (Wako Pure Chemical Industries), Millex-LG, 0.20 μm, 25 mm (Merck Millipore), 25 mM Rifampicin (× 1000) Stock (Rifampicin dissolved in DMSO and filter sterilized (Millex-LG) P450-Glo ™ CYP3A4 Assay with Luciferin IPA (Promega), Assay 96-well plate (Nunc), plate reader (ARVO MX-FLA, Perkin Elma), and cultured cells of Example 3 Using.
<Method>
(Induction of CYP3A4 enzyme activity)
The reaction conditions were a final concentration of 25 μM Rifampicin / 0.1% DMSO, 37 ° C., 48 hours.
1. The medium was changed one day after the start of the culture in Example 3 above. The medium was supplemented with 0.15 ml / well after adding 1 μl of 25 mM Rifampicin per ml of HCM (or 1 μl of DMSO as a solvent-only control group) and incubating in a 37 ° C. water bath.
2. Two days after the start of culture, the medium was changed in the same manner as one day after the start of culture.
3. CYP enzyme activity was evaluated 3 days after the start of culture (48 hours after drug exposure).

(Evaluation of CYP3A4 enzyme activity)
1. The medium was changed to 100 μl of HCM / Luciferin-IPA.
2. The same was added to 2 wells without cells as background samples.
3. Incubated for 1 hour at 37 ° C.
4). 40 μl (duplicate) of the supernatant was collected from each well onto an assay plate.
5). An equal amount of Luciferin Detection Reagent was added.
6). It was shielded from light with aluminum and allowed to stand at room temperature for 20 minutes.
7). Luminescence was measured using ARVOMX-FLA (Integration time: 1 second).

<Result>
The results are shown in FIGS. The basic enzyme activity of CYP3A4 in cells cultured with the collagen carrier of the present invention was about 2 to 3 times higher than the basic enzyme activity of CYP3A4 in cells cultured with a conventional collagen coat ( Figure 3B). For example, CYP3A4 of PHHs cultured with the collagen carrier of the present invention (collagen density 1 mg / cm ² ) was 3.4 times higher than CYP3A4 of PHHs cultured with a conventional collagen coat.
A CYP3A4 inducer (Rifampicin: rifampicin) for confirming drug responsiveness was added to the medium and cultured for 48 hours. As a result, the inducible enzyme activity of CYP3A4 only against the solvent (DMSO) was measured. The CYP3A4 inducing enzyme activity of the conventional collagen coat (0.04 mg / cm ² ) and the collagen carrier (2 mg / cm ² ) of the present invention is 11.8 ± 2.9 times (mean ± standard deviation) and 21.8 ± 1.9 times, respectively. (Figure 3C). That is, in the case of the collagen carrier of the present invention, the drug responsiveness (inducible enzyme activity of CYP3A4 against rifampicin) was about twice as high as that of the conventional collagen coat.
As described above, the collagen carrier of the present invention enhanced CYP3A4 enzyme activity and drug responsiveness, which are liver function markers of cells.

[Expression analysis of liver-related genes and cell adhesion factors]
PHHs are known to lose liver function during culture. Therefore, in this example, it was confirmed whether the collagen carrier of the present invention can maintain the liver function of PHHs during culture (or can suppress the decrease in function). Specifically, the expression of PHHs liver-related genes was examined by qRT-PCR.
Integrins are transmembrane cell adhesion proteins that link the cytoskeleton of cells and the extracellular matrix. Hepatocytes are known to interact with collagen through integrins α1 and β1. The discoidin domain receptor (DDR) is a known collagen receptor. Therefore, in order to investigate the influence of each collagen concentration during culture on the interaction between cells and extracellular matrix, cell adhesion factors {integrins α1, β1, DDR, etc.) that mediate the interaction between hepatocytes and collagen Expression was examined by qRT-PCR.

<Material>
SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Bio), primer (10 μM, sequence shown in Table 1 below), cDNA (diluted stock solution 2 times with distilled water), thermal cycler (Light Cycler ™ ST -300, Roche), capillary {Light Cycler Capillaries (20μl), Roche}, centrifuge (LCCarousel Centrifuge 2.0, Roche), HumanFetal Liver (Clontech), HumanAdult Liver {Clontech, PrimeScript ^™ RTreagent kit (Takara Bio) After reverse transcription according to the protocol attached to the product, the stock solution was diluted 6-fold with distilled water}, RNeasy Micro kit (Qiagen), PrimeScript ™ RT reagent kit (Takara Bio), and cultured cells of Example 3 were used. .

<Method>
Extraction of the total RNA from the cultured cells of Example 3 and reverse transcription reaction after 1, 3, and 7 days after the start of the culture in Example 3 according to the product attachment using the RNeasyMicro kit and PrimeScript ™ RTreagent kit, respectively. Carried out.
1. The qRT-PCR reaction premix was prepared using SYBR Premix Ex Taq (Tli RNaseH Plus) according to the method described in the product attachment (SYBR Premix 10 μl, Forward Primer 0.4 μl, Reverse Primer 0.4 μl, Distilled water 7.2 μl).
2. A 18 μl premix was dispensed into the capillary.
3. 2 μl of Template (6-fold diluted cDNA) was added.
4). QRT-PCR was performed in the Light Cycler. The reaction conditions were denaturation (95 ° C 60 seconds), PCR (95 ° C 5 seconds, 60 ° C 20 seconds, 40 cycles), dissociation (65 ° C 15 seconds), cooling (40 ° C 30 Second).
5). Comparative quantitative analysis was performed using the ΔΔCt method.
6). Statistical analysis was performed using One-ANOVA tukey test using GraphPad Prism7 or t-test using Microsoft Excel.

<Result>
The results of liver-related gene expression in cells cultured with the collagen carrier of the present invention are shown in FIG. The expression was expressed as a relative value with the expression 1 day after the start of culture in a conventional collagen coat (0.04 mg / cm ² ) as 1. The liver-specific secreted proteins albumin (ALB), α1-antitrypsin (AAT), and tryptophan 2,3-dioxygenase (TO) were significantly increased (2.1 ± 0.2-fold, 1.5 ± 0.1-fold, and 3.6 ± 0.3) Times). The drug-metabolizing enzymes Phase 1 CYP1A2, CYP2C9, CYP2C19 and CYP2D6 also increased significantly (3.0 ± 0.2 times, 2.0 ± 0.2 times, 1.7 ± 0.1 times and 3.0 ± 0.1 times, respectively). The expression of CAR and PXR, which are nuclear receptors, was also significantly increased.
Therefore, an increasing tendency of expression of these genes in cells cultured with the collagen carrier of the present invention was observed. This result means that the collagen carrier of the present invention enhanced the production of liver-related protein and CYP activity of cells.
The results regarding the maintenance of liver-related gene expression during cell culture are shown in FIG. The results of qRT-PCR of liver-related genes 1, 3, and 7 days after the start of culture are shown. In the collagen carrier {2 mg / cm ² (solid line)} of the present invention, the liver-specific secreted protein, the drug-metabolizing enzyme Phase 1 and the nuclear receptor indicated by a bold frame are the conventional collagen coat { The gene expression tended to be higher than 0.04 mg / cm ² (dotted line)}. For example, albumin gene expression decreased to 6.4 ± 0.4% 7 days after the start of culture with a conventional collagen coat. On the other hand, the albumin gene expression was maintained at 17.4 ± 1.5% after 7 days from the start of cultivation with the collagen carrier of the present invention. AAT and TO gene expression increased 2.2-fold (454.4 ± 97.5%) and 1.7-fold (20.7 ± 2.6%) 7 days after the start of culture with the collagen carrier of the present invention. CYP1A2, CYP2C9, CYP2C19 and CYP2D6 gene expression is 3.3 times (797.9 ± 73.0%), 1.4 times (13.9 ± 0.9%), 1.4 times (15.6 ± 0.8%) 7 days after the start of culture on the collagen carrier of the present invention And 6.3 times (9.6 ± 0.5%). The results show that the collagen carrier of the present invention was able to maintain liver function more efficiently than the conventional collagen coat.
Regarding Ahr gene expression, although it was low 7 days after the start of cultivation with the collagen structure of the present invention, it was similar to PHHs that had been cryopreserved immediately after thawing. That is, it was suggested that Ahr gene expression is close to in vivo gene expression kinetics when cultured on the collagen carrier of the present invention.
The expression of CAR, PXR and HNF4α genes 7 days after the start of culture with the collagen carrier of the present invention was significantly higher than that of the conventional collagen coat (respectively 6.6 times (6.1 ± 0.8%) and 1.5 times (42.1 ± 5.7%). And 1.4 times (57.0 ± 2.9%).
The result regarding the gene expression of the cell adhesion factor for investigating the cell-extracellular matrix interaction is shown in FIG. Integrins are transmembrane cell adhesion proteins that connect the cytoskeleton and extracellular matrix. It interacts with collagen through integrins α1 and β1. Before the start of culture, the expression levels of all genes except integrin α1 were low or below the detection limit. Integrin α2, α3, α5, α6, αV, β1 and β3, and DDR1 expression increased abnormally in the conventional collagen coat (0.04 mg / cm ² ) culture 3 days after the start of the culture. The low expression level was maintained in the culture with the collagen carrier.
The results show that the gene expression of the cell adhesion molecule is close to the in vivo gene expression kinetics when cultured on the collagen carrier of the present invention. Moreover, the culture | cultivation by the collagen carrier of this invention shows that the activation of FAK signal was suppressed by suppressing the expression of an integrin gene, and dedifferentiation and liver function decline were suppressed.

[Hepatocyte culture derived from iPS cells using the collagen carrier of the present invention]
In this example, iPS cell-derived hepatocyte culture using the collagen carrier of the present invention was performed and compared with culture using a conventional collagen coat.

<Material>
Corning ™ Matrigel ™ Growth Factor Reduced (GFR) Basement Membrane Matrix, * LDEV-Free, 10 mL (Corning) was used.

<Method>
Hepatocyte induction of human iPS cells (Tic) has been described by Si-Tayeb, K. etal and Mallanna, Sunil K. and
A four-step induction method with improved conditions was performed with reference to DuncanStephen, A.
Specifically, the proliferation of hepatocyte cultures of the collagen carrier of the present invention was compared with the conventional differentiation induction method (Conv.) Using a conventional collagen coat and Matrigel (trademark) by the following method.
1. Matrigel ™ was diluted 30-fold with DMEM (Gibco, 11965-092), added to a 48-well plate at 0.2 ml / well, and allowed to stand at 4 ° C. overnight to form a Matrigel ™ coat.
2. Human iPS cells (Tic) were seeded on a Matrigel ™ coat and cultured in a differentiation induction medium containing Activin A (R & D systems, 338-AC-050 / CF) for 5 days to induce differentiation of definitive endoderm .
3. On day 5 of differentiation induction, CXCR4 positive cells were positively selected by MACS (Miltenyi Biotech), seeded on Matrigel ™ coat, and BMP4 (Pepro Tech, 120-05ET) and FGF2 (Sigma, F0291-25UG) Hepatic endoderm was induced by culturing in the containing medium for 5 days.
4). Differentiation induction of hepatic stem progenitor cells was performed in a medium containing HGF (Pepro Tech, 100-39) for 5 days from the 10th day of differentiation induction.
5). On the 15th day of culture corresponding to hepatic progenitor cells, the cell mass was collected, and the cell mass (100,000 cells / well) was seeded on various collagen carriers or Matrigel ™ coats.
6). Mature culture was performed in 0.1 to 0.15 ml of a medium containing OSM (R & D systems, 295-OM-010 / CF) until the 31st day of differentiation induction to induce differentiation of hepatocyte-like cells. Hepatocyte-like cells (iPS-Hep) induced to differentiate from iPS cells are converted into conventional differentiation induction method (Conv.) Matrigel (trademark), collagen carrier (2 mg / cm ² ) of the present invention, or conventional collagen The cells were cultured on the coat (0.04 mg / cm ² ).
7). The evaluation period was 21, 26 and 31 days after the start of induction. The amount of albumin secretion in the culture supernatant was quantified in the same manner as in Example 5 (n = 4), and the enzyme activity of the drug metabolizing enzyme CYP3A4 was quantified in the same manner as in Example 6 (n = 4).
8). Liver-related gene expression was evaluated in the same manner as in Example 7 above (n = 4).

<Result>
The results are shown in FIG.
The albumin secretion amount and CYP3A enzyme activity of cells grown on the collagen carrier of the present invention were observed to increase over time compared to those of the conventional collagen coat and the conventional differentiation induction method (FIGS. 7A and 7B). .
The expression level of liver-related genes in cells grown on the collagen carrier of the present invention was significantly increased compared with the expression level of liver-related genes in cells in the conventional collagen coat and the conventional differentiation induction method. (FIG. 7C). More specifically, liver-specific proteins (ALB, AAT, TO) were significantly increased. In addition, UGT1A1 involved in glucuronidation was significantly increased in Phase2-related, MRP2 was significantly increased in Phase3-related, and AhR was increased in nuclear receptors.
As described above, in the hepatocyte culture on the collagen carrier of the present invention, in all cells-derived hepatocytes, or human primary cultured hepatocytes, human iPS-derived hepatocyte-like cells, ES-derived hepatocyte-like cells, Muse-derived Hepatocyte-like cells, MSC-derived hepatocyte-like cells, and human liver tumor-derived cells {eg, HepaRG (trademark), etc.] or primary human cultured hepatocytes and human iPS-derived hepatocyte-like cells. Can enhance, maintain, enhance function and / or enhance maturity.

[General]
As described above, the morphology and function of PHHs cultured on the collagen carrier of the present invention and the conventional collagen coat were compared, the surface was observed using SEM, and the cross section was observed using TEM.
In the collagen structure constituting the collagen carrier of the present invention of the above example, a collagen amount 50 times that of the conventional was used (collagen of the collagen structure of the present invention against the collagen density of the conventional collagen coat of 0.04 mg / cm ² ). Density 2 mg / cm ² ). The conventional collagen coat (collagen density 0.04 mg / cm ² ) consists of a fine and flat surface (FIG. 1 (a)), and no collagen fibrils were observed in the cross-sectional image (FIG. 1 (k)). On the other hand, the collagen structure of the present invention (collagen density 2 mg / cm ² ) exhibited a characteristic (micro) dimple surface ((M) DS} structure (FIG. 1 (d)). . This dimple was observed at 1 mg / cm ² , but not at 0.2 mg / cm ² atelocollagen. Thus, there is a clear difference between collagens below 0.2 mg / cm ² and above 1 mg / cm ² . This was consistent with the results of motif analysis (FIGS. 1 (e)-(j)) and obvious differences between 0.2 and 1 mg / cm ² in cell morphology and liver function in cell culture experiments. Therefore, the surface structure of the collagen structure of the present invention is considered to have a great influence on the behavior of cells.
Since the collagen structure of the present invention has characteristic dimples on the surface, it is clearly different from collagen gel or collagen vitrigel. The collagen structure of the present invention is a collagen scaffold characterized by having collagen fibrils and (micro) dimples.
The structure different from the conventional collagen coat of the collagen structure of the present invention affected the morphology and function of PHHs. More specifically, the collagen structure of the present invention enhanced the liver function of PHHs compared to the conventional collagen coat.
CYP3A4 is most related to drug metabolism. Thus, the culture model using the collagen carrier of the present invention can improve the sensitivity of drug metabolism and risk assessment of drug-induced liver damage. Primary rat hepatocytes using conventional collagen-coated or Matrigel sandwich culture models significantly reduced CYP1A2 expression during 72 hours of culture (Choi, HJ & Choi, D. Successful mouse hepatocyte culture with sandwich collagen gel formation. J Korean Surg. Soc. 84, 202-208 (2013).). However, the culture method using the collagen carrier of the present invention enhanced CYP1A2 expression compared to PHHs stored frozen immediately after thawing. Therefore, the collagen carrier of the present invention enables highly sensitive analysis of CYP1A2-related drug metabolism and toxicity. CYP1A2, CYP2C9, CYP2D6, CYP2C19, and CYP3A4 metabolize 90% of currently distributed drugs, and their activity is maintained in cells cultured using the collagen carrier of the present invention. Therefore, the collagen carrier of the present invention enables shortening of experimental periods in various fields such as pharmacology, toxicology, tissue engineering, and clinical hepatocyte transplantation.
Focaladhesion kinase (FAK) and its downstream signal are activated by cell adhesion to collagen via integrin. In mouse primary hepatocytes cultured on hard dried collagen, FAK was activated via Src and activated ERK and Akt signaling. Activation of the Ras / Raf / ERK pathway (MAP kinase) causes dedifferentiation and epithelial-mesenchymal transition, and activation of the PI3K / Akt pathway is known to result in resistance to apoptosis (Godoy, P. et al. al. Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factorb-induced apoptosis. Hepatology 49, 2031-2043 (2009). Cell culture with the collagen carrier of the present invention suppressed FAK activation compared to the findings in cell culture with a conventional collagen coat. The relationship between integrin and FAK activation is known to be that FAK phosphorylation is inhibited by inhibition of integrin β1, for example, in human lens epithelial cells (Liu, J. et al. Electric field exposure promotes epithelial- mesenchymaltransition in human lens epithelial cells via integrin β1-FAK signaling. Mol. Med. Rep. 16, 4008-4014 (2017).). FAK phosphorylation is known to be significantly reduced in β1 conditional knockout mice (Wang, Y. et al. Β1-Integrin deletion from the lens activates cellular stress responses leading to apoptosis and fibrosis. Investig. Ophthalmol. Vis Sci. 58, 3896-3922 (2017).). Therefore, it is considered that the collagen carrier of the present invention reduces FAK activation through integrin expression and suppresses dedifferentiation.
From the above, the results of this Example suggest that the structure of the collagen structure constituting the collagen carrier of the present invention caused a change in the function and form of PHHs. Furthermore, the collagen carrier of the present invention is useful for enhancing liver function as compared with a conventional collagen coat. The conventional co-culture and / or sandwich culture method is more complicated than the culture method of the present invention. The collagen carrier of the present invention is simple and useful, and becomes a culture carrier excellent in maintaining the function of PHHs.
Thus, the collagen carrier of the present invention is useful as a cell culture carrier for drug development, particularly evaluation of human liver drug metabolism, clearance, drug-drug interactions, transporter activity and hepatotoxicity.

  The collagen structure and collagen carrier of the present invention can provide a cell culture carrier for drug development, in particular human liver drug metabolism evaluation, clearance, drug-drug interactions, transporter activity and hepatotoxicity.

Claims (18)

A collagen structure containing collagen fibrils having dimples on the surface.
The collagen structure according to claim 1, wherein the dimple on the surface is a hollow space formed by overlapping a plurality of collagen fibrils.
The collagen structure according to claim 1 or 2, wherein the dimple has 1 to 4,000 per 475 µm ² of the surface.
The collagen structure according to any one of claims 1 to 3, wherein the number of motifs per surface 475 µm ² is 4,377 or less.
The collagen structure according to claim 4, wherein an average area of the motif is 0.12 µm ² or more.
The collagen structure according to any one of claims 1 to 5, wherein the surface has a random orientation.
The collagen structure according to any one of claims 1 to 6, wherein a space is formed between a plurality of collagen fibrils forming the collagen structure.
The collagen structure according to any one of claims 1 to 7, wherein a plurality of collagen fibrils forming the collagen structure are randomly oriented.
The collagen structure according to any one of claims 1 to 8, wherein the collagen fibrils are atelocollagen fibrils.
The collagen structure according to any one of claims 1 to 9, wherein a collagen density of the collagen structure is 0.21 mg / cm ² or more.
The collagen carrier containing the collagen structure of any one of Claims 1-10.
The collagen carrier according to claim 10 or 11, which is used for cell culture.
The collagen carrier according to claim 12, which has an effect of enhancing enzyme activity of cultured cells.
The collagen carrier according to claim 12 or 13, which has an action of enhancing drug responsiveness of cultured cells.
The collagen carrier according to any one of claims 12 to 14, which has a dedifferentiation inhibitory action on cultured cells.
A method for producing a collagen structure comprising collagen fibrils having dimples on the surface, comprising the following steps:
(1) A step of coating the surface of the object to be coated with a collagen solution having a concentration of 0.1 mg / ml to 100 mg / ml so that the collagen density is 0.21 mg / cm ² or more.
Furthermore, the manufacturing method of the collagen of Claim 16 including the following processes,
(2) A step of crosslinking the collagen structure.
  The method for producing collagen according to claim 16 or 17, wherein the collagen is atelocollagen.