JPS6176161A - Sterilization of collagen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for sterilizing collagen used for cell culture, medical materials, etc.

[Background technology]

Collagen is widely distributed in the animal kingdom, and is extracted from these animals and used for various purposes.

Its uses include, for example, cell culture substrate 1 artificial skin;
They include hemostatic agents and are often used in direct contact with living organisms or cells.

Therefore, regardless of whether the collagen is dried (solid) or in solution, it is required to be sterile or sterilized.

Conventionally, methods for sterilizing collagen include: (1) filtering and sterilizing a collagen solution through a 0.20-0.45 μm membrane filter; (2) sterilizing dry collagen by irradiating it with radiation or using gas. Two methods were used.

However, although the filtration/sterilization method described in (2) has the advantage of not applying heat, it does not remove viruses, and aggregates of collagen molecules are removed along with bacteria, so the collagen concentration after filtration decreases. However, this method had the disadvantage of causing loss. However, radiation sterilization and gas sterilization described in (2) have the drawback that a partial crosslinking reaction occurs in collagen, making it difficult to dissolve the collagen. Gas sterilization also poses the problem of residual gas.

[Object of the Invention] In view of the above, an object of the present invention is to provide a method for completely sterilizing collagen without impairing its properties.

[Disclosure of the invention]

In order to achieve the above object, the gist of the present invention is a method for sterilizing collagen, which is characterized by irradiating ultraviolet rays while the collagen is in a solution. This invention will be explained in detail below.

Collagen types I to II are known as types of collagen, and the collagen used in this invention is mainly collagen type I. Examples of the raw material include, but are not limited to, cow skin, cow skin, pig skin, pig skin, rat tail, etc. Collagen can be extracted from such raw materials by any known processing method.

For example, the raw material is washed, degreased, deashed, made fine, then dispersed in an acid solution (or dispersed and then made fine), and then the acid-soluble collagen is extracted, purified, and recovered. Alternatively, there are methods in which the collagen is dispersed in an acid solution, and then an acidic and active protease is added to extract the solubilized collagen, which is then purified and recovered, but is not limited to these methods. A collagen solution can be obtained by dissolving purified collagen in a solution such as hydrochloric acid. Alternatively, the raw material may be processed directly to obtain a purified collagen solution.

In this invention, when sterilizing collagen with ultraviolet light, it is important that the collagen be in a solution state. Collagen /8e1. This is because ultraviolet rays have low energy and have a low ability to penetrate substances, and when collagen is in a solid state, there are many blind spots (areas that are not exposed to ultraviolet rays), making sterilization insufficient. However, even if collagen is in a solution state, the transmittance of ultraviolet rays is not 100%. Since the transmittance varies depending on the type and concentration of the solute, it is preferable, from the viewpoint of the sterilizing effect, that the thickness of the collagen solution in the direction of UV transmission is 5 fl or less, although it cannot be generalized. The spread of the collagen solution in directions other than the ultraviolet transmission direction (for example, in the direction perpendicular to the ultraviolet transmission direction) depends on the form of the light source, the form of the apparatus used for sterilization, and the like. For example, a collagen solution can be made into a thin layer and irradiated with ultraviolet rays, but the method is not limited to this and may be set as appropriate.

Ultraviolet rays have weaker energy than gamma rays and other radiation, so they have almost no effect other than sterilizing the irradiated materials, and they are easy to operate. It is also advantageous that viruses can also be killed and the selectivity of killing is relatively low. Therefore, in this invention, it was decided to use it for sterilizing collagen. The bactericidal effect of ultraviolet rays exists over the entire wavelength range (inm=400 nm), but ultraviolet rays exhibit particularly strong sterilizing power at wavelengths around 260 nm. 253. Mercury lamps that emit wavelengths of '7 nm (nanometers, 10-9 m) are commercially available as germicidal lamps and can be used as a light source. Alternatively, other light sources may be used, and the type of light source is not particularly limited. The number of light sources also varies, such as cases in which one is sufficient and cases in which two or more are necessary, and may be selected as appropriate. The collagen solution may be irradiated with ultraviolet rays not only from one direction, but also from two or more directions, and may be selected as appropriate.

By the way, among various microorganisms, the one that is said to have the highest resistance to ultraviolet light is Aspergillus ni.
ger). For sterilization, 4400μW・mi
A UV radiation dose of n/crd is required. That is, in order to sterilize collagen in solution by UV irradiation, it is preferable to use an irradiation amount of 44 Q OuW-min /crA or more.

As described above, if collagen is sterilized by irradiating ultraviolet rays while the collagen is in solution, the collagen will be completely sterilized and its properties will not be impaired.

Collagen sterilized in this manner is used for various purposes in a solution state or dried by various methods such as freeze-drying. This dried collagen is
It has the same solubility as collagen before sterilization and can be made into a collagen solution again. Examples of the above-mentioned uses include various biomaterials, 1 artificial skin, hemostatic agents, and pharmaceutical sustained-release agents, and 3 diagnostic agents.

There are cosmetics, etc. It is also used to impart biocompatibility to medical materials, such as coating them.

One of the important uses of collagen is as a cell culture substrate used in the medical field, drug development field, etc. One of the methods for culturing animal cells is a culture method called culture in collagen gel, and this is the substrate used in this case. Since this matrix is used by gelling collagen, the gel strength of the collagen gel is important. In other words, when culturing in a collagen gel, the gel strength of the collagen gel is will be influenced.

Collagen gel culture is performed by dispersing cells in a collagen solution, neutralizing it, and then gelling it to disperse and maintain the cells in the gel. At this time, unless the collagen gel has a high gel strength and a fast gelation rate (generally, collagen solutions with high gel strength have a fast gelation rate), the cells will settle to the bottom of the gel and the collagen Cell culture in gel is not possible. In addition, when culturing is continued, it is necessary to frequently replace the culture medium on the collagen gel with a new culture medium, and when replacing the culture medium, if the gel strength of the collagen gel is low, it may be necessary to remove the culture medium together with the culture medium that should be removed. , the gel and cells may be discarded. Furthermore, if the gel strength is low, the collagen gel will contract during the stage of cell proliferation, making it impossible to proliferate and maintain the cells.

In this way, collagen used in cell culture substrates is
It is undesirable that ultraviolet sterilization reduces the gel strength to an extent that it cannot be used for practical purposes. Therefore, in order to sterilize collagen used as a cell culture substrate, the gel strength of the collagen gel prepared from the collagen solution after sterilization must be the gel strength of the collagen gel prepared from the initial (pre-sterilized) collagen solution. It is preferable to adjust the amount of ultraviolet irradiation within a range that does not cause a drastic decrease.

In order to see the effects of the collagen sterilization method of this invention, Examples and Comparative Examples will be shown below.

〔Example〕

(A) Preparation of collagen solution (A-1) Preparation of acid-soluble rat tail collagen solution Thoroughly washed, degreased, and decalcified rat tail elbow was mixed with hydrochloric acid solution (pH
2, 5), the collagen was extracted by stirring under cooling for 24 hours. After centrifuging this extract to remove the insoluble portion, add sodium hydroxide/8 solution to adjust the pH.
7, and the resulting precipitate was collected by centrifugation. This was redissolved in a hydrochloric acid solution (pH 3,0) and purified by precipitation treatment in the same manner. After thoroughly washing the obtained precipitate with water, it was dissolved in a pH 3 hydrochloric acid solution to give a concentration of 3.0.
A purified acid-soluble rat tail elbow collagen solution of mg/mA was obtained (A-2) Preparation of pepsin-solubilized rawhide fullergen solution. 4% amount of pepsin (manufactured by Wako Pure Chemical Industries, Ltd. 1: 10,
000) in a hydrochloric acid solution, and then homogenized to disperse the raw material cowhide. Thereafter, stirring was continued for 48 hours at 15°C to perform enzyme treatment. This treated solution was filtered through a glass filter to remove insoluble portions, and then a diluted soda solution was added to adjust the pH to 10 and left to stand for 6 hours to deactivate pepsin. Next, the mixture was adjusted to p117 and left to stand overnight, and the resulting precipitate was collected by centrifugation. After washing the obtained precipitate with water, it was dissolved in a hydrochloric acid solution of pH 3, and the precipitate obtained with pi (7) was washed thoroughly with water, and then dissolved in a hydrochloric acid solution of pH 3.
A purified pepsin-solubilized cow skin collagen solution of 3.0 mg/mE at 65 degrees was obtained.

(B) Ultraviolet irradiation sterilization treatment Place each of the above collagen solutions in a petri dish with an inner diameter of 9 cm to a thickness of about 21mm, and use a 15W ultraviolet lamp (sterilizing lamp GL15 manufactured by Toshiba O-sun) to apply UV rays from a distance of 20mm. Irradiation was performed.

As shown in Table 1, the amount of ultraviolet irradiation was varied to examine the effect on the bactericidal effect and gel strength. The amount of UV irradiation is
Measurement was performed using an ultraviolet intensity meter UVR-254 (Tokyo Kogaku Kikai Co., Ltd.).

Using each collagen solution sterilized with ultraviolet light in this way,
A collagen gel was prepared as follows, and the gel strength was measured.

To 24 ml of each of these collagen solutions, add 3 mg of 0.1 M-IJ acid buffer (pH 7,4) containing 1.4 M sodium chloride and 3 ml of sodium hydroxide solution under cooling, and then mix to prepare the final solution. After adjusting the pH to 7.4, 3
A gel was formed by heating at 7° C. for 1 hour.

The obtained gel was measured using a rheometer (tJRM-2002D:
(manufactured by Fudo Kogyo Co., Ltd.) using a 0.5 inch plunger,
Gel strength was measured under the conditions of insertion depth IQm (insertion speed 201 m/min). The results are shown in Table 1.

Further, using each collagen solution sterilized by ultraviolet rays as described above, a bacterial test was conducted according to the general bacterial testing method shown below.

Each of the above collagen solutions 1m7! Place each in a sterilized petri dish, neutralize to pH 5-7 with sterilized sodium hydroxide solution, and dissolve standard agar medium solution (standard agar medium: 23.8 g of agar manufactured by Eiken Chemical ■ in 11 pure water). and 121°C
After adding about 20 ml (obtained by autoclaving for 120 minutes) and mixing well, the mixture was cultured at 37°C for 48 hours, and the presence (+) or absence (-) of bacterial growth was determined. The results are shown in Table 1.

[Comparative Example 1] The same purified acid-soluble rat tail elbow collagen solution as in Example (A-1) 1 with a concentration of 3.0 mg/mi was used, and this collagen solution was sterilized by filtration with a 0.45 μm membrane filter. I did it. After filtration, the concentration is 1.2 mg/
ml.

Also, the same concentration as Example (A-2), 3.0 mg/
This collagen solution was sterilized by filtration using a 0.45 μm membrane filter using a purified pepsin-solubilized cow skin collagen solution. After filtration, the concentration is 2.4
mg/ml.

C Comparative Example 2] Same as Example (A-1), concentration 3.0 mg/
ml of purified acid-soluble rat tail elbow collagen solution was placed in a Mezoim bottle, tightly stoppered, and gamma (T
) rays were irradiated. As shown in Table 2, the absorbed dose was varied to examine the effect on the bactericidal effect and gel strength.

Using each collagen solution of Comparative Example 2, collagen gels were prepared in the same manner as in Examples, and the gel strength was measured. The results are shown in Table 2. In the sterilization treatment of Comparative Example 1, the 'lrN degree of the collagen solution decreased after the treatment, and naturally the gel strength also decreased, so the gel strength was not compared.

Using the sterilized collagen solutions of Comparative Examples 1 and 2, a bacterial test was conducted using the same general bacterial testing method as in the example. The results are shown in Table 2.

As a blank, each of the collagen solutions used in the above Examples and Comparative Examples was gelled without being sterilized, gel strength was determined as described above, and a bacterial test was conducted. The results are also shown in Table 1.

(Hereafter, extra white) Table 1 *2: Does not gel.

*3: Gelified during irradiation.

As shown in Table 1, it can be seen that collagen is completely sterilized by the collagen sterilization method of the present invention. The same table shows that even an ultraviolet irradiation amount of 290 μW-min/cal has a seedling growth effect, but this is not absolute because it depends on the contamination state of the collagen solution before sterilization. The preferred amount of ultraviolet irradiation is for black mold (Aspergillus niger)
This is the area where the sterilizing dose is 4400 μW-min/cr1 or more. According to this invention, in the case of acid-soluble collagen that yields a gel with high gel strength, 4400 μW-
Irradiation far exceeding min/cd ff1l 160
Even at 0 μW-min/cat, the decrease in gel strength due to ultraviolet irradiation is extremely small, as will be described in detail below. UV irradiation amount 11600 μW-
When a collagen solution sterilized at min/cni is gelled, the gel strength is 254 g, and compared to the gel strength of 288 g when a collagen solution that has not been irradiated with ultraviolet rays (blank collagen solution) is (254/288) x 100 ( %) =88
．． 2 (%), and the decrease in gel strength is ((288-254) /2881 X100 (%
)=L1.8 (%). When cells were cultured in the gel using this collagen gel solution, good results were obtained and it was found that there was no problem in practical use.

The inventors have confirmed that if the decrease in gel strength is less than 20% (in other words, the gel strength of the collagen gel prepared from the collagen solution after UV irradiation is lower than that of the collagen solution that has not been irradiated with UV rays). If the gel strength of the collagen gel to be prepared is maintained at 80% or more), it can be used practically without any problem.

Further, as shown in Table 1, the gel strength of the gel obtained from the enzyme-solubilized collagen solution is lower than that of acid-soluble collagen, but this is caused by the difference in the structure of collagen. The enzyme-solubilized collagen solution also had an ultraviolet irradiation amount of 11,600 μW-min/csA and 17
400 μW-min/cI]! The gel strength differs greatly between the two.

As shown in Table 2, the membrane filter of Comparative Example 1
When filter sterilized, the integrity of the resulting sterile collagen solution was reduced because some collagen molecules were filtered out. Furthermore, in the case of T-ray irradiation sterilization, even at a small irradiation dose, the gel strength of the resulting gel was significantly reduced, and at a sterilizing irradiation dose, the collagen solution gelled during irradiation, making it unusable.

〔Effect of the invention〕

The collagen sterilization method of this invention sterilizes the collagen by irradiating it with ultraviolet rays while it is in a solution state, so the collagen is completely sterilized and there is no loss that would occur with the filtration method. does not occur and the properties of collagen are not impaired. In other words, the collagen does not become insoluble, and the decrease in gel strength is suppressed to a level that does not pose a problem for practical use. By the collagen sterilization method of the present invention, the sterilized collagen becomes an excellent material for the various uses described above, regardless of whether it is in the form of a solution or a dried product.

Claims (2)

[Claims] (1) A collagen sterilization method characterized by irradiating ultraviolet rays while the collagen is in a solution. (2) Ultraviolet irradiation increases the gel strength of the collagen gel prepared from the collagen solution after sterilization by 8 times the gel strength of the collagen gel prepared from the initial collagen solution.
The collagen sterilization method according to claim 1, which is carried out at an irradiation dose that is maintained at 0% or more.