JPH07114953B2 - How to make beads - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing beads using collagen and / or gelatin.

[Conventional technology]

Conventionally, beads made of collagen and / or gelatin have been used as a support substrate for cell culture, a biomaterial (for example, a biomaterial for medical use), a carrier for a bioactive substance, and the like. Several methods for producing such collagen beads have been proposed.

For example, in JP-A-55-47130, an aqueous collagen solution is added dropwise to an organic solvent that is not easily miscible with water (hereinafter, referred to as an “immiscible organic solvent”), or an aqueous collagen solution is added to the organic solvent. A method has been proposed in which the collagen aqueous solution is made spherical by emulsification, and then the collagen in the spherical collagen aqueous solution is crosslinked to obtain collagen beads.

Further, in JP-A-60-174725, there is a method of producing collagen beads by dispersing an aqueous collagen solution in a poorly miscible organic solvent as a large number of droplets to emulsify and solidifying the droplets. Proposed. The obtained collagen beads are used as a support substrate for animal cells to be cultured in large quantities.

[Problems to be Solved by the Invention]

In the above-mentioned manufacturing method, a poorly miscible organic solvent is used to make the collagen aqueous solution into particles. Therefore, it is necessary to cross-link the collagen to maintain its shape and then remove the organic solvent. However, this organic solvent has a problem that it cannot be removed by water alone.

An organic solvent that mixes a poorly miscible organic solvent with water (hereinafter,
It is washed and removed with a "miscible organic solvent"), and this miscible organic solvent is further washed and removed with water or an aqueous solution. Therefore, cleaning is very troublesome.

Moreover, since the collagen beads are in the form of a gel containing a large amount of water, they are soft and break or crush during washing. Therefore, it is difficult to manufacture in large quantities.

Therefore, it is a first object of the present invention to provide a method for producing beads that are less likely to be crushed or broken by using collagen and / or gelatin and without using an organic solvent.

Further, a second object of the present invention is to provide a method capable of producing beads in which collagen and / or gelatin are hard to flow out.

Further, the present invention solves the first and second problems,
Moreover, the third object is to provide a method capable of producing beads made of collagen and / or gelatin.

[Means for Solving the Problems]

The method for producing beads according to the present invention comprises molding a bead-forming agent and a bead-forming agent aqueous solution containing a polysaccharide that forms a water-insoluble gel by binding with a metal ion, to a metal ion aqueous solution by dropping. At the same time as obtaining the granular bead forming agent aqueous solution, the granular bead forming agent aqueous solution is solidified by gelling the polysaccharide.

Further, the bead forming agent is cross-linked after the gelation of the polysaccharide.

Furthermore, the gelled substance of the polysaccharide is removed after the crosslinking.

[Action]

Beads can be obtained without using any organic solvent because the beads forming agent aqueous solution is dropped to form a granular form, and the polysaccharide is gelled with the metal ion aqueous solution to solidify the granular beads forming agent aqueous solution. . Therefore, it can be washed with water or an aqueous solution. Moreover, the beads are
As it contains a gelled substance of polysaccharides, it is strong, hard to break and hard to crush. The solidification can occur for a very short time, the so-called instant.

Cross-linking the bead forming agent prevents outflow due to diffusion of the bead forming agent.

By crosslinking the bead-forming agent and then removing the gelled product of the polysaccharide, beads composed of collagen and / or gelatin are easily obtained.

〔Example〕

In the present invention, a bead forming agent made of at least one of collagen and gelatin, and a bead forming agent aqueous solution containing a polysaccharide are used.

The type of collagen used in this invention is not particularly limited,
Various ones are available. Collagen is a fibrous protein that is ubiquitous in the body of animals, and is suitable for use as a support substrate for culturing animal cells, a biomaterial, a carrier for a bioactive substance, DDS, and the like.

The type of gelatin used in the present invention is not particularly limited, and various types can be used. Gelatin is a protein derived from collagen, and like collagen, is suitable for the above-mentioned uses.

In the present invention, any collagen and / or gelatin can be used. This is because the aqueous solution of the bead-forming agent solidifies in a very short time due to gelation of the polysaccharide, so that there are almost no restrictions on the physical properties of collagen and / or gelatin, and the hardness of the solidified product is the polysaccharide. This is because it depends on the gel.

The polysaccharide used in the present invention is not particularly limited as long as it can form a strong gelled product by gelling. For example, alginic acid, alginate (for example, sodium alginate, etc.), duran gum, pectin, carrageenan and the like can be mentioned.

The metal ion aqueous solution gels the polysaccharide and is appropriately selected and used according to the polysaccharide. For example, when one or more of alginic acid, alginate, duran gum, pectin and the like is used as the polysaccharide, a divalent metal ion solution is preferably used. Examples of the divalent metal ion solution include calcium chloride aqueous solution and barium chloride aqueous solution. Further, when one or more of carrageenan and duragan are used as the polysaccharide, a monovalent metal ion, for example, an aqueous potassium chloride solution is preferably used.

The bead-forming agent aqueous solution is dropped into the metal ion aqueous solution to perform molding and gelation to obtain granular beads.

The concentration of the polysaccharide in the aqueous bead-forming agent solution is not particularly limited as long as it is a concentration capable of forming a gel by binding with metal ions. For example, in the case of sodium alginate,
Although it depends on the concentration of metal ions such as calcium ions in the metal ion aqueous solution, it is preferably about 0.2% by weight or more, more preferably 0.5 to 3% by weight. If the concentration is higher than this, the viscosity of the aqueous solution of the bead forming agent becomes high, and it may be difficult to drop in a uniform amount.

Further, the concentration of the bead forming agent is not particularly limited as long as it is a concentration below which a solution in which dropping is smoothly performed can be obtained. If the concentration of the bead-forming agent is too low, the beads after removal of the polysaccharide will of course be very small. For example, when collagen is used alone and gelatin is not used, the collagen concentration of the aqueous bead forming agent solution is preferably in the range of 0.1 to 5.0% by weight.

The ratio of bead former to polysaccharide is related to the density of collagen and / or gelatin beads obtained when the polysaccharide is removed. That is, the higher the bead former / polysaccharide ratio, the higher the density of the beads obtained after removal of the polysaccharide, and thus the stronger the beads. The ratio of bead forming agent to polysaccharide can be appropriately selected depending on the required properties of the resulting beads.

The metal ion contained in the metal ion aqueous solution may be determined according to the polysaccharide used. For example, when sodium alginate is used, calcium ion is suitable, and an aqueous solution of calcium chloride, calcium sulfate or the like can be used. In this case, generally, the higher the calcium ion concentration, the faster the gelation rate and the stronger gel is obtained. For example, in the case of calcium chloride, 0.5-3% by weight
Aqueous solution is preferable. If the concentration is lower than this, gelation may be delayed, and if the concentration is higher than this, the effect is not improved because the calcium ion is excessive and the effect is useless.

When collagen is used, the temperature of the metal ion aqueous solution is
In order to avoid the thermal denaturation, it is preferably carried out at, for example, 15 ° C or lower.

The method of dropping the aqueous solution of the bead forming agent is not particularly limited, and for example, it may be dropped from an injection needle or a thin tube. The particle size of the obtained beads is related to the concentration of the bead forming agent and the polysaccharide in the bead forming agent aqueous solution used, but can be freely set mainly by changing the diameter of the injection needle or the thin tube to be dropped. it can. For example, when using a bead-forming agent aqueous solution containing 0.5% by weight of collagen and 1% by weight of sodium alginate and dropping it into an aqueous solution of 1% by weight calcium chloride, the beads obtained when using an injection needle with an outer diameter of 1 mm The average diameter is about 3 mm, and for thin tubes with an outer diameter of 5 mm it is about 9 mm.

The bead forming agent aqueous solution is dropped into the metal ion aqueous solution to be formed into particles, and at the same time, the polysaccharide contained therein is bonded with the metal ions and gels to solidify. In this solidified product, a gelled product of a polysaccharide is distributed in a three-dimensional network, and a bead forming agent is present between them. Therefore, it is stronger than the beads formed by gelling collagen or gelatin, is less likely to be crushed or broken, and is extremely easy to handle. On the other hand, in the molding method of molding in an organic solvent and crosslinking, it takes time to solidify, the beads are soft, and it is difficult to handle. Moreover, it takes time to clean.

The bead forming agent in the beads obtained as described above may be crosslinked. Cross-linking can prevent the bead-forming agent from diffusing out of the beads.

Crosslinking of collagen and / or gelatin after the formation of beads is performed, for example, by using a crosslinking agent or by irradiating with radiation, ultraviolet rays or the like. Examples of cross-linking agents include hexamethylene diisocyanate (HMDI
C), aldehydes such as glutaraldehyde and formaldehyde are used. As the radiation,
X-rays, γ-rays, etc. are used.

In the beads obtained by crosslinking the bead forming agent as described above, the crosslinked bead forming agent is firmly bound three-dimensionally. Therefore, if the gelled substance of the polysaccharide is removed, the resulting beads are composed of collagen alone, gelatin alone, or a crosslinked body of collagen and gelatin alone, so that they have good biocompatibility and good cell adhesion. Is. For this reason,
It is convenient to use as a supporting substrate for cells in large-scale culture of animal cells.

For example, when the gelled substance of the polysaccharide is formed by a bond between the polysaccharide and a divalent metal ion, the polysaccharide is treated with an aqueous solution using a chelating agent to dissolve the polysaccharide. It can be done by removing. As the chelating reagent, for example, EDTA or the like is used. The method for removing the gelled substance of the polysaccharide is not limited to this.

By this removal, beads made of collagen and / or gelatin are obtained. The beads stay wet,
Alternatively, it can be dried before use. Drying is performed, for example, by freeze-drying, but is not limited thereto.

The beads obtained by the production method of the present invention can be washed with water and / or an aqueous solution or sterilized before use. In the manufacturing method of the present invention, since it is not necessary to use an organic solvent, washing can be performed with water and / or an aqueous solution. Moreover, since the bead-forming agent aqueous solution can be molded and solidified by dropping it into the metal ion aqueous solution, the operation is simple and the operation can be performed in a very short time.

The use of the obtained beads is, for example, a support substrate for large-scale culture of animal cells, a biomaterial such as a biomaterial for medical use,
Examples include carriers such as bioactive substances and DDS (drug delivery system; so-called sustained release agents are also included in this). When used as a carrier, a bead-forming agent solution is preliminarily contained in a bead-forming agent solution to form beads.

Hereinafter, more specific examples and comparative examples will be shown, but the present invention is not limited to the following examples.

The embodiments of the present invention are the same as the embodiments 3, 6, 9, 12, and 15.
Is. Other Embodiments 1 and 2 or Embodiment 4
And 5, Examples 7 and 8, Examples 10 and 11, and Examples 13 and 14, are examples for explaining intermediate steps in the production method of the present invention.

-Example 1-Enzyme-solubilized collagen aqueous solution (pH 3.0, concentration 1.0% by weight)
Was adjusted to pH 7.0 and stirred well to obtain a collagen fiber dispersion liquid. Next, dissolve sodium alginate in water and
A 2.0 wt%, pH 7.4 solution was mixed with a collagen solution at a volume ratio of 1: 1 to obtain an alginic acid-collagen mixed solution. This was placed in an injection cylinder container and dropped into a 1% calcium chloride (dihydrate) aqueous solution at 15 ° C. from an injection needle having an outer diameter of 1 mm to form spherical beads. After standing for 20 minutes, the beads were collected by filtration with a nylon mesh and thoroughly washed with water at 15 ° C. The obtained beads of collagen-alginic acid were uniform beads having a diameter of about 3 mm, had extremely strong strength, and were extremely easy to handle.

-Example 2-The beads obtained in Example 1 were added to a 0.5% glutaraldehyde aqueous solution (pH 8.0), treated at 20 ° C for 10 hours with occasional stirring, and then thoroughly washed with water.

Example 3 1% of beads cross-linked in Example 2-EDTA aqueous solution (pH
It moved to 7.4) and stirred gently at 20 ° C. for 24 hours. The alginic acid in the beads was dissolved and removed, and beads consisting of collagen alone were obtained. Then, it was sufficiently washed with water to obtain collagen beads having a diameter of 3 mm. The collagen beads had sufficiently satisfactory physical properties.

-Example 4-In Example 1, 3.0 instead of the collagen fiber dispersion liquid was used.
Hard gelatin-alginate beads (diameter about 3 m) were treated in the same manner as in Example 1 using a weight% gelatin solution (pH 7.0).
m) was obtained. However, the dropping solution was kept at 40 ° C. and the temperature of the aqueous calcium chloride solution was room temperature.

-Example 5-The gelatin-alginate beads obtained in Example 4 were crosslinked by treating in the same manner as in Example 2.

-Example 6- By treating the crosslinked beads obtained in Example 5 in the same manner as in Example 3, beads consisting of gelatin and having a diameter of about 3 mm were obtained. These beads also had sufficient physical properties.

-Example 7- Instead of the collagen fiber dispersion liquid in Example 1, a collagen fiber dispersion liquid (pH 7.0, concentration 1.5% by weight) and an aqueous gelatin solution (pH 7.0, concentration 0.5% by weight) were mixed at a volume ratio of 1: 1. The same mixture as above was used and treated in the same manner to obtain hard beads composed of collagen-gelatin-alginic acid.

-Example 8-The beads obtained in Example 7 were treated in the same manner as in Example 2 to be crosslinked.

-Example 9- The crosslinked beads obtained in Example 8 were treated in the same manner as in Example 3 to obtain collagen-gelatin beads.

-Example 10- Using a mixture of an alkali-solubilized collagen aqueous solution (pH 8.0, concentration 1.0% by weight) and an aqueous solution of duragan gum (pH 7.4, concentration 2% by weight) at a volume ratio of 1: 1, a stainless steel inner diameter 3 mm
The mixture was added dropwise to a 1% aqueous solution of calcium chloride at 15 ° C. from the capillaries, and thereafter the same procedure as in Example 1 was carried out to obtain hard collagen-duranium gum beads having a diameter of about 8 mm.

-Example 11- The beads obtained in Example 10 were treated in the same manner as in Example 2 to be crosslinked.

-Example 12- The crosslinked beads obtained in Example 11 were treated in the same manner as in Example 3 to obtain collagen beads.

-Example 13-In Example 10, an aqueous pectin solution (pH 7.4, concentration 2.0% by weight) was used in place of the duragan gum aqueous solution, and the outer diameter was changed.
It was dropped into a 1% calcium chloride aqueous solution at 15 ° C. from a 1 mm injection needle to obtain solid collagen-pectin beads having a diameter of about 3 mm.

-Example 14-The beads obtained in Example 13 were crosslinked by treating in the same manner as in Example 2.

-Example 15- The crosslinked beads obtained in Example 13 were treated in the same manner as in Example 3 to obtain collagen beads.

-Comparative Example 1-8 volumes of acid-soluble collagen, phosphate buffer (0.1M, 1.4M
-NaCl) 1 volume, 0.05N-NaOH solution 1 volume are mixed at 4 ° C, put in an injection cylinder container, and dropped from an injection needle having an outer diameter of 1 mm into a 10 mmM phosphate buffer aqueous solution kept at 37 ° C. did. Collagen diffused into the aqueous solution before it gelled, and as a result, it was not possible to obtain granular beads.

〔The invention's effect〕

The method for producing beads according to the present invention can produce beads that are not easily crushed or broken without using an organic solvent.

Further, by crosslinking the bead forming agent, it is possible to prevent the bead forming agent from diffusing and flowing out from the beads. If the bead-forming agent is not crosslinked, the bead-forming agent may be removed by flow-out during the subsequent polysaccharide removal step, or some of the beads may be removed when the beads are used for various purposes. There will be problems such as leakage.

Furthermore, since the gelled substance of the polysaccharide is removed after the bead forming agent is cross-linked, the beads finally obtained are composed of only the target bead forming agent.
As a result, it is possible to prevent the polysaccharide from adversely affecting the chemical or biological properties when using the beads.

When removing the polysaccharide in this way, if the bead forming agent is not crosslinked, even the bead forming agent may be removed along with the removal of the polysaccharide, or the shape of the beads may not be maintained. . Therefore, it is important to perform the bead forming agent crosslinking step prior to the polysaccharide removal step.

Claims (1)

[Claims] 1. A method for producing beads, which comprises a step of shaping an aqueous bead-forming agent solution containing a bead-forming agent composed of at least one of collagen and gelatin into granules and solidifying the aqueous solution, wherein the aqueous bead-forming agent solution contains metal ions. The step of preliminarily containing a polysaccharide that forms an insoluble gel in water by combining with the step of adding the bead forming agent aqueous solution to an aqueous solution containing a metal ion for gelling the polysaccharide. A step of solidifying the granular bead forming agent aqueous solution by gelling the polysaccharide with the metal ion aqueous solution while performing molding to granulate the bead forming agent aqueous solution, and solidifying the bead forming agent aqueous solution Cross-linking the bead-forming agent after the step, and removing the gelled substance of the polysaccharide after the step of cross-linking the bead-forming agent A method of producing beads containing.