JPH08283666A - Method for treating collagen - Google Patents

Abstract

PURPOSE: To prepare a collagen gel whose form does not change during the dehydration process, in which water is sufficiently removed in the hydration process, which is perfectly hardened in an epoxy resin so as to be enabled to be cut out as specimen by a microtome in the embeding in the epoxy resin and to enable the electron microscope observation of the morphology of cell, etc. CONSTITUTION: A collagen gel is treated with a protein-fixing solution to react to form cross linkages. An aqueous solution of inorganic salt is brought into contact with the cross-linked gel to remove gel preparing components in the gel by replacement. Subsequently, this is treated with pure water to remove the inorganic salt, and then a mixed solution of an amphipatic organic solvent and water are brought into contact with the water treated product to to remove the water. Further, a mixed solution containing the amphipatic solvent in a higher ratio is brought into contact with it to replace the mixed solution containing water in a higher ratio. Finally, the amphipatic organic solvent of dry state is brought into contact with it to remove water by substitution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a treatment of a collagen gel mainly used as a culture substrate, which is used in an epoxy resin embedding process when preparing a sample for observation with a transmission electron microscope. It is about the method.

[0002]

2. Description of the Related Art In recent years, function-maintaining culture of cells has become popular, and as one of the techniques, cell culture has been carried out on or in collagen gel. Since it is necessary to examine the correlation between the functional expression of cells and the morphology of cells at a micro level, observation by an electron microscope is performed.

Generally, collagen gel used in cell culture has a high water content and is very soft. Therefore, even if an attempt is made to embed it in an epoxy resin to prepare a sample for electron microscope observation, dehydration in the collagen gel does not occur. It doesn't work,
It is difficult to maintain the structure of the collagen gel during cell culture because the epoxy resin does not cure or the volume of the collagen gel decreases during the dehydration treatment performed after fixation.

Water-soluble epoxy resins are commercially available so that the epoxy resin can be spread in the collagen gel even if some water remains, but in order to cure the resin, the water content in the collagen gel must be removed. It is necessary to remove it by evaporation. When water remains, the epoxy resin does not harden and cannot be embedded in the epoxy resin. On the other hand, when the collagen gel dries, the volume decreases and the internal structure of the collagen gel changes.

In the culture on extracellular matrix such as collagen, the mutual relationship between extracellular matrix and cells is important, and only cells are taken out from the extracellular matrix by using an enzyme and observed by an electron microscope. Even if it does, the effect of extracellular matrix on cells cannot be observed.

[0006] When cells are cultured on a collagen gel layer having a thickness close to that of collagen, dehydration from the collagen gel is relatively easy, and epoxy embedding in sample preparation for electron microscope observation is possible. However, in the case of culturing cells on a collagen gel having a certain thickness or culturing cells embedded in collagen gel, dehydration from collagen gel is performed while maintaining the structure of collagen gel during culturing. Or, it could not be embedded with epoxy resin or the like, and it was difficult to observe with an electron microscope.

[0007]

The present invention solves such a problem in preparing a sample for observation with a transmission electron microscope in cell culture on or in a collagen gel. It is an object of the present invention to provide a method for treating a collagen gel, which makes it possible to embed the entire collagen gel with an epoxy resin without breaking the internal structure of the collagen gel.

[0008]

Means for Solving the Problems As a result of intensive studies conducted by the present inventor on related phenomena and causes, the components of the medium used during cell culture have high affinity with collagen and water, and Since it is difficult to exchange substances in the structure of the collagen gel, the components of the medium are stored in the collagen gel, which is the cause of difficult dehydration, and as a result, it was found that the epoxy resin is hard to cure. . further,
Due to insufficient curing of the epoxy resin, the collagen gel shrinks in the dehydration process, and based on the idea that the structure changes, a study for removing the medium component stored in the collagen gel was conducted, It came to completion.

That is, according to the present invention, a collagen gel is brought into contact with a protein immobilization solution to react with it to crosslink, and then an aqueous solution of an inorganic salt is brought into contact therewith to replace and remove gel preparation components in the collagen gel, and then to purify it. Substituting and removing the inorganic salts by contacting with water, then contacting a mixed solution of an amphipathic organic solvent and water to remove and replace water, and water with a higher ratio of the amphipathic organic solvent. Treatment of collagen gel characterized by dehydration treatment by contacting with a mixed solution of the above to replace and remove a mixture containing a large amount of water, and finally by contacting with a dried amphiphilic organic solvent to replace and remove water. Is the way.

Further, collagen is prepared by adding a medium for cell culture and a neutralizing solution to an acidic collagen solution to carry out cell culture using a collagen gel, removing the culture solution, and then performing dehydration treatment in the same manner as above. It is a method of treating the gel, and furthermore, the collagen gel dehydrated in this way is immersed in propylene oxide to replace the amphipathic organic solvent, and then a liquid epoxy resin is added,
A method for preparing a sample for electron microscope observation, which comprises substituting propylene oxide in a collagen gel structure, heat-curing the mixture, and embedding the collagen gel in an epoxy resin.

The present invention utilizes the diffusion of the solution due to the difference in concentration between the inside and the outside of the collagen gel structure. The outside solution penetrates into the inside of the collagen gel, and conversely the solution inside the collagen gel is in the outside solution. It is exchanged, that is, replaced, in such a manner that it diffuses into the medium and expels the solution such as the medium stored inside the collagen gel. Further, since it is necessary to promptly move within the collagen gel structure, it is necessary that the solute of the solution has a small molecular weight and the charge of the whole solution is neutral. Most suitable to use. However, since it is necessary to finally remove these solutes and water outside the system, as described above, using an amphipathic organic solvent that can be freely mixed with water,
Substitution and dehydration are performed in stages.

The inorganic salts used are required to be those which do not cause precipitation of metal ions, and are preferably low in toxicity. Examples of such metals include sodium and potassium. When these are comprehensively viewed, sodium chloride or potassium chloride is suitable as the inorganic salt used. In addition, the aqueous solution of the inorganic salt to be brought into contact with the collagen gel has a function of rapidly infiltrating into the collagen gel and diffusing the gel preparation components etc. stored in the collagen gel to the outside. It is efficient to make the concentration as high as possible and the concentration gradient as large as possible. Therefore, it is preferable to use a saturated solution.

As described above, it is necessary that the collagen gel and the cell proteins cultured in or on the collagen gel are fixed as a premise that a high-concentration solution can be contacted. Generally, those that cause cross-linking by reacting with amino groups or carboxyl groups in proteins are used, and such cross-linking agents include glutaraldehyde, hexamethylene diisocyanate, carbodiimide, etc. Considering up to
Glutaraldehyde is suitable. If the treatment with a high-concentration aqueous solution of inorganic salt is performed before the immobilization treatment with the protein immobilization solution, the structure of the collagen gel is destroyed and the cell morphology is also changed.

The gel preparation components stored inside the collagen gel or the inorganic salts that have penetrated into the collagen gel in the form of being replaced with the medium can be easily removed by replacement with the outside of the collagen gel by contacting with water. You can Further, in order to remove the water contained in the collagen gel, it is brought into contact with a mixed solution of an amphipathic organic solvent and water.

In order to completely remove the water content from the collagen gel, the water-mixed solution having a higher proportion of the amphipathic organic solvent is brought into contact with the solution to replace and remove the water-rich mixed solution. In this way, the ratio of the amphipathic organic solvent is increased stepwise, and depending on the thickness of the collagen gel layer, the operation of contacting with the mixed solution is repeated 2 to 7 times, preferably 3 to 5 times, and then the last step. Then, it is brought into contact with a dry amphiphilic organic solvent that does not mix water, and the water is sufficiently replaced and removed. If the number of times of contact with the mixed solution is too large, the efficiency is poor, and if it is too small, a sufficient effect cannot be obtained. As the amphipathic organic solvent, methanol, ethanol, acetone or the like can be used.

Next, regarding the procedure of the method for treating a collagen gel of the present invention, the treatment of the collagen gel and the embedding with the epoxy resin during the preparation of the sample for observation with a transmission electron microscope after cell culture will be described in detail. To describe.

First, a collagen gel forming solution comprising an acidic collagen solution, a gel preparation component and a neutralizing solution is prepared, dispensed into a plate or a well of a petri dish, and heated at 37 ° C. to form a collagen gel. As a plate to be used, a 24-well plate is suitable because it does not require a large sample for observation with an electron microscope, and from the viewpoint of gel handling. A medium for cell culture may be used as the gel preparation component.

Next, the collagen gel formed in the plate wells is seeded with a cell suspension in which cells are suspended in a medium for culturing, or the cells are suspended in the above collagen gel forming solution. The cell suspension for culturing collagen gel-embedded culture is dispensed and heated at 37 ° C. to form a collagen gel in which cells are embedded, and then a medium is added to perform culturing. At this time, it is preferable to use a medium as a gel preparation component for gel formation.

After culturing the cells, the medium is removed by suction with a pipette or the like, and the protein immobilization solution is dispensed and allowed to stand to crosslink and immobilize the cells and the collagen gel. As a protein immobilization solution, an aqueous glutaraldehyde solution generally adjusted to pH 7.4 is used. After the immobilization is completed, the protein immobilization solution is removed by suction, if necessary, stained with osmic acid or the like, and then a saturated aqueous solution of inorganic salts is dispensed. As the inorganic salt used at this time, sodium chloride or the like is suitable as described above. Dispense a saturated aqueous solution of sodium chloride and let stand for several hours. It is desirable that the saturated aqueous solution of sodium chloride be dispensed at least twice the volume of the collagen gel. By this operation, the medium components stored in the collagen gel are easily removed outside the collagen gel and replaced with the sodium chloride solution.

Then, the sodium chloride solution is removed by suction with a pipette or the like, pure water is added, and the mixture is allowed to stand. In this process, sodium chloride that has entered the collagen gel instead of the medium is replaced with water this time and removed outside the collagen gel. Further, the pure water is removed by suction, and the operation of dispensing a mixed solution of ethanol and water and allowing it to stand is sequentially repeated using a mixed solution of water with an increased ethanol concentration in a stepwise manner, whereby collagen gel and Replaces and removes water in cells. In this process, the sodium chloride remaining in the collagen gel is completely removed, and the amount of water gradually decreases. And finally, 100% without adding water
By the same treatment with ethanol, dehydration is performed until the water content in the collagen gel is close to zero.

Since the medium components such as sugars remaining in the collagen gel cannot be removed unless the treatment with an aqueous solution of an inorganic salt such as sodium chloride is performed, the treatment with ethanol changes the structure of the collagen gel. , The volume of collagen gel is reduced. It is presumed that when contacted with ethanol, water in the collagen gel is deprived to the ethanol side, but since it is not replaced with ethanol, voids are generated in the structure of the collagen gel and the collagen gel contracts. However, even this ethanol treatment cannot completely remove the water content in the collagen gel.

Next, the collagen gel sufficiently dehydrated in this manner is embedded in an epoxy resin. First, the dehydrated collagen gel is transferred to a solvent-resistant container with tweezers, propylene oxide is poured and allowed to stand, and ethanol in the collagen gel is replaced with propylene oxide. Then, a liquid epoxy resin is poured and allowed to stand, the epoxy resin is permeated into the structure of the collagen gel, and then the epoxy resin is hardened by heating to complete the epoxy resin embedding.

Finally, a section is cut out by a microtome and used as a sample for electron microscope observation. The completely cured epoxy resin-embedded collagen gel can be cut into sections, and retains a good collagen gel structure when observed with an electron microscope.

[0024]

EXAMPLES Next, the present invention will be specifically described with reference to Examples and Comparative Examples. [Example 1] 0.3% acid-solubilized collagen type I solution and 1
A 0-15 concentration L-15 medium and a neutralizing solution were mixed at a ratio of 8: 1: 1 to prepare a collagen gel forming solution,
24-hole plate (MS-8024 manufactured by Sumitomo Bakelite Co., Ltd.
0) Dispense into each well and heat at 37 ° C to a thickness of 3
Form a collagen gel layer of mm. Next, 1 ml / well of L-15 medium was dispensed into each well, and C
After standing still for 17 hours in an O ₂ incubator, L-1
5 medium was removed and 2% glutaraldehyde solution was added to 1 ml /
Dispensing was carried out at a ratio of wells, and the mixture was allowed to stand at 4 ° C for 1 hour for cross-linking and immobilization. After removing the glutaraldehyde solution in the well, dispense saturated saline at a rate of 1 ml / well, leave it at room temperature for 2 hours, remove the saturated saline, and dispense 1 ml / well of pure water. It was left at room temperature for 30 minutes.
After this, ethanol concentration 50%, 70%, 90%, 95
% Dehydration solution (mixture of ethanol and water) was sequentially added for treatment, and 100% ethanol was further added for sufficient dehydration treatment, which was then subjected to an epoxy resin embedding test.

Example 2 The same 24-well plate as in Example 1 was used, and 0.3% acid-solubilized collagen type I solution and 1 were used.
A cell suspension prepared by mixing hepatocytes collected from a rat with a collagen gel solution prepared by mixing 0-fold concentrated L-15 medium and a neutralizing solution at a ratio of 8: 1: 1 was dispensed into each well. As a result, a collagen gel was formed in which rat hepatocytes were embedded. Next, 1 ml / well of L-15 medium containing 10% fetal bovine serum and hormones was dispensed in each well,
After culturing in a CO ₂ incubator at 37 ℃ for 1 week, remove the medium and add 1 ml of 2% glutaraldehyde solution.
/ Well was dispensed, and the mixture was allowed to stand at 4 ° C for 1 hour for cross-linking and immobilization. Subsequently, dehydration treatment was performed in the same procedure as in Example 1, and the sample was subjected to an epoxy resin embedding test and a morphological observation test using a transmission electron microscope.

Comparative Example 1 A collagen gel layer was formed in the same manner as in Example 1, crosslinked and immobilized with glutaraldehyde, and immediately treated with 1 ml of a saturated saline solution.
/ Well of pure water was dispensed into each well, and then dehydration treatment was performed by the same procedure as in Example 1, and the obtained collagen gel was subjected to an epoxy resin embedding test.

[Comparative Example 2] In the same manner as in Example 2, after forming a collagen gel in which hepatocytes collected from a rat were mixed and crosslinked and immobilized with glutaraldehyde, immediately without treatment with saturated saline, 1 ml / well of pure water was dispensed into each well, and then dehydration treatment was carried out in the same procedure as in Example 1, and the obtained collagen gel was subjected to an epoxy resin embedding test and an observation test using a transmission electron microscope. .

[Epoxy Resin Embedding Test] The collagen gels obtained in Examples 1 and 2 and Comparative Examples 1 and 2 were transferred to a polyethylene container, propylene oxide was poured into the container, and the mixture was allowed to stand at room temperature for 1 hour. The ethanol in the gel was replaced. Then, the epoxy resin for embedding was poured and allowed to soak at room temperature for 12 hours to allow it to penetrate into the structure of the collagen gel, and then heated at 35 ° C. for 12 hours and at 60 ° C. for 24 hours to cure the epoxy resin. . Curing status of epoxy resin and change in collagen gel thickness (100% immediately after immobilization with glutaraldehyde
The thickness after completion of the epoxy resin embedding operation) was examined. The results are shown in Table 1.

[Morphological Observation Test by Transmission Electron Microscope] With respect to the collagen gel embedded with epoxy resin in Example 2 and Comparative Example 2, it was possible to cut out a section with a microtome and whether to observe the morphology of cells with an electron microscope. I checked. The results are shown in Table 1.

[0030]

[Table 1]

From the results shown in Table 1, the cross-linking and immobilization with glutaraldehyde followed by the treatment with a saturated saline solution completes the curing of the resin when embedding the epoxy resin, so that it is possible to cut out a section with a microtome. It can be seen that, since the structure of the collagen gel is not changed, the morphology of cells in the collagen gel is maintained, and the method for treating a collagen gel according to the present invention is effective.

[0032]

EFFECTS OF THE INVENTION According to the method of the present invention, substances that are difficult to remove, such as gel preparation components and cell culture media, contained in collagen gel are replaced and removed, so that the subsequent dehydration is smooth and sufficient. As a result, the structure and morphology of the collagen gel do not change during the dehydration process, and the resin is completely cured when it is embedded in the epoxy resin.Therefore, cut out a section for transmission electron microscope observation using a microtome. In addition, the cell morphology during cell culture can be observed with an electron microscope.

Claims (8)

[Claims] 1. A collagen gel is contacted with a protein immobilization solution,
The reaction is allowed to crosslink, and an aqueous solution of an inorganic salt is brought into contact therewith to replace and remove the gel preparation component in the collagen gel,
Next, pure water is contacted to replace and remove the inorganic salts, and then contact is made to a mixed solution of an amphipathic organic solvent and water to replace and remove water, and the ratio of the amphipathic organic solvent is further increased. Collagen characterized by being dehydrated by contacting it with a mixed solution with water to replace and remove a mixture containing a large amount of water, and finally contacting it with a dried amphiphilic organic solvent to replace and remove water. How to treat the gel. 2. After culturing cells using a collagen gel prepared by adding a medium for cell culture and a neutralizing solution to an acidic collagen solution, the medium is removed and the protein immobilization solution is contacted and reacted. Cross-linking, by contacting it with an aqueous solution of an inorganic salt to replace and remove the gel preparation component in the collagen gel, and then contact with pure water to replace and remove the inorganic salt,
Then, contact with a mixed solution of an amphipathic organic solvent and water to replace and remove water, and further contact with a mixed solution of water with an increased ratio of this amphiphilic organic solvent to form a mixed solution with a large amount of water. A method for treating a collagen gel, which comprises performing a dehydration treatment by removing the water by replacing it and finally contacting it with a dried amphiphilic organic solvent to remove the water content. 3. The method for treating collagen gel according to claim 1, wherein the protein immobilization solution is an aqueous glutaraldehyde solution. 4. The method for treating a collagen gel according to claim 1, wherein the aqueous solution of the inorganic salt is a saturated solution. 5. The method for treating a collagen gel according to any one of claims (1), (2) and (4), wherein the inorganic salt is sodium chloride or potassium chloride. . 6. An operation of contacting a mixed solution of an amphipathic organic solvent and water to replace and remove water, and increasing the ratio of the amphipathic organic solvent stepwise, and repeating the operation 2 to 7 times. The method for treating a collagen gel according to claim (1) or (2). 7. The amphipathic organic solvent is one selected from methanol, ethanol, and acetone, wherein (1), (2), and (6).
The method for treating a collagen gel according to any one of 1. 8. A collagen gel treated by the method according to any one of claims (2) to (7),
After immersing in propylene oxide to replace the amphipathic organic solvent, add liquid epoxy resin to replace the propylene oxide in the collagen gel structure and heat-cure the collagen gel into the epoxy resin. A method for preparing a sample for electron microscope observation, which comprises embedding.