JP4915693B2 - Cell culture carrier - Google Patents

Description

  The present invention relates to a cell culture carrier using collagen as a raw material, more specifically to a method for producing a chondrocyte culture carrier.

The meniscus of the knee joint is a fibrocartilage tissue in the joint, and it has many functions such as shock absorption, load distribution, improved slidability, joint stability, and is easily damaged by sports trauma and daily living activities Injury causes knee pain and movement limitation, but is difficult to heal. Traditionally, surgical treatment has been performed for injured meniscuses that have not been cured by conservative treatments such as drug therapy and exercise therapy, and meniscus resection or partial resection has been performed. Damaged. In recent years, with the advancement of endoscopic technology, arthroscopic meniscus suturing has been performed, and function preservation has been achieved.
However, injuries with defects, complex injuries, degenerative ruptures, etc. are not suitable for suturing, and the meniscal function cannot be repaired. This problem is not limited to the meniscus and can be said to be a general problem of cartilage with little blood vessel running.
Regenerative medicine has been actively studied as a solution for this, but cells, carriers, and activators are said to be three elements in regenerative medicine, and culture carriers are regarded as important.
It is necessary to select an optimal carrier such as shape and physical properties depending on the cells to be cultured. However, in order to culture chondrocytes, it is desirable that the carrier has physical properties or shapes that are close to the in vivo cartilage tissue.

  However, it is difficult to say that conventional carriers are similar in physical properties and shape. Therefore, the carrier itself does not have cartilage-like strength, it is difficult to match the shape unique to the cartilage tissue, the cartilage matrix component is not produced at the time of culturing, the material contains materials other than biological components, etc. was there.

According to Patent Document 1, a tissue equivalent for transplantation is disclosed in which the density of chondrocytes on the surface and inside is changed using a collagen gel, and fixation to the tissue peripheral part after transplantation is performed at an early stage.
According to Patent Document 2, it is disclosed that chondrocytes are grown in a medium on a membrane filter in collagen.
According to Patent Document 3, a scaffold implant for reconstructing cartilage using a mixture of collagen I and collagen II as a raw material is disclosed.
According to Patent Document 4, a tissue regeneration base material and a transplant material in which a three-dimensional shape is maintained by combining a collagen sponge and an absorbable synthetic polymer are disclosed.
JP2002-233567 JP 2003-135056 A JP 2003-180815 A Tokurei WO2003-011353

However, in Patent Document 1, Patent Document 2, and Patent Document 3, three-dimensional culture similar to that in a living body is possible, but physical properties similar to those of a living tissue cannot be obtained. In Patent Document 4, it is possible to produce a carrier having a strength similar to that of a tissue, but it is difficult to say that it is optimal as an implant because it contains a compound other than a matrix component of a living body.

  An object of the present invention is to provide a cell culture carrier that uses collagen, which is a matrix component of cartilage tissue, as a raw material, has physical properties similar to those of living cartilage tissue from the beginning of culture, and has the shape of a transplant site. The carrier must have a pore for the cells to enter. Furthermore, if the culture for the addition of chondrocytes and cartilage tissue in vivo is possible using the cell culture carrier, it is possible to obtain an implant more similar to the living tissue by culturing.

  The gist of the present invention is that a material having a physical property close to cartilage tissue in a living body is produced by freeze-drying using a high-concentration collagen dispersion, solution or mixture thereof as a raw material, and further, its physical strength, in vivo It is a cell culture carrier having a stress of 10 to 30 kPa at a load of 10% by performing an insolubilization treatment for adjusting the absorption rate.

  By using the carrier for cell culture, after the chondrocytes are seeded on the carrier, the transplantation or the transplantation after the culture is integrated with the own cartilage tissue, and after seeding the cells, Cultivation with the addition received by cartilage tissue is also possible, and an implant more similar to living tissue can be obtained by culturing.

  The carrier for cell culture of the present invention is produced using collagen as a raw material. Collagen used is insoluble collagen collected from living tissues, such as tendon collagen derived from Achilles tendon, collagen derived from skin, soluble and solubilized collagen, such as enzyme-solubilized collagen (atelocollagen), alkali-solubilized collagen, acid-soluble collagen, salt Soluble collagen or the like can be used, but atelocollagen is particularly desirable. There is no particular limitation on the animal species, and there is no problem as long as the collagen has a denaturation temperature that does not cause heat denaturation during culture. Specifically, it can be derived from mammals such as cows and pigs, birds such as chickens, fishes such as tuna and sea bream. Recombinant collagen can also be used. Chemically modified products of the side chains of the constituent amino acids of collagen, specifically, acylation such as acetylation, succinylation and phthalation, esterification such as methylation and ethylation, and the like can be used.

Prepare a collagen dispersion or solution before lyophilization. In the case of insoluble collagen, a dispersion is obtained, and in the case of soluble collagen, the solution or the dispersion can be adjusted. There is no particular limitation on the pH of either the dispersion or the solution, but it is preferably near neutral, specifically pH 4-10.

In order to approach the cartilage-like strength by freeze-drying, it is conceivable to increase the density by pressing the dried product obtained after freeze-drying, but in that case, the pores formed by freeze-drying are crushed, It is difficult to seed cells into the carrier.
Therefore, the collagen concentration in the insoluble and soluble collagen dispersion, solution, or mixture thereof as a raw material for lyophilization needs to be 50 mg / ml or more . If 5 0 mg / ml or more can be obtained carrier of similar physical properties to cartilage tissue. In the case of a low concentration, for example, 30 mg / ml, there is a large difference in physical properties from cartilage in the living body, so that it is difficult to immediately transplant or to transplant after culture after seeding chondrocytes on this carrier. Later, it becomes difficult to carry out culture in which a load applied to chondrocytes and cartilage tissue in vivo is applied .

Any of a dispersion, solution or mixed solution having the above collagen concentration can be used, but a dispersion is particularly desirable. The dispersion means a state where the collagen is not dissolved but is dispersed, precipitated or swelled at a pH other than the pH at which the collagen is dissolved.
If bubbles are contained in the solution or dispersion, it is not desirable because air bubbles are formed inside the lyophilized product. Therefore, it is necessary to remove the bubbles from the solution and dispersion before lyophilization. The method of removal is not particularly limited, but a temperature increase such that collagen undergoes heat denaturation is not desirable. Specifically, heating, long time or powerful ultrasonic treatment cannot be used.

For freeze-drying, the prepared collagen solution, dispersion or mixed solution is first filled into a mold having a desired shape and frozen. As a desired shape, a cube can be made and cut into a desired shape at the time of use, or any method of using a mold having a desired shape from the beginning can be used.
The method of using a mold having a desired shape from the beginning is not particularly limited. For example, after culturing using the culture carrier of the present invention, the culture carrier containing the cells may be directly transplanted into a cartilage defect part. In some cases, it is desirable to make the culture carrier itself according to the shape of the cartilage defect.
As a specific method, a mold having the shape of the defect can be made by stereolithography based on the CT or MRI data of the patient himself.

The mold is filled with a collagen dispersion, solution, or a mixture thereof, and lyophilized, and the bubbles may be removed before or after filling.
Freezing methods include rapid or slow freezing, but there is a possibility that the pore size of the dried product may differ depending on the freezing method, and it is necessary to select a freezing method that can achieve the desired pore size.
There is no restriction | limiting in particular in the method of drying, The technique of freeze-drying performed normally can be used.

The dried product that has been freeze-dried is then insolubilized. By performing the insolubilization treatment, the physical strength can be increased and the remaining period in the transplanted tissue can be adjusted.
When performing the insolubilization treatment, the insolubilization treatment must be performed uniformly without breaking the shape of the resulting dried product.

There is no particular limitation on the method of insolubilization treatment, but during the insolubilization treatment, if water is used as a solvent, the dried product swells, making it difficult to maintain the desired shape obtained by drying. It is desirable to do. If an organic solvent is used to suppress swelling, the insolubilization treatment inside the dried product is difficult to proceed, and only the surface is insolubilized, which is not desirable.
As the insolubilization treatment of the present invention, a dry heat treatment, γ-ray irradiation, a water-soluble chemical cross-linking agent, a vaporizable chemical cross-linking agent, and the like that can be insolubilized to the inside of the dried product are desirable. More specifically, aldehyde compounds and epoxy compounds can be used as water-soluble chemical crosslinking agents, and formaldehyde and the like can be used as vaporizable chemical crosslinking agents.

Specifically, the insolubilization treatment varies depending on the method used. For example, if it is a dry heat treatment, it can be performed by letting it stand in a heating atmosphere at about 120 ° C. for 30 minutes or more after giving the humidity to the dried product to such an extent that it does not swell with gamma irradiation. It can be performed by irradiation of 10 krad or more. For insolubilization treatment with a water-soluble chemical cross-linking agent, for example, glutaraldehyde can be achieved by immersing the dried product in an aqueous solution containing glutaraldehyde at a concentration of 0.5%. In addition, the dry product may be subjected to a dry heat treatment in advance, and then insolubilized with a water-soluble chemical crosslinking agent.
In the insolubilization treatment with a vaporizable chemical crosslinking agent, the insolubilization treatment is performed with formaldehyde vaporized in the sealed container by placing a dried product and a chemical crosslinking agent, for example, a formalin solution, in the sealed container.

This carrier can be seeded with chondrocytes, and can be transplanted as it is or transplanted after culturing.
It is also possible to perform the culture while applying a load similar to the load received by the living cartilage tissue.
Further, when cells are seeded inside the pore, only the cells may be seeded or they may be seeded after being suspended in a collagen solution which is the same material as the culture carrier.

Hereinafter, the content of the present invention will be described with reference to examples, but the present invention is not limited thereto.
Example 1
Enzyme-solubilized collagen (atelocollagen) derived from bovine dermis is added to water at pH 9 with NaOH. After slowly stirring overnight to fully swell the collagen, this solution is centrifuged to obtain a collagen dispersion. The collagen concentration in this dispersion is measured by the burette method. Adjust the collagen concentration in the dispersion to 80 mg / ml. If the concentration is lower than 80 mg / ml, additional centrifugation is performed to increase the concentration. If the concentration is higher than 80 mg / ml, add water at pH 9 above, stir again slowly, and adjust the concentration according to the rotation speed and time of centrifugation.

The obtained collagen dispersion is dispensed onto a 24-well plate for culture (CulturPlate ^™ 24 cell culture microplate manufactured by PerkinElmer). After dispensing, the plate is placed in a laboratory vacuum device to remove bubbles in the dispersion. Thereafter, the plate is placed in a freeze dryer in which the shelf is cooled to −20 ° C., and the dispersion is frozen, followed by drying under reduced pressure. The shelf was not heated during drying.
The dried product after drying is taken out from the plate, put into a desiccator containing a beaker containing formalin solution, and left at room temperature overnight.
The collagen carrier that has been insolubilized is removed from the desiccator, placed in another desiccator that does not contain formalin solution, and left under reduced pressure with an aspirator for 3 hours to obtain a carrier capable of culturing chondrocytes.
A cross-sectional photograph of the obtained carrier is shown in FIG. The lower part in FIG. 1 represents a scale, and the interval of one scale is 1 mm.
The physical strength of the obtained carrier was measured. The measurement was performed at 100 micrometers / second, and the force and displacement were measured by applying a compression load up to 35 kPa.
As a result, it was 15 kPa at 5% load , 22.5 kPa at 10% load, and 33 kPa at 20% load.
Further, the tangent modules were obtained from this result and found to be 224 kPa at 5% load and 160 kPa at 10% load.
The measured values were obtained by one-way distribution analysis.

Example 2
When adjusting the dispersion in the same manner as in Example 1, the collagen concentration is adjusted to 100 mg / ml, and a culture carrier is produced in the same manner as in Example 1.

Example 3
A culture carrier is produced in the same manner as in Example 1 using enzyme-solubilized collagen (atelocollagen) derived from porcine dermis layer instead of bovine in Example 1.

Example 4
In Example 2, a culture carrier is produced in the same manner as in Example 1 using enzyme-solubilized collagen (atelocollagen) derived from porcine dermis layer instead of bovine.

After freeze-drying to obtain a dried product in the same manner as in Example 1, this dried product is further dried at 70 ° C. under reduced pressure for 2 hours. After drying, heating is performed at 120 ° C. for 2 hours under normal pressure, and insolubilization is performed by dry heat. Thereafter, glutaraldehyde is added to water adjusted to pH 9.0 with NaOH at a concentration of 0.5 ml / 100 ml to prepare a solution, and the dried product which has been insolubilized by dry heat is added thereto, and slowly added at room temperature for 1 hour. Stir.
After the treatment, the culture carrier is taken out, washed thoroughly with water, and then placed in a culture medium for culture.

2 is a cross-sectional photograph of the carrier obtained in Example 1. FIG.

Claims (6)

Dispersion of collagen Collagen concentration is 50 mg / ml or higher, the solution or after the freeze-drying the mixture, subjected to insolubilization has a stress 10~30kPa at 10% load, one lifting the pore structure surface and the inside Cell culture carrier. 2. The cell culture carrier according to claim 1, wherein the collagen is enzyme-solubilized collagen. The cell culture according to any one of claims 1 to 2 , wherein the insolubilization treatment is any one of heating, γ-ray irradiation, water-soluble chemical cross-linking agent, vaporizable chemical cross-linking agent, or a combination thereof. Carrier. The cell culture according to any one of claims 1 to 3 , wherein a collagen dispersion, a solution or a mixture thereof is injected into a closed container having a gap of a desired shape at the time of freeze-drying and dried after freezing. Carrier. The carrier for cell culture according to claim 4 , wherein the sealed container having a gap of a desired shape is produced based on CT and MRI data. The cell culture carrier according to any one of claims 1 to 5 , which is used for culturing chondrocytes.

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