JP6429490B2 - Collagen fiber cross-linked porous body - Google Patents

Description

  The present invention relates to a cross-linked collagen fiber porous body, and in particular, a cell culture substrate and a medical material (for example, a scaffold material for regenerative medicine, a transplant material, a cosmetic material, a wound dressing material, an adhesion prevention material, It is a material suitable for a drug transport carrier and the like.

  Collagen is an important protein that accounts for 30% of proteins in the body and has functions such as skeletal support and cell adhesion. For example, bone, cartilage, ligaments, tendons, corneal stroma, skin, liver, muscle and other tissues Is made of collagen fibers. A molded body using this collagen (hereinafter referred to as “collagen molded body”) is a cell culture substrate and a scaffold material for regenerative medicine (for example, cartilage, bone, spine, nucleus pulposus, ligament, corneal stroma, skin, blood vessel, Nerve / liver tissue regeneration material), transplantation material (wound dressing material, bone filling material, hemostatic material, anti-adhesion material, etc.) or drug delivery carrier, especially large tissue regeneration by regenerative medicine, prevention of cell dispersion And essential for inducing cell differentiation.

  However, since the collagen molded body has insufficient mechanical properties, it is difficult to operate, and its use in clinical settings is limited. In general, a collagen molded body is likely to swell in water or a cell culture medium, and when this is used as a cell culture substrate, the form changes when the culture period is over several days, Or collagen may decompose | disassemble gradually and may elute. Therefore, when transplanted, the seeded cells are dispersed and it is difficult to stay at a desired tissue site.

  In Patent Document 1, bubbles are removed from a collagen dispersion, solution or mixture thereof having a collagen concentration of 50 mg / ml or more, and then freeze-dried, followed by insolubilization treatment by physical crosslinking or chemical crosslinking, A technique relating to a carrier for cell culture having a stress of 10 to 30 kPa at 10% load and having a pore structure on the surface and inside is disclosed.

  Patent Document 2 discloses a technique relating to a collagen structure that is composed of collagen fibers having an average diameter of 1 to 5 μm.

Japanese Patent No. 4915693 International Publication No. 2013/105665 Pamphlet

  Since collagen is present in a fibrous form in the living body, it is considered that the collagen used as a carrier in cell culture is preferably in a fibrous form instead of an amorphous form.

  On the other hand, in order to enable three-dimensional cell culture, it is necessary to be a porous body composed of continuous pores having a pore size capable of cell migration so that cells can enter the substrate. .

  However, with the conventional technology, it has been difficult to produce a porous body (hereinafter also referred to as “fibrous macroporous body”) that is composed of collagen fibers and has a porous structure capable of three-dimensional cell culture. . By the way, there are various theories about the size of the pore diameter that enables three-dimensional cell culture, and it is said to be at least 50 μm or 70 μm. On the other hand, 100 μm and 150 μm are also proposed, but this is considered to realize sufficiently smooth cell migration.

  In order to fibrillate collagen, for example, as described in JP-A-2010-273847, a neutral buffer solution is used, and the ionic strength of the solubilized collagen solution is appropriately increased so that the pH is close to neutral. It is well known that this is important, and in this publication, salt aqueous solutions having buffering capacity such as phosphate, acetate, carbonate, citrate and Tris are exemplified as neutral buffers. Phosphate buffer is mentioned as preferred.

The present applicant performed the following methods (a) to (c) as an attempt to obtain a fibrous macroporous material.
(a) A method in which phosphate buffered saline (PBS) is added to a solubilized collagen solution to fibrillate collagen, and then freeze-dried.
(b) After PBS was added to the solubilized collagen solution to fibrillate the collagen, the fibrillated collagen was mixed with ethanol series (mixed solution of ethanol and water whose ethanol concentration was increased stepwise. Ethanol concentration: 50%, 70%, 90%, 100%), followed by dehydration and desalting, then replacing the solvent with t-butanol, freezing, and freeze-drying.
(c) A method of freeze-drying a solubilized collagen solution and then fibrillating the collagen with PBS.

  However, no fibrous macroporous material was obtained by any of the methods (a) to (c).

  That is, in the method (a), the fibrillated collagen becomes non-fibrotic (defibrinated) by a high concentration salt concentrated during freeze-drying, and the pore diameter is small. Since the cell culture carrier described in Patent Document 1 is also produced by the same method as the method (a), it is difficult to say that the collagen is fibrous.

  In the method (b), a porous body composed of collagen fibers was obtained, but it was unsuitable for three-dimensional cell culture because of its small pore size. This is probably because the frozen crystals of t-butanol were not as large as the frozen crystals of water. By the way, when the solubilized collagen solution in an aqueous solvent is freeze-dried as it is, a porous body having a pore size that allows three-dimensional cell culture can be obtained, but non-fibrous (amorphous) dissolved collagen can be obtained. It is known that it is non-fibrous (amorphous) because it is freeze-dried as it is.

  Further, in the method (c), a stable shape could not be obtained.

  The collagen structure described in Patent Document 2 is suitable for three-dimensional cell culture because the pore diameter is small enough to be less than 50 μm as judged from FIGS. 3 and 4 of the electron microscope image according to Example 1. It was hard to say.

  An object of the present invention is to provide a collagen molded body composed of collagen fibers and having a porous structure capable of three-dimensional cell culture.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have surprisingly found that fibrillar collagen precipitated by alkali metal bicarbonate is freeze-dried and cross-linked so that the collagen is fibrillated. It was found that a collagen molded body having a porous structure composed of continuous pores having a pore size capable of cell migration while maintaining the structure and capable of effectively functioning as a cell culture substrate can be obtained. It has been completed.

That is, the present invention is as follows.
[1] A collagen fiber crosslinked porous body comprising a collagen fiber, having a porous structure capable of three-dimensional cell culture, and subjected to a crosslinking treatment.
[2] In an electron microscope image of the collagen fiber cross-linked porous body, when the maximum number of the holes is n in a fixed section where the number of holes observed on the outermost surface is at least 30, the average pore diameter = [1] The average pore diameter calculated by the formula {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n) is in the range of 50 to 300 μm [1] The collagen fiber cross-linked porous body described.
[3] The collagen fiber cross-linked porous body according to the above [1] or [2], wherein collagen fibers having D-periodic horizontal stripes are observed in at least a part of the porous structure.
[4] When the mouse fibroblast cell line L929 is cultured for 10 days using the collagen fiber cross-linked porous body as a cell culture substrate, the cultured cell culture substrate has a strength that can be held by metal tweezers. The collagen fiber cross-linked porous body according to any one of the above [1] to [3], which is a product.
[5] A transplant material formed by cell culture using the collagen fiber crosslinked porous material according to any one of the above [1] to [4] as a cell culture substrate.
[6] A cell culture method using the collagen fiber cross-linked porous body according to any one of [1] to [4] as a cell culture substrate, wherein a cell suspension is applied to the dried or dehydrated porous body. A cell culture method characterized in that cells are three-dimensionally distributed in the porous body by absorbing liquid.
[7] The method includes the first step of mixing the solubilized collagen solution and the alkali metal bicarbonate to precipitate fibrotic collagen, the second step of freeze-drying, and the third step of crosslinking treatment. The method for producing a collagen fiber crosslinked porous material according to any one of [1] to [4].
[8] The collagen fiber crosslinked porous material according to the above [7], wherein the crosslinking treatment in the third step is treatment by any one of the following (1) and (2) or a combination of (1) and (2) Manufacturing method. (1) A process of physical crosslinking by γ-ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation or thermal dehydration. (2) A process of chemically crosslinking with a water-soluble chemical crosslinking agent or a chemical crosslinking agent having vaporization ability.
[9] Collage in alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ^{2 to} 3 × 10, when the amount of alkali metal bicarbonate applied is 300,000. ^The method for producing a crosslinked collagen fiber according to [7] or [8], which is in the range of ⁴ .

  The collagen fiber cross-linked porous body of the present invention has a fibrous structure and further undergoes a cross-linking treatment, so it is hardly water-soluble, that is, has a certain strength even when contacted with water for a certain period of time, and its shape changes almost It has the characteristic of not. In addition, it has excellent cell affinity and biocompatibility, and has the advantage that cells can survive not only on the surface of the porous body but also inside. In a more preferred embodiment, a certain strength is maintained even after cell culture, and the shape is relatively stable in terms other than contraction. Therefore, not only as a cell culture substrate, but also in vivo as a medical material such as scaffolding materials for regenerative medicine, transplant materials, cosmetic materials, wound dressings, anti-adhesion materials, drug transport carriers, etc. It is also suitable for use.

It is a photograph when the collagen fiber cross-linked porous body in Example 1 of the present invention is pinched with metal tweezers in a wet state after being immersed in water at 37 ° C. for 24 hours. It is an electron microscopic image (magnification: 100 times) of the collagen fiber crosslinked porous material in Example 1 of the present invention. It is an electron microscopic image (magnification: 500 times) of the collagen fiber crosslinked porous material in Example 1 of the present invention. It is an electron microscopic image (magnification: 10,000 times) of the collagen fiber cross-linked porous body in Example 1 of the present invention and an enlarged view thereof (right figure). The inside of the outermost layer hole is filled so that only the outermost layer hole of FIG. 2 can be identified. It is a photograph when the non-fibrotic collagen porous body in Comparative Example 1 of the present invention is pinched with metal tweezers. It is a photograph when the collagen fiber porous body in the comparative example 2 of this invention is pinched with metal tweezers. It is an electron microscopic image (magnification: 100 times) of the collagen fiber porous body in the comparative example 2 of this invention. It is a photograph when the collagen fiber cross-linked porous body in Example 1 of the present invention is pinched with metal tweezers in a wet state stained with Giemsa after being subjected to a cell culture test.

Hereinafter, the collagen fiber cross-linked porous body of the present invention which is a fiber macroporous body (hereinafter referred to as “the porous body of the present invention”) will be described in detail.
The porous body of the present invention is composed of collagen fibers, has a porous structure capable of three-dimensional cell culture, and is subjected to a crosslinking treatment.

  A feature of collagen fibers is that they have D-periodic horizontal stripes. D periodic horizontal stripes are characteristically observed in fibrous collagen, and there are descriptions of 64 nm and 70 nm depending on the literature, but in general there are periodic horizontal stripes that are recognized at intervals of 67 nm. (Refer to “Electron Microscope” vol.26, No.1, p.2-9, 1991, etc.) When the porous body of the present invention is observed with an electron microscope, a three-dimensional network structure is observed at a low magnification, but D periodic horizontal stripes are observed at an appropriate magnification. However, in an electron microscope image, it is difficult to observe D periodic horizontal stripes in all parts of the image due to the focus position and the like. However, if collagen fibers having D-periodic horizontal stripes are observed in at least a part of each of the electron microscopic images obtained by photographing a plurality of locations, it can be assumed that the constituent elements of the porous structure are collagen fibers. .

  As a porous structure capable of three-dimensional cell culture, a sponge-like (sponge-like) structure having continuous pores having a pore size suitable for smooth cell migration so that cells can enter the inside of the substrate can be used. There is no limit. A preferred embodiment relating to the pore diameter is an electron microscopic image of the porous body of the present invention, wherein the maximum number of holes is n in a fixed section where the number of holes observed on the outermost surface is at least 30. Average pore diameter = {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n), the average pore diameter is in the range of 50 to 300 μm. Is. A more preferable range of the average pore diameter is 70 to 250 μm. In addition, as said hole, a complete hole is object and the incomplete hole by which the hole was divided | segmented by the division boundary line is not included.

  The crosslinking treatment is not particularly limited as long as crosslinking is performed between collagen molecules or between collagen fine fibers formed by collagen molecules. Specific crosslinking treatment methods include, for example, (1) physical crosslinking by γ-ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation or thermal dehydration, and (2) a water-soluble chemical crosslinking agent or vaporization ability. Examples of the chemical cross-linking treatment include a chemical cross-linking agent. Either one of (1) and (2) may be used, or (1) and (2) may be used in combination. Of course, a plurality of cross-linking treatments may be adopted in each of the cross-linking treatments (1) and (2). For example, γ-ray irradiation may be performed after UV irradiation. Moreover, what is necessary is just to set a crosslinking degree suitably according to the objective.

  Since the porous body of the present invention has been subjected to a crosslinking treatment, it is excellent in mechanical properties and has a high tendency for poor solubility. A preferred embodiment of the porous body of the present invention has a large resistance to external forces such as compression and extension, and is hardly damaged in appearance even when handled with metal tweezers. When used as a cell culture substrate, a certain mechanical strength can be maintained even after the cell culture period ends even if there is some shape change such as contraction. As a good example, when the mouse fibroblast cell line L929 is cultured for 10 days using the porous body of the present invention as a cell culture substrate, the cultured cell culture substrate has a strength that can be held by metal tweezers. Is.

  In addition to cell culture substrates, the porous body of the present invention can be used for medical materials (for example, scaffold materials for regenerative medicine, transplant materials, cosmetic materials, wound dressings, adhesion prevention materials, drug transport carriers). Etc.) can be exemplified, but is not limited thereto. As an aspect related to the transplant material, in addition to the method of using the porous body of the present invention as it is, for example, by utilizing the mechanical properties of the porous body of the present invention, cells using the porous body of the present invention as a cell culture substrate Examples include a method of culturing and using a substrate containing cultured cells as a transplant material as it is. The cell type, culture conditions, etc. may be appropriately selected and designed so as to obtain an optimal transplant material.

  In the case where the porous body of the present invention is used as a cell culture substrate, a cell culture method for efficiently distributing cells in the substrate three-dimensionally utilizing the mechanical properties is as follows. .

  First, the porous body of the present invention is dried or dehydrated. If the porous body of the present invention is already in a dry or dehydrated state, it may be used as it is. However, when the porous body of the present invention is wet because it is stored in a liquid, it is once dried or dehydrated. When drying or dehydrating, it is preferable to use a technique of replacing the solvent with water or a medium (preferably the same medium used in the next cell culture step) and then drying or dehydrating. The drying is preferably performed at a low temperature so that the collagen is not denatured, and may be dried by low temperature ventilation. In addition, centrifugal dehydration may be performed for dehydration. However, when mechanical strength is sufficient, a method of performing dehydration by compressing with fingers is simple.

  Next, the cell suspension is absorbed into the porous body of the present invention in a dry or dehydrated state. When the cell suspension is absorbed, the cells enter the substrate together with the medium, and the cells can be distributed three-dimensionally in the substrate. In addition, you may perform absorption operation of a cell suspension in multiple times. After performing this operation, normal cell culture may be performed.

Next, the manufacturing method of the porous body of this invention is demonstrated.
The method for producing a porous body of the present invention comprises a first step of mixing a solubilized collagen solution and an alkali metal bicarbonate to precipitate fibrotic collagen, then a second step of freeze-drying, and further a crosslinking treatment. A third step is included. In addition, since the mixed liquid in which the collagen fibers are precipitated in the first step exhibits a gel shape, the mixed liquid is hereinafter referred to as “fibrotic collagen gel”.

  A solubilized collagen solution is an aqueous solution in which collagen is dissolved. The collagen is preferably water-soluble collagen having a triple helical structure. The solubilized collagen solution may partially contain peptides, amino acids, gelatin, etc., but these are preferably excluded as much as possible.

  Water-soluble collagen having a triple helix structure can be obtained by known methods from collagen-containing tissues of biological materials such as mammals, seafood, birds and reptiles. For example, [1] dilute acid Examples include acid-solubilized collagen obtained by the extraction method, [2] enzyme-solubilized collagen obtained by a method of solubilizing with an enzyme, and [3] alkali-solubilized collagen obtained by a method of solubilizing with an alkali. . Acid-solubilized collagen and enzyme-solubilized collagen are soluble under acidic conditions, and alkali-solubilized collagen is soluble under alkaline conditions. It is known to fibrosis.

  As the type of collagen, collagen derived from fish that does not have a virus common to humans is particularly suitable, and those having a relatively high denaturation temperature are preferable from the viewpoint of applicability to various uses. It is derived collagen. Of the genus Oreochromis, tilapia, which is cultivated mainly for food from China to Southeast Asia and is readily available, is particularly preferred. Furthermore, atelocollagen from which telopeptide which is an antigenic determinant of collagen is removed is preferable. There is no particular limitation on the type of collagen, but type I, which is abundant in living organisms, is preferred.

  Here, the method for obtaining the enzyme-solubilized collagen of [2] will be described. The acquisition method is not particularly limited, and may be a conventional method. For example, the method described in JP 2006-257014 A or JP 2010-193808 A can be exemplified. One aspect of the acquisition method will be briefly explained with an example of scales. By treating scales decalcified with acid with a protease such as pepsin, the collagen is atelolated and purified as necessary to produce enzyme. Solubilized collagen can be obtained. For the purification treatment, for example, salting out or a method using activated carbon having a pH of 7 or less described in JP2013-116875A can be applied.

  As the alkali metal bicarbonate, sodium bicarbonate and potassium bicarbonate are preferable. The present invention does not exclude the mixing of alkali metal carbonates as long as the effects of the present invention are not impaired.

The amount of alkali metal bicarbonate applied is not particularly limited as long as fibrotic collagen is precipitated and the porous body of the present invention is finally obtained. If the amount of alkali metal bicarbonate is too small, collagen fibrillation will be insufficient, while if too large, collagen may become non-fibrotic (defibrillated) during lyophilization in the second step. Therefore, it is desirable to appropriately set the amount of alkali metal bicarbonate so that the porous body of the present invention can be obtained. As an example of a preferred embodiment relating to the amount of alkali metal bicarbonate applied, collagen in the alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ² when the molecular weight of collagen is 300,000. The amount is in the range of ˜3 × 10 ⁴ .

  The method for mixing the solubilized collagen solution and the alkali metal bicarbonate in the first step is not particularly limited. For example, from the viewpoint of workability, an alkali metal bicarbonate aqueous solution is used as the alkali metal bicarbonate. It is preferable.

  Next, the second step of freeze-drying the fibrillated collagen gel obtained in the first step is performed. A known method may be employed as the freeze-drying method. The lyophilization conditions may be appropriately set so that a porous material can be obtained by a conventional method. For example, the freezing temperature is preferably in the range of -20 to -60 ° C, and the lyophilization time is preferably 1 to 60 hours. .

  In order to obtain the porous body of the present invention, it is important to prevent fibrous collagen from becoming non-fibrotic (defibrotic) during lyophilization. For example, when PBS is used for fibrosis of collagen, collagen is non-fibrotic (defibrotic) by PBS concentrated at the time of freeze-drying. Therefore, if the amount of PBS in collagen fibrillation is reduced so as not to be non-fibrotic (defibrotic) during lyophilization, fibrosis becomes insufficient.

  On the other hand, alkali metal bicarbonate is presumed not to inhibit the crystal growth of frozen crystals (ice) of water, and alkali metal bicarbonate can fibrosis collagen even at a low concentration compared to PBS, A porous body having a porous structure composed of collagen fibers and capable of three-dimensional cell culture can be obtained.

  Next, a third step is performed in which the freeze-dried porous body of fibrillated collagen obtained in the second step (hereinafter referred to as “fibrous porous body”) is subjected to a crosslinking treatment.

  As the crosslinking treatment, (1) physical crosslinking by γ-ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation or thermal dehydration, (2) chemical crosslinking with a water-soluble chemical cross-linking agent or a chemical cross-linking agent having vaporization ability. A cross-linking treatment is a good example, and only one of (1) and (2) may be used, or (1) and (2) may be used in combination. Of course, a plurality of cross-linking treatments may be adopted in each of the cross-linking treatments (1) and (2). For example, γ-ray irradiation may be performed after UV irradiation. Moreover, what is necessary is just to set a crosslinking degree suitably according to the objective.

  Among the physical cross-linking of (1), the cross-linking treatment by the irradiation method of γ-ray irradiation, electron beam irradiation, plasma irradiation and UV irradiation can be performed at the same time as the cross-linking treatment by appropriately setting the irradiation conditions. If a package is appropriately selected so as to maintain a sealed state during and after cross-linking, it can be distributed to the market as a sterilized product. Of the above irradiation methods, γ-ray irradiation is particularly preferable because of its high permeability and uniform crosslinking. In γ-ray irradiation, a predetermined irradiation dose can be easily obtained by using a radiation source with a fixed dose rate and appropriately setting conditions such as irradiation time. For example, when a cobalt 60 radiation source is used, the crosslinking treatment can be performed at an absorbed dose of 5 to 75 kGy, preferably 5 to 50 kGy, more preferably 10 to 50 kGy, and further preferably 15 to 30 kGy.

  Moreover, you may perform the crosslinking process by an irradiation method in presence of a liquid. Here, the presence of liquid refers to a state in which the entire surface of the fiber porous body is covered with the liquid during the cross-linking treatment, and may be in a wet state, for example, but preferably the entire fiber porous body is in the liquid. It is the state immersed in. Therefore, the volume of the liquid is not limited as long as the entire surface of the fiber porous body is covered with the liquid, but the volume of the liquid is preferably 2 to 100 with respect to the volume of the fiber porous body. ~ 50 is more preferred. The liquid is not limited as long as it contains water, and an aqueous solvent such as water or a buffer can be exemplified. Furthermore, an aqueous solvent obtained by adding an organic solvent to water or a buffer solution can also be used. Specific examples of the buffer solution include phosphate buffer solution, Tris buffer solution, HEPES buffer solution, acetate buffer solution, carbonate buffer solution, citrate buffer solution, and the like, and are physiological saline thereof. It may be PBS, D-PBS, Tris buffered saline, or HEPES buffered saline.

  As the water-soluble chemical cross-linking agent or the chemical cross-linking agent having vaporization ability, a known one may be used. For example, glutaraldehyde, polyepoxy compounds (ethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc.), carbodiimide compounds (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide / hydrochloride, etc.), reducing sugar (ribose, etc.) and the like may be mentioned, and crosslinking may be performed according to a conventional method.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In Examples, “%” means “% by mass” unless otherwise specified. In addition, an analytical scanning electron microscope JSM-6010LA manufactured by JEOL Ltd. was used as the electron microscope, and a solid and straight stainless steel tweezers were used as the metal tweezers.

[Example 1]
As a solubilized collagen solution, a colorless and transparent solution prepared by dissolving “Cell Campus FD-08G” sponge manufactured by Taki Kagaku Co., Ltd. in tilapia scales in an HCl solution of pH 3 and adjusting the collagen concentration to 1.1% ( Hereinafter, “collagen solution A”) was used.

Add 1 part by volume of sodium bicarbonate aqueous solution to 9 parts by volume of collagen solution A so that the collagen (molar ratio) in sodium bicarbonate / collagen solution A is 1.5 × 10 ³ to precipitate fibrotic collagen. It was. Next, 2 ml each of the fibrillated collagen gel containing the fibrillated collagen was dispensed into a 12-well plate, and then freeze-dried at −35 ° C. for 3 hours to form a collagen porous body composed of collagen fibers (hereinafter, “ Fiber porous body 1 ") was obtained.

  Next, by irradiating 25 kGy of γ-rays in a state where the fibrous porous body 1 was immersed in a 0.05 M aqueous sodium bicarbonate solution, a γ-ray crosslinked fibrous porous body, which is a porous body of the present invention, was obtained.

  FIG. 1 shows a photograph of the porous γ-ray fibrous body immersed in water at 37 ° C. for 24 hours and pinched with metal tweezers in a wet state. Damage due to metal tweezers was not observed in appearance. Further, even when the gamma-ray crosslinked fibrous porous material was immersed in water for 1 week, the shape was maintained in appearance.

  Electron microscope images of the gamma-ray crosslinked fiber porous body are shown in FIGS. The magnification is 100 times in FIG. 2, 500 times in FIG. 3, and 10,000 times in FIG. In FIG. 4, D periodic horizontal stripes were observed everywhere.

  FIG. 5 shows the inside of the outermost layer hole filled so that only the outermost layer hole of FIG. 2 can be identified. Using FIG. 5, the maximum width and the minimum width were measured for all the complete holes (holes not divided by the partition boundary line), and the average hole diameter was calculated from the above formula. As a result, the average hole diameter was 116.0 μm. .

[Example 2]
The fiber porous body 1 was thermally dehydrated and cross-linked at 110 ° C. for 10 hours under reduced pressure to obtain a heat-crosslinked fiber porous body that is a porous body of the present invention.

  Even if the thermally crosslinked fiber porous body was immersed in water at 37 ° C. for 24 hours and then pinched with metal tweezers in a wet state, damage due to the metal tweezers was not visually observed. In addition, even when the thermally crosslinked fibrous porous material was immersed in water for 1 week, the shape was maintained in appearance.

[Comparative Example 1]
9 parts by volume of collagen solution A and 1 part by volume of PBS prepared to a concentration 10 times higher were mixed to precipitate fibrotic collagen. Next, 2 ml of fibrillated collagen gel containing the fibrillated collagen was dispensed into a 12-well plate, and then lyophilized at −35 ° C. for 3 hours to obtain a porous body.
As shown in FIG. 6, the obtained porous body was deformed in a shape such as being curved. Observation with an electron microscope showed that the crystallized salt was attached and the fibrosis state of the collagen could not be discriminated, so that it was a non-fibrotic collagen porous body.

[Comparative Example 2]
9 parts by volume of collagen solution A and 1 part by volume of PBS prepared to a concentration 10 times higher were mixed to precipitate fibrotic collagen. Next, the solvent of the fibrillated collagen gel containing the fibrillated collagen was successively desalted and dehydrated with an ethanol series (ethanol concentration: 50%, 70%, 90%, 100%) by mixing ethanol and water. Thereafter, the solvent was replaced with t-butanol and freeze-dried at −35 ° C. for 3 hours to obtain a collagen fiber porous body.

  FIG. 7 is a photograph when the collagen fiber porous body is pinched with metal tweezers. In the collagen fiber porous body, D periodic horizontal stripes were observed everywhere by observation with an electron microscope. As shown in the electron microscope image (magnification: 100 times) in FIG. It was a porous body composed of pores that were too small to be recognized. That is, since the presence of pores having a size of at least 50 to 300 μm was not observed, it was not suitable for three-dimensional cell culture.

[Cell culture test]
As a cell culture substrate, the gamma-ray crosslinked fiber porous body of Example 1 was used. In addition, DMEM + 10% FBS was used as the medium.

After injecting 150 μl of a cell suspension containing 1.0 × 10 ⁶ cells of mouse fibroblast cell line L929 into a floating cell culture 12-well plate, this was placed with a dehydrated γ-ray cross-linked fiber porous body, The cell suspension was absorbed from the lower surface direction. This operation was repeated once more, and 300 μl of the cell suspension was absorbed by the γ-ray crosslinked fiber porous material. Next, the γ-ray crosslinked fiber porous body was inverted and cultured at 37 ° C. overnight. Subsequently, 700 μl of a medium was additionally added, and thereafter, the medium was cultured for 9 days while appropriately changing the medium according to a normal cell culture method. At this time, the culture period is 10 days by overnight +9 days.

  After culturing, when the γ-ray crosslinked fiber porous body was picked up with metal tweezers in a wet state, it could be lifted almost as it was, so that the γ-ray crosslinked fiber porous body could be held by metal tweezers. It was found to have strength. The gamma-ray crosslinked fibrous porous body was hardly deformed except that some contraction was observed at the initial stage of culture.

  In addition, the gamma-ray crosslinked fibrous porous material after completion of the culture was washed with PBS and immersed in 10% formaldehyde at room temperature overnight to fix the cells. Next, after washing with PBS and staining with Giemsa staining solution diluted 20 times with PBS for 15 minutes, the cells were washed with PBS and observed with a microscope. As a result of observing while changing the focus of the microscope from the upper surface to the lower surface of the gamma-ray crosslinked fiber porous body, the cells were uniformly distributed at any focal position. That is, it was found that the cells were uniformly distributed three-dimensionally within the gamma-ray crosslinked fiber porous body.

  In addition, as shown in FIG. 9, the gamma-ray crosslinked fiber porous body retains a high strength that can be pinched with metal tweezers in an almost intact form even in a wet state after Giemsa staining. Was.

Claims (8)

A collagen fiber cross-linked porous body comprising a porous spongy structure composed of collagen fibers and capable of three-dimensional cell culture, and subjected to a cross-linking treatment.
However, in the electron microscope image of the collagen fiber cross-linked porous body, in a fixed section where the number of holes observed on the outermost surface is at least 30, when the maximum number of the holes is n,
Average hole diameter = {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n)
The average pore diameter calculated by the mathematical formula is in the range of 50 to 300 μm.
The following porous sponge-like structure is excluded from the category of the porous sponge-like structure. That is, it has a controlled pore diameter in the range of 50 to 2000 μm, and the pore communicates in a straight line from one surface to the other surface, and each pore exists substantially independently of each other. Has a porous spongy structure. The collagen fiber cross-linked porous body according to claim 1, wherein collagen fibers having D-periodic horizontal stripes are observed in at least a part of the porous structure. When the collagen fiber cross-linked porous body is used as a cell culture substrate and the mouse fibroblast cell line L929 is cultured for 10 days, the cultured cell culture substrate has a strength that can be held by metal tweezers. The collagen fiber cross-linked porous body according to claim 1 or 2. The transplant material formed by carrying out cell culture using the collagen fiber bridge | crosslinking porous body of any one of Claims 1-3 as a cell culture base material. A cell culture method using the collagen fiber cross-linked porous body according to any one of claims 1 to 3 as a cell culture substrate, wherein the cell suspension is absorbed into the dried or dehydrated porous body. A cell culture method characterized by distributing cells three-dimensionally within the porous body. A first step of mixing solubilized collagen solution and alkali metal bicarbonate to precipitate fibrotic collagen;
A second step of freeze-drying,
The method for producing a collagen fiber crosslinked porous material according to any one of claims 1 to 3, further comprising a third step of crosslinking treatment. The method for producing a collagen fiber crosslinked porous material according to claim 6, wherein the crosslinking treatment in the third step is treatment by any one of the following (1) and (2) or a combination of (1) and (2).
(1) A process of physical crosslinking by γ-ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation or thermal dehydration.
(2) A process of chemically crosslinking with a water-soluble chemical crosslinking agent or a chemical crosslinking agent having vaporization ability. When the amount of alkali metal bicarbonate applied is 300,000, the collagen in the alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ^{2 to} 3 × 10 ⁴ The method for producing a crosslinked collagen fiber according to claim 6 or 7.

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