WO2004112860A1

WO2004112860A1 - Material for cell adhesion and proliferation

Info

Publication number: WO2004112860A1
Application number: PCT/JP2003/007731
Authority: WO
Inventors: Yoshihito Osada; Jian Ping Gong
Original assignee: Hokkaido Technology Licensing Office Co., Ltd.
Priority date: 2003-06-18
Filing date: 2003-06-18
Publication date: 2004-12-29
Also published as: AU2003242464A1

Abstract

A material for cell adhesion and proliferation which is made of a polyacrylic acid hydrogel and appropriately usable in culturing cells, preferably culturing cells to a confluent or subconfluent state.

Description

Specification

Cell adhesion and proliferative materials

The present invention relates to a bioadhesive / proliferative material suitable for cell culture, preferably, capable of culturing cells to a confluent or subconfelt state, in particular, a biomaterial comprising a hydrogel made of a synthetic polymer as a raw material The present invention relates to the above-mentioned material which can be used as a medical device. Background art

In recent years, Hyde-mouth gel has attracted attention as an important application as a support for cell culture and tissue regeneration in advanced medical fields such as tissue engineering and regenerative medical engineering. Since biosafety and biocompatibility are required for use in these applications, hydrogels mainly using collagen derived from living organisms are mainly used. In addition, Japanese Patent Application Laid-Open No. 2002-1866874 proposes a hydrogel similarly using a naturally occurring silk fiber mouth protein as a raw material. This silk-fiber mouth protein is a material that is also used as a surgical suture, and is a natural polymer with excellent biocompatibility and produced from silkworms.

However, when using natural products as raw materials, there are problems in industrial production, such as uneven quality and increased production costs. For example, it is said that collagen is produced by extraction from animal tissues and cannot be sterilized in an autoclave, which may cause contamination with unknown pathogens. On the other hand, although the use of synthetic macromolecules has been studied, no examples have been reported so far in which endothelial cells were cultured to a confluent or subconfelted state on synthetic polymer gels.

The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a cell adhesion / proliferation material composed of a hydrogel using a synthetic polymer as a raw material. Disclosure of the invention

The present invention (1) is a cell adhesion / proliferation material comprising a polyacrylic acid octaidogel.

The present invention (2) is the cell adhesion / proliferation material according to the above-mentioned invention (1), wherein the degree of crosslinking is in the range of 0.5 to 2.5 mol%.

The present invention (3) is the cell adhesion / proliferation material according to the above invention (1) or (2), wherein the polyacrylic acid hydrate mouth gel has a swelling degree of 20 to 30.

The present invention (4) is a medical device using the cell adhesion / proliferative material according to any one of the inventions (1) to (3).

The present invention (5) is the above-mentioned invention, which is an artificial blood vessel or a template for cell culture.

It is a medical device of (4). BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the results obtained by rolling the human vascular endothelial cells for the polyacrylic acid hydrated mouth gel of Example 1 (crosslinking degree: 2 mol%) and the polymethacrylic acid hydrated mouth gel of Comparative Example 1 (crosslinking degree: 2 mol%) after 6 hours. 1 shows the cell morphology. In the figure, the black part is in the extended state (the cells are attached to the substrate gel and is extended), the gray part is weakly adsorbed (the cells are attached to the substrate gel but the shape is round), and white The part shows floating (the cells are not attached to the gel of the substrate). FIG. 2 shows the polyacrylic acid hydrate mouth gel (degree of crosslinking: 2 mol%) of Example 1, the polyacrylic acid hydrate mouth gel (degree of crosslinking: 3 mol%), and the polymethacrylate hydrogel (degree of crosslinking: (2 mol%), showing the results of counting the cells in an extended state over time after rolling human vascular endothelial cells. In the figure, the vertical axis indicates the number of extending cells, and the horizontal axis indicates time (h). FIG. 3 shows the results of the cells on the polyacrylic acid hydrated mouth gel (cross-linking degree: 2 mol%) of Example 1 at 6 hours (a) and 192 hours (b) after the human vascular endothelial cells were wound. This is an electronic photograph of the situation. FIG. 4 shows the extension rate of various polyacrylic acid hydrogels having different degrees of swelling in the culture medium after the human vascular endothelial cells were wounded for 6 hours. In the figure, the vertical axis shows the extension rate (%), and the horizontal axis shows the degree of swelling. Figure 5 Fig. 3 shows the cell morphology (after 6 hours) of the BC-AA double network gel (cross-linking degree: 2 mo 1%) of Example 3. In the figure, the black part is in the extended state (the cell is attached to the gel on the substrate and stretched), the gray part is weakly adsorbed (the cell is attached to the gel on the substrate but the shape is round), The shaded area indicates floating (the cells are not attached to the gel on the substrate). FIG. 6 is an endothelial cell growth curve of the BC-AA double network gel (cross-linking degree: 2 mol%) of Example 3. FIG. 7 shows the cell morphology (after 6 hours) of Examples 5 and 6 {AA—A Am—AA triple (TN, triple mesh) gel}. In the figure, the gray part is in the extended state (the cell is attached to the gel of the substrate and stretched), the black part is weakly adsorbed (the cell is attached to the gel of the substrate but the shape is round), The white part indicates suspension (the cells are not attached to the gel on the substrate). FIG. 8 is an endothelial cell growth curve of an AA-AAm-AA triple (TN) gel of Example 6. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described. A feature of the present invention is that a polyacrylic acid hydrate gel using synthetic polyacrylic acid as a raw material is used as a cell adhesion / proliferation material. That is, this polyacrylic acid hydrate mouth gel has cell adhesion and proliferation properties similar to a natural polymer, unlike a hydrogel obtained from another synthetic polymer, although it is made of a synthetic polymer as a raw material. This feature was first discovered by the present inventors. It is unknown at this time why this gel has such properties, but one possible reason is that the gel surface provides optimal tension for cells to spread, There is a very high possibility that the adsorption of related adhesive proteins (eg, laminin, fibronectin, etc.) is extremely high.

The polyacrylic acid octaidogel used in the present invention preferably has a degree of crosslinking of 1 to

It is in the range from 4 mol%, particularly preferably from 0.5 to 2.5 mol%.

Here, the “crosslinking degree” refers to a value expressed as a percentage of the molar concentration of the crosslinking agent to the molar concentration of the monomer charged. Actually, there may be a few monomers that did not participate in the polymerization and some crosslinking agents that did not participate in the crosslinking. The degree of crosslinking of the gel in this specification is as described above. The polyacrylic acid hydrate mouth gel used in the present invention preferably has a degree of swelling in the range of 20 to 30. Here, “swelling degree” is represented by the following formula:

Degree of swelling = a value obtained by swelling gel weight (W _w ) / dry gel weight (W _D ). The “hide mouth gel” as used herein refers to a gel in which the solvent is water, but may contain a water-soluble solvent (eg, alcohol) in an amount that does not affect the gel.

Further, the polyacrylic acid hydrate mouth gel used in the present invention may be an interpenetrating network hydrogel or a semi-interpenetrating network hydrogel from the viewpoint of improving the strength. For example, in the case of a gel having an interpenetrating network structure with a mouth opening, the other network structure is uniformly entangled with the base network structure (crosslinked product of polyacrylic acid) throughout the gel. In addition, in the case of a semi-interpenetrating network structure mouth-opening gel, the linear network is uniformly entangled with the base network structure (crosslinked product of polyacrylic acid) throughout the gel. The other network structure and linear polymer are not particularly limited as long as they have biocompatibility, and may be natural products such as collagen or bacterium cellulose (BC), or PN a SS (styrene sulfone). It may be a synthetic polymer (a crosslinked product thereof) such as a sodium acid polymer. In addition, "interpenetrating network structure octaid gel" and "semi-interpenetrating network structure hydrating gel" are concepts that include not only double network type but also gels with triple or quadruple network structure. .

Next, the cell adhesion / proliferation material, which is the use of the polyacrylic acid hydrated mouth gel, is a cell in the advanced medical field such as tissue engineering or regenerative medicine engineering, which requires cell adhesion and cell proliferation. It refers to a material used as a culture support or a tissue regeneration support, and includes, for example, materials used for medical devices such as artificial blood vessels or cell culture templates. Here, “cell adhesion” refers to a property to which anchorage-dependent cells can adhere, and “cell proliferative” means that the proliferated cells are in a state of a confluent or subconfelted state. .

In addition, if the cells to be proliferated are anchorage-dependent cells, regardless of established cells or primary cells, for example, fibroblasts, smooth muscle cells, vascular endothelial cells, and epithelial cells are listed. I can do it.

Next, the method for producing polyacrylic acid hydrogel according to the present invention will be described. The polyacrylic acid hydrate mouth gel according to the present invention can be produced, for example, by adding acrylic acid, a polymerization initiator and a cross-linking agent to a suitable solvent (for example, water) and subjecting to thermal polymerization. As the polymerization initiator, for example, a water-soluble thermal catalyst such as potassium persulfate, and a redox initiator such as potassium persulfate-sodium thiosulfate can be used. As the crosslinking agent, for example, N, N'-methylenebisacrylamide can be used. The solvent (7J) finally contained in the gel may be replaced with water by using water as a solvent from the production stage or by exchanging the solvent after production.

Finally, the method of using the polyacrylic acid hydrogel according to the present invention will be described. The polyacrylic acid hydrogel according to the present invention can be used as a cell adhesion-proliferating medical device, and can be said to be particularly suitable for an artificial blood vessel. Artificial blood vessels currently in use are prone to clots and do not have the permeability of substances to the outside of blood vessels, so they have a large effect on blood vessels. The material comprising the polyacrylic acid hydrogel according to the present invention is flexible, has a selective substance permeability, and has cell adhesion and proliferation properties. The reason is that cells can be constructed. It is also suitable for use as a template for cell culture. Example

Hereinafter, the present invention will be described more specifically with reference to examples. It goes without saying that the present invention is not limited to the examples. Example 1 (Example of producing PAA gel having a degree of crosslinking of 2 mol%)

40 ml of a 2 mol / L aqueous solution of acrylic acid (AA) as a monomer and a crosslinking agent

Combine 4 ml of a 0.4 mol / L aqueous solution of Ν, Ν'-methylenebisacrylamide (Μ Β ΑΑ) with 1 ml of a 0.1 mo 1 / L aqueous solution of 2-oxodaltalic acid, which is an initiator, and mix with water. Adjustment gave 80 ml of aqueous solution. This aqueous solution was deoxygenated using nitrogen gas. Subsequently, this deoxygenated aqueous solution was added to one glass of a polymerization vessel similar to that in Example 1. After pouring into the opening of the silicon plate placed on the plate, overlaying the other glass plate on the silicon plate and sealing around the opening, a UV lamp with a wavelength of 365 nm (22 W, 0.34 A) was used. The polymer was irradiated with ultraviolet rays at room temperature for 6 hours to polymerize it, thereby producing a PAA gel having a degree of crosslinking of 2 mol%. The swelling degree of the obtained PAA gel was 30. Example 2 (Example of manufacturing PAA gel having a degree of crosslinking of 3 mol%)

In the same manner as in Example 1, the amount of the crosslinking agent was changed by 0.6 mol / L to produce a PAA gel having a degree of crosslinking of 3 mo 1%. The degree of swelling of the obtained PAA gel was 26. Comparative Example 1 (Production example of PMAA gel with a degree of crosslinking of 2 mol%)

A PMAA gel having a degree of crosslinking of 2 mol% was prepared in the same manner as in Example 1 except that the monomer was changed to methacrylic acid. Test Example 1 (Adhesion test of human vascular endothelial cells)

The osmotic pressure of each of the hydrated gels (swollen gels) of Example 1 and Comparative Example 1 was adjusted with a HEP ES buffer solution and then with an Earle's balanced salt solution. Then, after sterilization by autoclave, the sterilization gel in serum-free medium, placed in C_〇 ₂ incubator in one of 37 ° C, allowed to equilibrate swell again. The gel was spread on a cell, and endothelial cells suspended in a medium containing serum were seeded on the gel ( ⁴ × 10 ⁴ human vascular endothelial cells / 1.9 lcm ² gel). After 6 hours, the morphology of endothelial cells on the gel was examined. Figure 1 shows the results. As can be seen from the figure, the PAA hydrated gel of Example 1 had nearly 80% of the cells in the extended state and almost no cells in the suspended state, whereas the PMAA hydrogel of Comparative Example 1 The gel was found to have less than 50% stretched cells. Test Example 2 (Proliferation test of human vascular endothelial cells)

In each of Examples 1 and 2 and Comparative Example 1 (swelled gel), The osmotic pressure was adjusted with an impingement solution and then with an Earl's balanced salt solution. Then, after sterilization by autoclave, the sterilization gel in serum-free medium, placed into the C_〇 ₂ incubator 37 ° C, allowed to equilibrate swell again. The gel was spread on a cell, and endothelial cells suspended in a medium containing serum were seeded on the gel (4 × 10 ⁴ human vascular endothelial cells / 1.9 lcm ² gel). Then, the number of extending cells was counted over time. Figure 2 shows the results. As can be seen from FIG. 2, in the PAA hydrogels of Examples 1 and 2, the cells in the extended state grew steadily over time, and the PAA hydrated gel of Example 1 However, in the PMAA hydrated mouth gel of Comparative Example 1, the number of cells decreased over time, and all cells died after 100 hours. In the case of the PAA hydrogel of Example 1, cells were observed for a further 192 hours (8 days). Figure 3 shows images of the cells on the gel at 6 hours (a) and 192 hours (b) after rolling. Test Example 3 (Relationship test between degree of swelling and extension)

According to the method described in Example 1, various PAA hydrogels having different degrees of swelling were produced, and the morphology of endothelial cells on the gel 6 hours after winding was examined in the same manner as in Test Example 1 above. Fig. 4 shows the results. As can be seen from Fig. 4, a particularly high elongation was observed when the degree of swelling was in the range of 20 to 30. Example 3 {Production of BC-PAA gel (DN gel) having a degree of crosslinking of 2 mol%}

Bacto Pepton 0.5%, Yeast Extract 0.5%, disodium hydrogen phosphate 0.27%, citric acid 0.115%, glucose 2% were dissolved in deionized water to obtain HS medium. . Next, the medium was divided into about 15-30 ml portions of an Erlenmeyer flask, the flask was capped, and autoclaved at 120 ° C. for 20 minutes. Thereafter, the acetic acid bacteria (ATCC 53582) stored at -80 were removed and transferred to the medium. Then, when allowed to stand at 28-30 ° C for about 2-3 days, bacterial cellulose starts to be produced from the air interface side of the culture medium. Further culturing was continued until the thickness became about 2 bandits. The obtained bacterial cellulose was washed with a 1% &# 11 aqueous solution for 1 day, and further subjected to solvent exchange with pure water for 2 days. Incidentally, the swelling degree of this pacteria cellulose was 46.

40 ml of lmol / L acrylic acid (AA) aqueous solution as monomer and crosslinker

4 ml of an aqueous solution of 0.2 mol / L of N, Ν'-methylenebisacrylamide (と) and 1 ml of an aqueous solution of 0.1 mol / L of 2-oxodaltalic acid are combined and adjusted with water to obtain an 80 ml aqueous solution. Obtained. This aqueous solution was deoxygenated using nitrogen gas. Subsequently, bacterial cellulose is impregnated with the deoxygenated aqueous solution, and irradiated with ultraviolet rays at room temperature for 6 hours using a 365-nm wavelength UV lamp (22 W, 0.34 A) to polymerize, so that the degree of crosslinking is 2 mol% { (MBAA amount: 0.2 mol / lX 4 ml / AA amount: lmol / lX 40 ml) = 2 mol} was prepared. The same container and procedure as in Example 1 were used for the polymerization of the gel. The swelling degree of this double network gel was 17. Test Example 4 (Adhesion and proliferation test of human vascular endothelial cells)

The morphology of endothelial cells on the gel after 6 hours had passed since ⁴ × 10 ⁴ human vascular endothelial cells were seeded on the gel according to the same procedure as in Test Examples 1 and 2 for the DN gel of Example 3 Was investigated. Figure 5 shows the results. As can be seen from this figure, as much as 30% of the cells were extended on this gel. When observation was continued for a long time, it reached subconfluent after about 9 days. Example 5 (Production of triple (TN) gel of AA—AAm AA)

Area 100 OmmX 100 orchid, thickness 2 thighs perimeter with cutter from silicon plate

A frame of 80mmX 80 face, width 5rai was cut out, and a groove of three bandits was made in one place of the frame. The silicon frame was sandwiched between two glass plates of 10 Omm x 100 mm and 3 thicknesses, and a polymerization container was assembled.

40 ml of 2 mol / L acrylic acid (AA) aqueous solution as monomer and cross-linking agent 0.1 mol / L of N, N'-methylenebisacrylamide (MBAA) aqueous solution (4 ml) and 0.1 mol / L of 2 oxodaltalic acid aqueous solution (1 ml) are combined and adjusted with water to obtain 80 ml of the aqueous solution. Obtained. This aqueous solution was deoxygenated using nitrogen gas. Subsequently, the deoxygenated aqueous solution is poured into an opening of a silicon plate placed on one glass plate of the polymerization vessel, and the other glass plate is stacked on the silicon plate to seal around the opening. UV-rays (22 W, 0.34 A) with a wavelength of 365 nm were irradiated with ultraviolet rays at room temperature for 6 hours to polymerize, producing an 88-gel with a cross-linking degree of 0.511101%.

50 ml of a 2 mol / L acrylamide (AAm) aqueous solution as a monomer, 0.1 ml / L of 架橋, Ν'-methylenebisacrylamide (ΜΒΑΑ) aqueous solution as a cross-linking agent, and 0.1 lmol / L as an initiator L and 1 ml of 2-oxodaltaric acid aqueous solution were combined and adjusted with water to obtain 200 ml of an aqueous solution (immersion solution). This immersion solution was deoxygenated using nitrogen gas.

Next, 4 g of the immersion solution and the single network type gel were placed in a seal container having a sufficiently larger capacity than the gel. This container was placed in a refrigerator at 4 ° C. for 24 hours, and the monomer, crosslinking agent and initiator in the immersion solution were diffused and permeated into the gel. In this process, the container was occasionally gently shaken in order to equalize the concentration of the immersion liquid.

Next, the gel was taken out of the immersion liquid, cut into an appropriate size, and then sandwiched between two glass plates having a width of 100 dragons, a length of 10 Omm, and a thickness of 3 mm so that air bubbles would not be mixed. . After sealing the four sides of the two glass plates, ultraviolet rays were irradiated for 6 hours at room temperature using a 365 nm wavelength UV lamp (22 W, 0.34 A). At this time, the AAm monomer diffused in the gel was polymerized to obtain a double network gel. The degree of crosslinking of the second network structure of this double network gel was 0.1 mol%.

20 ml of a 2 mol / L acrylic acid aqueous solution as a monomer and 4 ml of a 0.1 mol / L N, N'-methylenebisacrylamide (MBAA) aqueous solution as a crosslinking agent Was combined with 1 ml of 0.1 mo 1 / L aqueous 2-oxodaltaric acid solution as an initiator, and adjusted with water to obtain 200 ml of an aqueous solution (immersion solution). This immersion solution was deoxygenated using nitrogen gas.

Next, the dipping solution and 4 g of the double network type gel were placed in a sealed container having a sufficiently larger capacity than the gel. This container was placed in a refrigerator at 4 ° C. for 24 hours, and the monomer, crosslinking agent and initiator in the immersion solution were diffused and permeated into the gel. In this step, the container was occasionally gently shaken in order to equalize the concentration of the immersion liquid.

Next, the gel was taken out from the immersion liquid, cut into an appropriate size, and then sandwiched between two glass plates having a width of 10 Omm, a length of 100, and a thickness of 3 mm to prevent air bubbles from being mixed. After sealing the four sides around the two glass plates, ultraviolet light was irradiated for 6 hours at room temperature using a 365 nm wavelength UV lamp (22 W, 0.34 A). At this time, the AA monomer diffused in the gel was polymerized to obtain a triple gel. The degree of crosslinking of the third network structure of the triple gel was 1 mol%. Example 6 (Preparation of triple (TN) gel of A A—A Am—A A)

The title gel was produced in the same manner as in Example 5, except that 6 ml of MBAA was used in the production process of the triple gel. The degree of crosslinking of the third network structure of this triple gel was 1.5 mol%. Test Example 5 (Human vascular endothelial cell adhesion / proliferation test)

For the TN gels of Examples 5 and 6, following the same procedure as in Test Example 1, the morphology of endothelial cells on the gel 6 hours after ⁴ × 10 ⁴ human vascular endothelial cells were seeded on the gel Was investigated. Figure 7 shows the results. As can be seen from this figure, as much as 40% of the cells were extended on this gel. Furthermore, when the TN gel of Example 6 was applied and the observation was continued for a long time according to the same procedure as in Test Example 2, the confluent reached 144 hours later.

Claims

The scope of the claims

1. Cell adhesion and proliferation material consisting of polyacrylic acid hydrated mouth gel.

2. The cell-adhesive / proliferative material according to claim 1, wherein the degree of crosslinking is in the range of 0.5 to 2.5 mol%.

3. The cell adhesion / proliferation material according to claim 1 or 2, wherein the polyacrylic acid hydrate mouth gel has a swelling degree of 20 to 30.

4. A medical device using the cell adhesion / proliferative material according to any one of claims 1 to 3.

5. The medical device according to claim 4, which is an artificial blood vessel or a template for cell culture.