JP5224440B2 - Three-dimensional cultured elastic fiber tissue and method for producing three-dimensional cultured elastic fiber tissue - Google Patents

Description

The present invention relates to a three-dimensional cultured elastic fiber tissue having a three-dimensional thickness and expressing an elastic fiber component essential for the elastic fiber tissue such as elastin and fibrillin, and production of the three-dimensional cultured elastic fiber tissue. Regarding the method.

Recent advances in cell engineering technology have enabled the culturing of numerous animal cells, and research on regenerative medicine that uses these cells to reconstruct human tissues and organs is rapidly progressing. Attempts have been made to regenerate various tissues such as skin and blood vessels. By the way, the flexibility of the skin and blood vessels of the living tissue is maintained by elastic fibers, and in order to regenerate the living tissue having sufficient flexibility, the construction of the elastic fiber tissue is also important.

In vitro, it is known that fibroblasts produce elastic fiber components by culturing fibroblasts at high density using a serum-added medium on a petri dish. However, it was difficult to peel the elastic fiber tissue thus obtained from the petri dish while maintaining the tissue shape.
In contrast, a method has been proposed in which a cultured tissue is peeled into a sheet using a special culture substrate such as a thermosensitive response culture dish (Patent Document 1, etc.). If this method is applied, the elastic fiber tissue formed on the petri dish can be peeled into a sheet.

However, the elastic fiber tissue sheet thus obtained is only a single-layer cell sheet. Considering application in the field of regenerative medicine, in order to construct tissues such as skin and blood vessels, a single-layer cell sheet is insufficient in strength as a tissue and is difficult to handle for transplantation. Even if transplantation is possible, even if an elastic fiber tissue sheet having almost no thickness is transplanted, the inherent performance of the elastic fiber tissue that imparts flexibility cannot be exhibited. In order to construct a thick tissue using the cell sheet produced by such a method, an operation of peeling off the produced cell sheets and bonding the cell sheets together is necessary. When the formed tissue becomes thick, there is a problem in that the regenerated tissue produced cannot be fed to the inside and necrotized.

As a three-dimensional cell culture method, a method of culturing cells in a collagen gel is known. However, so far, there has been no report that even if fibroblasts are cultured in a collagen gel at a high density, elastic fiber components are expressed.
International Publication No. 2002/010349 Pamphlet

In view of the above-described situation, the present invention has a three-dimensional culture elastic fiber tissue having a three-dimensional thickness and expressing an elastic fiber component indispensable for elastic fiber tissue such as elastin and fibrillin, and the three-dimensional culture It aims at providing the manufacturing method of elastic fiber tissue.

The present invention includes a step 1 of seeding only fibroblasts at a density of 1 × 10 ³ / cm ² or more with respect to a porous base material having an average pore diameter of 1 to 30 μm made of heat-crosslinked collagen; a three-dimensional culture elastic fibrous tissue fibroblasts obtained in step 1 were seeded porous substrate produced by the production method and a step 2 of culturing in serum-containing medium, wherein the porous base The material has a residual weight ratio of 40-60% after being immersed in an aqueous collagenase solution at a concentration of 0.5 units / mL at 37 ° C. for 100 minutes, has a thickness of 5 μm or more, and contains elastin and fibrillin. It is a cultured elastic fiber tissue.
The present invention is described in detail below.

As a result of intensive studies, the present inventors seeded fibroblasts with high density on a porous substrate made of collagen and having an average pore size of 1 to 30 μm, and then cultured in a serum-supplemented medium for a certain period. Thus, elastic fiber components such as elastin and fibrillin are secreted from fibroblasts, and an elastic fiber tissue having a three-dimensional thickness is formed by further culturing, and the present invention has been completed. It was.

In order for the elastic fiber component to be secreted from the fibroblasts, it is considered that the fibroblasts need to be cultured in a high density state sufficiently close to each other. For example, in the case of plate culture on a petri dish, such high-density culture is possible, and it seems that an elastic fiber tissue was formed. On the other hand, in the culture method in collagen gel or the method of seeding and culturing fibroblasts on a collagen sponge having a relatively large pore diameter, which is generally used in conventional regenerative medicine, fibroblasts can be used no matter how the culture is continued. It is considered that they are not sufficiently close to each other, and therefore elastic fiber tissue is not formed.
In contrast, a porous substrate having an average pore diameter of 1 to 30 μm has a sponge property and a film property. That is, when fibroblasts are seeded on a porous substrate having an average pore size of 1 to 30 μm, the density is high enough that fibroblasts are sufficiently close to each other as in the case of seeding cells on a film (on a petri dish). Can be cultured in a stable state, and an elastic fiber tissue is formed. On the other hand, the base material is degraded by the degrading enzyme secreted by the cell itself, and the pores of the base material gradually increase, and some of the seeded fibroblasts enter the pores of the porous base material that has become larger due to the degradation. Can spread in three dimensions. Furthermore, since the porous substrate is excellent in the permeability of the medium components, it can sufficiently supply nutrients to fibroblasts cultured in high density and in large quantities. Thus, when the culture is continued, an elastic fiber tissue having a three-dimensional thickness is formed.

The three-dimensional elastic fiber tissue of the present invention comprises a step 1 of seeding fibroblasts at a density of 1 × 10 ³ / cm ² or more on a porous base material having an average pore diameter of 1 to 30 μm made of collagen. And a step 2 of culturing the porous substrate seeded with the fibroblasts obtained in the above step 1 in a serum-added medium.

The porous substrate used in the above step 1 is made of collagen. Since collagen is excellent in fibroblast adhesion, it can be cultured with high density by attaching a large amount of fibroblasts. In addition, the porous base material made of collagen is gradually decomposed along with the culture, and a part thereof is replaced with the elastic fiber component secreted from the fibroblasts, thereby forming an elastic fiber tissue.
The porous substrate may be any material to which fibroblasts adhere. For example, in addition to collagen, natural polymers such as proteins such as gelatin and polysaccharides such as hyaluronic acid; Synthetic polymers that can be decomposed and absorbed in the body can also be used.

The above-mentioned porous base material has a lower limit of the average pore diameter of 1 μm and an upper limit of 30 μm. If it is less than 1 μm, the seeded fibroblasts cannot enter the porous substrate, and an elastic fiber tissue having a three-dimensional thickness cannot be obtained. Since the major diameter of human fibroblasts is approximately 50 to 70 μm, when the pore diameter of the substrate exceeds about 30 μm, most of the seeded fibroblasts fall into the porous substrate, and the density of fibroblasts Becomes insufficient and the elastic fiber component is not secreted. A preferred lower limit is 5 μm and a preferred upper limit is 25 μm.

Although it does not specifically limit as thickness of the said porous base material, A preferable minimum is 0.1 mm and a preferable upper limit is 3 mm. If the thickness is less than 0.1 mm, an elastic fiber tissue having a sufficient thickness may not be formed, or the handleability at the time of transplantation may be inferior. If it exceeds 3 mm, nutrition supply to the seeded fibroblasts May be inferior.

The porous base material has a preferred lower limit of 40% and a preferred upper limit of 60% of the residual weight after being immersed in an aqueous collagenase solution with a concentration of 0.5 units / mL at 37 ° C. for 100 minutes. If it is less than 40%, the porous substrate is quickly decomposed by the enzyme secreted by the cells after seeding of fibroblasts, and the cell scaffold is lost before the three-dimensional structure is formed. May not be possible. If it exceeds 60%, the fibroblasts cannot penetrate into the pores of the culture substrate, and may become a single-layer cell sheet as in the case of culturing on a petri dish. The decomposability of such a porous substrate can be controlled by devising the temperature and time when performing the crosslinking treatment.
In the present specification, the weight residual ratio means the weight measured after the porous substrate remaining after being immersed in the collagenase aqueous solution is filtered using a filter and sufficiently dried, before the collagenase aqueous solution is immersed. It is calculated as a ratio to the weight of the porous substrate measured in advance.

The method for producing the porous substrate is not particularly limited. For example, a collagen sponge obtained by adding a fat-soluble organic solvent to a collagen aqueous solution, homogenizing and foaming, and then vacuum lyophilizing the sponge. Alternatively, a collagen sponge obtained in this way may be further pressed to adjust the pore diameter.

In the said process 1, a fibroblast is seed | inoculated with respect to the said porous base material. The lower limit of the seeding density is 1 × 10 ³ / cm ² . If it is less than 1 × 10 ³ / cm ² , the density of fibroblasts is insufficient and elastic fiber components are not secreted. A preferred lower limit is 1 × 10 ⁴ / cm ² . Although there is no particular limitation on the upper limit of the seeding density, seeding exceeding 1 × 10 ⁷ / cm ² only increases the number of cells that cannot adhere to the porous substrate and has little substantial effect.

In step 2, the porous substrate seeded with the fibroblasts obtained in step 1 is cultured in a serum-added medium.
The serum-added medium is not particularly limited, and examples thereof include those obtained by adding about 1 to 10% by weight of fetal bovine serum to a common culture solution such as MEM and DMEM.
The culture period varies depending on the pore size of the porous substrate, the cell seeding density, the type of the serum-added medium, and the like, but is not particularly limited, but an elastic fiber tissue is formed by culturing for about 1 to 4 weeks.

The three-dimensional cultured elastic fiber tissue of the present invention manufactured by such a manufacturing method contains at least elastic fiber components such as elastin and fibrillin. Presence of elastin, fibrillin, etc. can be confirmed by immunostaining.
The production method for producing the three-dimensional cultured elastic fiber tissue of the present invention is also one aspect of the present invention.

The lower limit of the thickness of the three-dimensional cultured elastic fiber tissue of the present invention is 5 μm. When the thickness is less than 5 μm, the strength as a tissue is insufficient at the time of transplantation and handling is difficult, and even if transplanted, the inherent performance of an elastic fiber tissue that imparts flexibility cannot be exhibited. A preferred lower limit is 10 μm, and a more preferred lower limit is 30 μm.

The three-dimensional cultured elastic fiber tissue of the present invention may be combined with a reinforcing material made of a bioabsorbable material for the purpose of increasing strength.
Non-woven fabric made of synthetic bioabsorbable polymers such as poly-L-lactide, polyglycolide, L-lactide-ε-caprolactone copolymer, especially natural polymers such as aliphatic polyester, collagen and gelatin as the reinforcing material And sponge.

The three-dimensional cultured elastic fiber tissue of the present invention has a three-dimensional thickness while containing elastic fiber components such as elastin and fibrillin. Thereby, it has sufficient strength even during transplantation and the like, and is excellent in handleability. Furthermore, after transplanting into a living body, the inherent performance of elastic fiber tissue, which imparts flexibility to the tissue, can be exhibited.

According to the present invention, a three-dimensional cultured elastic fiber tissue having a three-dimensional thickness and expressing an elastic fiber component essential for the elastic fiber tissue such as elastin and fibrillin, and the three-dimensional cultured elastic fiber tissue The manufacturing method of can be provided.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
(1) Preparation of porous substrate Type I collagen in a 0.3% aqueous solution (pH 3) was diluted 3-fold with 15% ethanol to obtain a 0.1% collagen and 10% ethanol aqueous solution. Further, 15 g of this solution was poured into a petri dish having a diameter of 9 cm, frozen at −135 ° C., and freeze-dried under the conditions of vacuum degree: 0.1, drying temperature: 40 ° C., drying time: 24 hours to obtain a collagen sponge. . Then, the porous base material was obtained by performing thermal crosslinking at 105 degreeC for 24 hours under vacuum.
The obtained porous substrate had an average pore diameter of 15 μm and a thickness of 1 mm.

The resulting porous substrate was subjected to an enzymatic degradation test according to the following procedure.
The produced porous substrate was immersed in a solution obtained by adding collagenase to Tris buffer (pH 7.4) so as to be 0.5 units / mL at 37 ° C. After a certain time, the porous substrate was taken out, washed with distilled water and dried. The weight of the dried porous substrate was measured, and the weight residual ratio was calculated by comparing with the weight before the test.
The test results are shown in FIG. Under the present test conditions, the obtained porous substrate showed a linear decrease in weight, and the residual weight ratio after 100 minutes was about 50%.

(2) Cell seeding and culture Human foreskin-derived fibroblasts are seeded on the resulting porous substrate to a seeding density of 1 × 10 ⁵ / cm ² , and then 10% bovine serum-added DMEM / F12 Cultured in medium. The culture was continued for 4 weeks after the start of the culture.

(3) Detection of elastic fiber tissue Four weeks after the start of the culture, the cultured tissue was fixed in formalin to prepare a paraffin block. The presence of elastin and fibrillin 1 was confirmed by immunostaining using the obtained paraffin sections. The respective immunostained images are shown in FIGS. 1 and 2, it was confirmed that the elastic fiber tissue was formed with a thickness of about 50 μm.

(Comparative Example 1)
(1) Preparation of porous substrate Type I collagen in 0.3% aqueous solution (pH 3) was stirred with a homogenizer and then frozen at -40 ° C. Furthermore, freeze-drying was performed under the conditions of vacuum degree: 0.1, drying temperature: 40 ° C., drying time: 24 hours, and a collagen sponge was obtained. Thereafter, thermal crosslinking was performed at 105 ° C. for 24 hours under vacuum, and further, chemical crosslinking reaction was performed in a 0.2% glutaraldehyde aqueous solution for 24 hours. The obtained cross-linked collagen sponge was sufficiently washed with water to remove glutaraldehyde, and then immersed in 15% ethanol and frozen at -135 ° C. Furthermore, freeze-drying was performed under the conditions of vacuum degree: 0.1, drying temperature: 40 ° C., drying time: 24 hours, and a porous substrate was obtained.
The obtained porous substrate had an average pore diameter of 90 μm and a thickness of 3 mm.

(2) Cell seeding and culture Human foreskin-derived fibroblasts are seeded on the resulting porous substrate to a seeding density of 1 × 10 ⁵ / cm ² , and then 10% bovine serum-added DMEM / F12 Cultured in medium. The culture was continued for 4 weeks after the start of the culture.

(3) Detection of elastic fiber tissue Four weeks after the start of the culture, the cultured tissue was fixed in formalin to prepare a paraffin block. The presence of elastin and fibrillin 1 was confirmed by immunostaining using the obtained paraffin sections. The respective immunostained images are shown in FIGS. 3 and 4, it was confirmed that no elastic fiber tissue was formed in this comparative example.

According to the present invention, a three-dimensional cultured elastic fiber tissue having a three-dimensional thickness and expressing an elastic fiber component essential for the elastic fiber tissue such as elastin and fibrillin, and the three-dimensional cultured elastic fiber tissue The manufacturing method of can be provided.

2 is an elastin-stained image of the tissue prepared in Example 1. 2 is a fibrillin 1 stained image of the tissue prepared in Example 1. FIG. 2 is an elastin-stained image of the tissue prepared in Comparative Example 1. 2 is a fibrillin 1 stained image of a tissue prepared in Comparative Example 1. 2 is a graph showing the results of an enzymatic degradation test of a porous substrate produced in Example 1. FIG.

Claims (2)

Obtained in Step 1 in which only a fibroblast is seeded at a density of 1 × 10 ³ / cm ² or more with respect to a porous substrate having an average pore diameter of 1 to 30 μm made of thermally cross-linked collagen, and obtained in Step 1 above. A three-dimensional cultured elastic fiber tissue produced by a production method comprising culturing a porous substrate seeded with the obtained fibroblasts in a serum-added medium,
The porous substrate has a residual weight ratio of 40-60% after being immersed in an aqueous collagenase solution having a concentration of 0.5 units / mL at 37 ° C. for 100 minutes,
A three-dimensional cultured elastic fiber tissue having a thickness of 5 μm or more and containing elastin and fibrillin. Step 1 of seeding only fibroblasts at a density of 1 × 10 ³ / cm ² or more with respect to a porous substrate having an average pore diameter of 1 to 30 μm made of heat-crosslinked collagen;
A step of culturing the porous substrate seeded with fibroblasts obtained in step 1 in a serum-added medium, and a method for producing a three-dimensional cultured elastic fiber tissue,
The three-dimensional culture elasticity characterized in that the porous substrate has a residual weight ratio of 40-60% after being immersed in an aqueous collagenase solution having a concentration of 0.5 units / mL at 37C for 100 minutes. A method for producing fibrous tissue.