JP2003219865A - Method for making spheroid, the resultant spheroid and spheroid-containing composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide spheroids each of a specified size by using various kinds of cells without being limited to the kinds, and to produce such spheroids uniformly and easily in large quantities. <P>SOLUTION: The spheroids comprise carriers having cell adhesivity and cells. Such spheroids are produced by the step of mixing cells with a population of carriers having cell adhesion activity under the conditions of not integratedly coagulating the population of carriers and the step of culturing the mixture for a specified period of time. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing spheroids (cell aggregates) obtained by a cell culture operation, a spheroid, and a spheroid-containing composition containing the spheroid.

[0002]

2. Description of the Related Art Unlike two-dimensional flat culture, spheroids (cell aggregates) that construct a three-dimensional structure similar to the in-vivo environment during culture maintain the functions of cells such as hepatocytes in long-term culture. It is used to

As a conventional method for producing spheroids,
By culturing the cells in a culture vessel coated with a non-cell-adhesive substrate such as agarose or agar, or a semi-adhesive substrate such as proteoglycan, or a culture vessel with a positive charge, the cells are accidentally or spontaneously cultured. A method of aggregating to form a spheroid is known. This method
It is known as a method for producing spheroids in cells that easily aggregate such as cancer cells, fetal cells, and hepatocytes.

In addition, after suspending a cell sheet or a spheroid precursor in a culture solution containing a sol-gel transition substance having no cell adhesiveness, the culture solution is transferred from a sol state to a gel state, A method for producing a spheroid by culturing a cell sheet or a spheroid precursor with the above method has been devised (Japanese Patent Laid-Open No. 08-140673: a method for producing a spheroid). This method has the advantage that the size of spheroids can be controlled.

On the other hand, an incubator whose surface is coated with a hydrophilic polymer such as polyhydroxyethyl methacrylate,
A spheroid containing an osteocyte, a calcium phosphate compound, and an extracellular matrix produced by the osteocyte in some cases has also been proposed by seeding an osteocyte and a calcium phosphate-based compound suspension (Japanese Patent Laid-Open No. Hei 10 (1999) -242242). 10
-243996).

[0006]

However, in the method using a specific cell culture vessel, cancer cells, fetal cells,
Limited to cell types that tend to aggregate, such as hepatocytes,
It was difficult to control the size (cell number) of spheroids. Further, in the method using a calcium phosphate-based compound, it is necessary to control the cell adhesiveness on the bottom surface of the dish by coating with a hydrophilic polymer. On the other hand, in the method of using a culture solution containing a sol-gel transition substance having no cell adhesiveness, the step of the production method requires the labor of previously producing a cell sheet or a spheroid precursor, which is not always easy. Not a manufacturing method. In particular, since a large amount of preparation requires a considerable amount of labor, it has been difficult to use it as a secondary use by mixing it with an external preparation such as an ointment or an injection.

In view of the above-mentioned problems of the prior art, the present invention provides a spheroid capable of uniformly and easily producing a large number of spheroids of a predetermined size by various cell types without being limited to the cell type. It is a technical object to provide a method for producing the spheroid, and a spheroid-containing composition containing the spheroid, which can be used as an external preparation.

[0008]

The method for producing spheroids according to claim 1, wherein a carrier population composed of a plurality of carriers having cell adhesiveness and adhesion-dependent cells, wherein the carrier population is cells. In addition, it is characterized by including a mixing step of mixing under a mixing condition that does not aggregate together and a culture step of culturing the mixture for a predetermined period. The method for producing a spheroid according to claim 2 is characterized in that the carrier is a collagen fiber. The method for producing spheroids according to claim 3 is characterized in that, in the method, the carrier is obtained by insolubilizing a collagen solution of 0.1 to 0.3 W / V%. The method for producing a spheroid according to claim 4, wherein in the method, the mixing condition is that cells are mixed with the carrier at a cell seeding density of 1 × 10 ³ to 1 × 10 ⁵ cells / ml. There is.

The spheroid according to a fifth aspect is characterized by being produced by any one of the methods for producing a spheroid. The spheroid according to claim 6 is characterized in that it contains collagen fibers and adhesion-dependent cells. The spheroid described in claim 7 is characterized in that, in the spheroid, the diameter of the spheroid is in the range of 50 to 500 μm.

The spheroid-containing composition according to claim 8 is characterized by containing any one of the spheroids and a substrate capable of holding the spheroid. The spheroid-containing composition according to claim 9 is characterized in that the base material is any of an ointment, an injection and a culture carrier.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing spheroids (cell aggregates) of the present invention comprises a carrier population composed of a plurality of carriers having cell adhesiveness and adhesion-dependent cells, wherein the carrier population is composed of cells. And a culturing step of culturing the mixture only for a predetermined period of time.

[0012] According to this method, the carrier population having cell adhesiveness and the cells are mixed under specific mixing conditions, so that uniform spheroids can be separately prepared from various cell types regardless of the agglutinability of the cells. It can be made directly and easily without making a body. Therefore, spheroids can be easily produced in large quantities.

The carrier used in the production method of the present invention may be any known carrier as long as it has cell adhesiveness, such as collagen, polyrotaxane and polylactic acid (P
LA), polylactic acid glycolic acid copolymer (PLGA), and the like. Among them, collagen, particularly collagen fiber is preferable. In this case, by insolubilizing the collagen solution, collagen fibers can be easily produced in large quantities. Further, the collagen that can be used in the present invention is all applicable as long as it has molecular characteristics, and even if it is naturally derived from any organism, it is artificially produced. It may be. Further, the form of the carrier in the present invention does not matter. For example, various shapes such as filamentous (linear), spherical, rod-like, and granular shapes, those composed of individual independent molecules, and those formed by aggregating a plurality of individual molecules (for example, collagen) are mentioned. To be

The cells used in the production method of the present invention may be adhesion-dependent cells, and various cells such as chondrocytes, fibroblasts, epithelial cells, hepatocytes, nerve cells and pancreatic islet cells may be used alone or It is possible to use them in combination, and it is particularly preferable to use mesoendodermal cells such as chondrocytes and fibroblasts.

In the mixing step, the cells and the carrier population are mixed under a mixing condition in which the carrier population does not aggregate together with the cells. The cells and the carrier population are usually mixed by a method used for such mixing, for example, by mixing a cell suspension containing a predetermined number of cells with a carrier-containing solution containing the carrier.

In the present invention, the carrier population is mixed with the cells under a mixing condition that does not aggregate together, so that when the carrier population and the cells are mixed, it is possible to avoid that the carrier population aggregates together with the cells. it can. Here, "the carriers are aggregated together" means that a carrier group composed of a plurality of carriers is
It means that the whole carrier population is aggregated into one mass by the cells, and the present invention requires that such aggregation does not occur. As a result, in the present invention, it becomes possible to easily and in large quantity obtain spheroids controlled to have a predetermined size by allowing cells to proliferate by using each carrier as a scaffold for cell proliferation.

As the above-mentioned mixing conditions, if the carrier population does not aggregate together with the cells, any conditions can be used, such as adjusting the mixing temperature or mixing with stirring. It is preferable to adjust the concentration depending on the number of cells and the number of seeded cells.

In particular, as the above-mentioned mixing conditions, it is preferable to mix the cells with the carrier population at a predetermined cell seeding density. The cell seeding density depends on the cell type, and for example, in the case of chondrocytes or fibroblasts, 1 × 10 ³ to 1 × 10 ⁵ cells /
It can be ml. At this time, it is necessary to adjust the seeding amount to a small amount for cell types that readily form spheroids, and to increase the seeding amount for cell types that hardly form spheroids. By selecting and adjusting such cell types and cell seeding densities, the carrier population can be efficiently prevented from coagulating together with cells.

The carrier used in the spheroid-producing method of the present invention is preferably one obtained through a carrier preparing step before the mixing step. The carrier obtained in this carrier preparation step is a carrier having uniform properties and morphology, and has a high ability to form spheroids with cells, and thus is preferably used.

The carrier preparation step involves preparing a suspension that cannot gel independently from the substructure that can gel alone, insolubilize the suspension, and centrifuge. By removing the supernatant after centrifugation, the target uniform carrier can be easily recovered as a precipitate consisting of an aggregate of a plurality of substructures. In general,
Suspensions can be prepared by diluting the solution containing the substructure to a dilute concentration that is difficult to gel.

When the carrier is collagen fiber, it is preferably obtained by insolubilizing a collagen solution,
It is particularly preferable that a collagen solution having a concentration of 0.1 to 0.3 W / V% is insolubilized. The carrier is insolubilized by neutralization. When insolubilized under such conditions, a plurality of collagen molecules aggregate to form collagen fibers. That is, this collagen fiber is formed by aggregating a plurality of collagen molecules, and such a collagen fiber can be easily recovered as a group of carriers by centrifugation thereafter.
The size of the collagen fiber may be any size as long as it does not impair the adhesiveness to cells. In the present invention, "collagen molecule" indicates a helical structure composed of three polypeptide chains, and "collagen fiber" means a fibril (Fibril) in which a large number of collagen molecules are gathered or a fiber (Fiber) in which a large number of fibrils are gathered. , Which have the same meanings as those skilled in the art use in the art.

In the carrier preparation step and the mixing step, any solvent that can be used in the field of cell culture can be used as a solvent for preparing a carrier or a solution of cells. Culture liquid (medium)
Is preferably used. As the medium that can be used in this case, any medium known in the art can be used.

The culturing step includes culturing the cells and the carrier mixed in the mixing step for a predetermined period. here,
Since the number of cells that make up spheroids increases due to culture, the size of spheroids can be adjusted by adjusting the culture period. The size of the spheroids can be adjusted also by the composition of the medium, the frequency of medium exchange, and the like.

The culturing period varies depending on the cell type and cell growth ability, but is preferably about 2 weeks for human chondrocytes and about 1 to 2 weeks for human fibroblasts. The medium used for the culture is appropriately selected from those usually used in this field according to the cell type used. In the culturing step, the mixture of the cells and the carrier is cultivated under the culturing conditions usually used in this field to form spheroids.

The spheroids obtained by this spheroid production method are obtained as a spheroid suspension in which a large amount of individual spheroids are suspended. In this spheroid suspension, cells are present as spheroids, and therefore handling such as cell transportation is facilitated. Further, since it can be easily blended with other blending components, the effect of spheroids can be easily applied to various uses.

The spheroid (cell aggregate) of the present invention is
It is characterized by being manufactured by any one of the above manufacturing methods. According to this spheroid, since the carrier has cell adhesiveness, it can contain almost all adhesion-dependent cell types regardless of cell cohesiveness, and has a size-controlled moderate size. Spheroids can be provided. Another spheroid of the present invention is characterized in that it contains collagen fibers and contact-dependent cells. According to this spheroid, collagen fibers can easily provide spheroids of uniform size by various cell types regardless of the aggregating property of cells.

In the spheroid, adhesion-dependent cells proliferate using the carrier as a scaffold in the early stage of culture. Therefore, it has a form in which one cell is joined with two or more carriers, and one carrier is joined with two or more cells. The shape of the spheroid is indefinite, but it can be considered that the spheroid is formed into a substantially spherical mass composed of individual cells. Although the size of the spheroid varies depending on the cell type and the culture period, it has a diameter of 50 to 500 μm, preferably 50 to 300 μm when it is considered to have a substantially spherical shape. The diameter of the spheroid in this case corresponds to the spherical diameter when the spheroid has a substantially spherical shape. If the size is within this range, cells forming spheroids, particularly cells located inside the spheroids can also have sufficient cell activity.

The spheroid-containing composition of the present invention comprises the above-mentioned spheroid composed of a cell-adhesive carrier and cells, and a substrate capable of holding the cells of the spheroid. In such a spheroid-containing composition, handling of the spheroids is facilitated by combining the base materials, and since the cells are retained while having biological activity, the effect that the spheroids have in the application depending on the base material. Can be demonstrated.

The base material capable of holding the spheroid cells may be any material as long as it can hold the spheroid cells, but it is used as an external preparation such as an ointment or an injection, or as a culture carrier. Those having a flowability are preferable, and those having a fluidity are particularly preferable. By using a fluid substance, it can be easily applied to various uses while maintaining the spheroid form.

The ointment which can be used here includes substances such as collagen, fibrin, gelatin, agarose, alginic acid, etc. which are hardened by the temperature or the added substances. Injectables include substances such as saline, PBS and the like. When such an external preparation is used, application,
Since injection and the like can be easily performed, spheroids can be easily handled, and cell therapy with spheroids can be performed after application or injection. Further, since the spheroid form is retained in the composition, the effect as a spheroid can be maintained for a long time in the external preparation.

The culture carrier preferably has a three-dimensional structure, and it is particularly preferable to embed the spheroids in a gelable culture carrier. In this case, the culture carrier includes collagen, fibrin, gelatin, agarose,
Alginic acid or the like can be used. In this case, since it is possible to start culturing cells in a spheroid state with respect to those embedded with individual cells, it is possible to start culturing at an early stage and shorten the culturing period.

The spheroid-containing composition may contain, in addition to the spheroid and the base material, other components depending on the use of the spheroid-containing composition. The other component used here may be any component that is usually used in the selected application, as long as it does not impair the cell activity of the cells contained in the spheroid.

The spheroid-containing composition can be produced by combining the spheroid as a spheroid suspension in which the spheroid is suspended and the substrate. Here, it is necessary to combine the individual spheroids contained in the spheroid suspension to such an extent that they are not destroyed. This makes it possible to obtain a spheroid-containing composition in which a spheroid composed of a plurality of cells is included in a base material. At this time, it is preferable that the spheroids are uniformly dispersed in the composition, but even if the spheroids are not strictly uniformly dispersed, if the spheroids are retained and dispersed in the composition, the present composition The effect of the thing can be obtained. The present invention is described below with reference to examples, but these examples are merely examples and do not limit the present invention.

[0034]

[Examples] <Example 1: Chondrocyte spheroids> Shoulder cartilage was collected from the knee, hip, and shoulder joints of Japanese white rabbits, subjected to enzyme treatment with trypsin EDTA solution and collagenase solution, and chondrocytes were separated and collected. . After washing the obtained chondrocytes, 10% FBS (fetal bovine serum) -containing DMEM (Dulbecco's modified Eagle medium) was added to give a cell density of 1 × 1.
0 Cell suspensions were prepared so that the ^four / ml.

On the other hand, acidified 0.01N HCl2.
After diluting by adding 0.1 ml of 3% atelocollagen implant (manufactured by Koken Co., Ltd.) to 3 ml, D
A 0.1% collagen solution was prepared by adding 0.6 ml of MEM. The collagen solution does not gel at this concentration.
After dissolution, the pH of this collagen solution was adjusted with 1N NaCl to neutralize it. After insolubilization by neutralization, this collagen solution was centrifuged at 1500 rpm for 5 minutes to precipitate a large number of collagen fibers, and the supernatant was removed to obtain a large number of collagen fiber populations.

200 μl of the above cell suspension was mixed (embedded) with 8 mg of this population of collagen fibers and seeded on the bottom surface (4.3 cm ² ) of the culture vessel. At this time, the collagen fiber concentration was 0.8 w / v%, and the cell concentration was 2 × 10 5.
³ pieces / ml. Then, as a liquid medium, 50 μg /
DMEM containing 10 v / v% FBS prepared to contain ml ascorbic acid and 100 μg / ml hyaluronic acid
It was poured into the culture vessel, 37 ° C., 2 under the conditions of 5% CO ₂
Culture was performed for a week. The medium was replaced appropriately. After culturing for 2 weeks, a spheroid suspension containing a large number of spheroids was obtained. In this spheroid suspension, since the spheroids are attached to each other, the culture vessel was lightly shaken to separate individual spheroids in the medium. As a result, spheroids having a diameter of about 50 μm to 150 μm could be obtained (see FIG. 1).

<Example 2: Fibroblast Spheroids> DMEM containing 10% FBS was added to mouse fibroblasts to prepare a cell suspension having a cell density of 1 × 10 ⁴ cells / ml. Subsequently, the cell suspension was mixed (embedded) with a large number of collagen fibers prepared in the same manner as in Example 1 and seeded on the bottom surface of the culture vessel. After the seeding, the same liquid medium as in Example 1 was injected into the culture container, and the culture was performed for 1 week under the conditions of 37 ° C. and 5% CO 2. After culturing for 1 week, a spheroid suspension containing a large number of spheroids was obtained. In this spheroid suspension, since the individual spheroids are attached to each other, the culture vessel was gently shaken to separate the individual spheroids in the medium. As a result, spheroids having a diameter of about 50 μm to 150 μm could be obtained.

[0038]

As described above, according to the method for producing spheroids of the present invention, spheroids of a predetermined size can be uniformly and easily produced in large quantities by various cell types without being limited to the cell types. can do. Moreover, the spheroid of the present invention is not limited to cell types, and can provide spheroids of various sizes with various cell types. Furthermore, according to the spheroid-containing composition of the present invention, the spheroids described above can be easily applied to various applications such as external preparations.

[Brief description of drawings]

FIG. 1 is an enlarged view of a spheroid according to an embodiment of the present invention.

Claims (9)

[Claims] 1. A method for producing spheroids, wherein a carrier population composed of a plurality of carriers having cell adhesiveness and adhesion-dependent cells are mixed under mixing conditions so that the carrier population does not aggregate together with the cells. And a culture step of culturing the mixture for a predetermined period of time. 2. The method for producing a spheroid according to claim 1, wherein the carrier is collagen fiber. 3. The method for producing a spheroid according to claim 2, wherein the carrier is obtained by insolubilizing a 0.1 to 0.3 W / V% collagen solution. 4. The mixing condition is that the cells are 1 × 10 ³ -1.
The method for producing spheroids according to any one of claims 1 to 3, wherein the carrier population is mixed at a cell seeding density of × 10 ⁵ cells / ml. 5. A spheroid produced by the production method according to claim 1. 6. A spheroid containing collagen fibers and adhesion-dependent cells. 7. The spheroid has a diameter of 50 to 500 μm.
The spheroid according to claim 5 or 6, which is in the range of m. 8. A spheroid-containing composition comprising the spheroid according to claim 5, and a substrate capable of holding the spheroid. 9. The spheroid-containing composition according to claim 8, wherein the base material is any one of an ointment, an injection and a culture carrier.