JP2009247334A - Carrier for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem that, though particle-like beads, hollow fibers and the like are brought into practical use to secure surface areas for adhesion in a large scale culture of adherent cells, those methods are in lack of simplicity with bad efficiency in sowing cells, requirement of specific processes for sowing the cells and the like and are accompanied with difficulty in industrially using the adherent cells. <P>SOLUTION: A carrier for cell culture is provided. The carrier is a solid body made by cutting external portions of a minute hollow body having the external shape of a sphere, an ellipsoid, a polyhedron, a cylinder or a pyramid and a 50-2,000 μm maximum major diameter and a maximum length perpendicular to the maximum major diameter, has an inner wall to a hollow part of which adherent cells can adhere, 5-40% opening on the external shape surface connecting the external space with the hollow part. A method for culturing adherent cells is also provided using the carrier for culturing cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell culture carrier and a method for culturing cells using the cell culture carrier of the present invention.

  In recent years, with the progress of cell isolation technology, cell culture technology, and cell storage technology from living organisms, studies are underway to utilize cells for various purposes. The technology that is most widely used in industry is bioproduction in which cells are used to produce cell growth products. Examples include the production of interferon, the production of monoclonal antibodies, the production of various viral vectors, and the like, and various industrial applications are made. In addition, in the development of medicines and research on life activities, assays using cells are performed as a method for screening drugs with high throughput instead of expensive experimental animals. Attempts have also been made for cell therapy using cells themselves for medical treatment.

  Thus, industrial applications of cells are being promoted in many fields, but many of these attempts are performed by non-adherent cells that can maintain cell survival in a floating state. Adherent cells that do not belong to this cell need to adhere to the scaffold to maintain cell survival, and are more convenient than non-adherent cells in terms of maintaining cell survival, culturing and handling cells at high density. Inferior to For example, in bioproduction, it is desirable to culture cells at as high a density as possible. In this respect, non-adherent cells that can be cultured in a suspended / suspended state are preferred.

  In recent years, with the progress of research on genome analysis and proteome analysis, many useful cell growth products have been discovered from the inside of multicellular organisms such as mammalian cells and insect cells, but in order to produce these cell growth products Adhesive cells derived from multicellular organisms may be required, and the agitation culture method used for non-adherent cells cannot be used directly for the production of these cell growth products.

  Adhesive cells can be handled in the same way as non-adherent cells if they can be maintained and cultured in a suspended state. To achieve this, a cell culture carrier has been devised. (Patent Document 1).

  As such cell culture carriers, carriers composed of various materials have been devised so far. By adhering adherent cells on the carrier, growing on the surface of the particles, and culturing the particles together, the surface area of the cells in the culture tank where the cells can adhere can be increased, and the cell culture density can be improved.

  The cell culture carrier originally devised was a simple spherical carrier, but the surface of such a carrier has a convex adhesive surface, and the cells adhering to it have to take a widely extended form. It is known that such a state creates an abnormal state in the cell and reduces the cell growth rate, the survival rate, and the production amount of the cell growth product. In addition, the initial adhesion efficiency of the cells to such a convex surface is poor, and it takes time to grow the cells to the whole particles after seeding the cells.

  In addition, many of the normal mammalian cells removed from the living body require many self-produced soluble substances such as cytokines for cell adhesion, survival, and proliferation, such as the cell culture carriers used in the early days. In the culture method in which the density of cells becomes extremely low at the time of cell seeding and the self-produced cytokine is dispersed, it is difficult to seed, efficiently proliferate, and culture the cells.

  Furthermore, these cell culture carriers that adhere cells to the surface of the carrier are unavoidably subject to physical damage to the cells due to contact between the carrier and the culture tank or between the carriers during suspension culture. There was a problem that speed and culture density were limited.

  Cell culture carriers have been further improved, and porous cell culture carriers (Patent Document 2) formed from foamed materials using polyurethane or the like for high-density culture of cells (Patent Document 2), cellulose fibers, carbon fibers, etc. ( A cell culture carrier composed of small pieces having a large number of gaps into which cells are introduced has been devised.

  In these carriers, some of the adhered cells enter the pores inside the carriers, so compared to spherical cell culture carriers, the negative effects on the cells due to the obtuse angle of the adhesion surface, between the carriers, between the carriers and the culture tank Physical damage to the cells due to contact with can be suppressed.

  However, with this type of carrier, it is difficult to strictly control the pores that enter the inside of the carrier where cells can enter, and even if the carrier has a wide inner wall surface, the pores leading to the inner wall surface are disorderly. Only a part of the inner wall surface that is formed and is present during culture is used. In addition, the cell introduction route needs to be performed through randomly formed pores, and a common method is to mix a cell dispersion and a carrier and introduce a part into the pores when suspended. . In order to improve these efficiencies, the intermittent stirring method and Ficoll method (Non-patent Document 1) are known, but these methods do not have satisfactory introduction efficiency, and the cells are efficiently applied to all the inner wall surfaces of the carrier. It was not the method that can be introduced.

  In order to efficiently introduce cells into a cell culture carrier that is difficult to introduce such cells, a method of repeatedly infiltrating and removing the solution from the inside of the carrier (Patent Document 4) and a chamber under negative pressure are used. Several methods have been devised, such as a method for forcibly introducing cells into the carrier (Patent Document 5), but a device for introduction is required, or some damage to the cells is caused by the introduction. As expected, both methods increased the number of work steps in the initial stage of culture.

  As a method for culturing adherent cells at high density, there is a culture using hollow fibers. Cell growth products can be produced by cells densely integrated and cultured by filling cells inside or outside the bundled hollow fibers and circulating the culture solution (Patent Document 6). There is also an attempt to realize a “hollow fiber type artificial liver” that cultivates a large amount of hepatocytes in a hollow fiber and circulates patient blood using this mechanism.

Although the cell culture method using these hollow fibers is a method capable of realizing a high culture density, cell filling into a long hollow fiber requires a highly controlled apparatus and method that requires cells to be fed together with the liquid feeding. It was necessary. In addition, in order to cultivate cells cultured in hollow fibers, it is necessary to circulate the cell culture solution forcibly. Due to this limitation, bioproduction plants and artificial organ modules are complicated, including peripheral auxiliary devices. However, there is a problem that it must be made large-scale.
JP-A-4-360682 JP-A-4-281784 JP 2004-135668 A JP 2006-25635 A JP2006-81468 JP 2003-180334 A Supervised by Michio Oishi "Animal cell culture technology and substance production" 2002 CMC Publishing

  The above-described conventional cell culture carrier has the following problems. That is, in the conventional carrier, since the adhesion surface of the carrier surface is convex, the initial adhesion of the cells is poor, and the cell condition is adversely affected because the cells take an unnaturally extended shape. Furthermore, since it is an environment in which self-produced cytokines are easy to disperse, it is difficult to adhere cells close to normal to carriers.

  Further, in the conventional cell culture carrier, since the cells are adhered to the surface of the carrier, physical damage is inevitable when the carriers are in contact with each other or when the carrier is in contact with the culture tank. Although there is an improved cell culture carrier made of foam material and fiber material aiming to solve these problems, because the internal voids are disordered structure formed using foam or fibrous material, There are problems in that space utilization efficiency is poor and it is difficult to introduce cells uniformly with high efficiency.

  In addition, although the culture method of adherent cells using hollow fibers enables high-density culture, special measures are required for the uniform introduction of adherent cells into the hollow fibers. In some cases, forced circulation of the culture solution is required, so that a separate liquid supply facility is required in addition to the cell culture portion.

  In order to solve the above-described problems, the present invention has devised a novel cell culture carrier shown in the present invention.

That is, the present invention has the following configuration.
(1) A solid body having an outer shape of a sphere, an ellipsoid, a polyhedron, a columnar body, or a cone, and the outside of a micro hollow body having a longest diameter and a maximum length in a direction perpendicular to the longest diameter of 50 to 2000 μm. A cell culture carrier having an inner wall capable of adhering adhesive cells in a hollow part, and an opening connecting the outer part and the hollow part of 5 to 40% on the outer shape surface.
(2) The cell culture carrier according to (1), wherein the hollow portion is continuous.
(3) The cell culture carrier according to (1) or (2), which has one or two openings.
(4) The cell culture carrier according to any one of (1) to (3), wherein the external shape is a columnar body.
(5) The cell culture carrier according to any one of claims 1 to 4, which is produced by cutting a hollow fiber.
(6) A cell culture method for culturing adhesive cells using the cell culture carrier according to any one of (1) to (5).
(7) The cell culture method according to (6), wherein the cell culture carriers are arranged in a plane in a medium.
(8) The cell culture method according to (7), wherein the cell culture carriers are arranged in a plane on a medium by bundling and cutting the hollow fibers.

  The cell culture carrier of the present invention has an effect close to that of a normal cell in terms of initial adhesion efficiency, cell survival rate, proliferation rate, protein production ability, etc., compared with a cell culture carrier that retains cells on the existing outer periphery. Is obtained.

  The cell culture carrier of the present invention is a solid obtained by cutting the outside of a micro hollow body having a longest diameter of 50 μm to 2000 μm having a spherical, ellipsoidal, polyhedral, columnar or pyramidal outer shape, and an adhesive cell is attached to the hollow part. May have a predetermined shape having an inner wall that can be bonded, and having an opening connecting the outer space and the hollow portion in a portion of 5 to 40% of the outer shape surface.

  Here, the “shape” indicates the shape of a portion where the constituent material of the carrier exists. “External shape” refers to the three-dimensional shape of the “internal space” of the carrier. What is the inner space? If there is a carrier that intersects at both ends of (A) among any three-dimensional straight lines passing through (A) at any space point (A), that point is designated as “ It is defined as “inner space”. A portion that is not an “inner space” is referred to as an “outer space”.

  The external shape before cutting of the cell culture carrier of the present invention is a sphere, an ellipsoid, a polyhedron, a columnar body or a cone, but the external shape is also a shape whose shape is flexibly changed by an external stress. Included in the external shape. The “columnar body” here is defined as the shape of a cylinder, an elliptical column or a polygonal column, and the “weight” is defined as the shape of a cone, an elliptical column or a polygonal column.

  A portion of the “inner space” that is not a carrier is referred to as a “hollow portion”. The hollow part is a space in which the culture solution or cells can enter from the outside of the carrier, but the degree of freedom when moving to the outer space again can be limited.

  In the cell culture carrier of the present invention, there are hollow portions in this inner space, and the shape of the carrier is complicated. However, when referring to the external shape, the portions excluded by these hollow portions are not considered. That is, the internal space combining the carrier portion and the hollow portion is referred to as “external shape”.

  “Micro hollow body” indicates a micro solid shape having a portion in which the constituent material of the carrier does not exist in the internal space, and “micro” indicates here that the size is 2000 μm or less. There may be one or a plurality of hollow portions where no constituent material exists.

  A plurality of hollow portions means that there are a plurality of non-continuous hollow spaces in which cells cannot move back and forth inside the carrier. Even if there is a continuous part, if cells cannot move through the continuous part, it is considered that there are a plurality of hollow spaces.

  The “opening” refers to a site through which cells can pass among the boundary between the hollow part and the outer space.

  Although there are no restrictions on the porosity inside the carrier as long as other conditions are satisfied, it is preferable that the porosity is high in order to achieve high-density cell culture that is an effect of using the cell culture carrier of the present invention. , Preferably 50% or more, most preferably 80% or more.

  Here, the “void ratio” indicates the proportion of the “hollow portion” in the “inner space”. For example, when the volume occupied by the constituent material of the cell culture carrier is 10, the volume of the hollow portion is 90, and the volume of the inner space is 100, the porosity is calculated to be 90%.

  The “inner wall” indicates the surface of the carrier constituting material existing in the hollow portion.

  An “adhesive cell” refers to a cell that adheres to an adhesive surface when an adhesive surface is present at an adjacent site of the cell. It is mainly found in living organisms of multicellular organisms, such as hepatocytes, skin keratinocytes, hair matrix cells, oral epithelial cells, esophageal epithelial cells, gastric mucosal epithelial cells, small intestinal absorption epithelial cells, large intestine absorption epithelial cells, Bile duct epithelial cells, pancreatic insulin secreting cells, pancreatic glucagon secreting cells, bone cells, chondrocytes, smooth muscle cells, cardiomyocytes, muscle satellite cells, nerve cells, hormone secreting cells, white fat cells, brown fat cells, bone marrow, etc. Examples include, but are not limited to, cultured cells, ES cells, iPS cells, or adhesion-derived cells prepared by induction of adult stem cells. It is preferable to apply a culture carrier, among which hepatocytes, oral epithelial cells, esophageal epithelial cells, gastric mucosal epithelial cells, small intestine absorbing epithelial cells, large intestine absorbing epithelial cells Biliary epithelial cells, pancreatic insulin secreting cells, pancreatic glucagon secreting cells, primary culture cells of hormone-secreting cells, ES cells, adhesion system cells differentiated from iPS cells or adult stem cells are preferred. In addition, cell lines created by selecting cells that exist in nature, and cells created artificially by gene transfer and gene deletion in normal cells or cell lines derived from living organisms are the same as cells that exist in nature. It can be used suitably. Examples of established adherent cells include CHO cells derived from Chinese hamster ovary cells, MDCK cells derived from canine kidney epithelial cells, NIH3T3 cells derived from mouse fetal skin, PC12 cells derived from rat adrenal medulla, and S2 cells derived from Drosophila. , Sf9 cells derived from sputum, Vero cells derived from African green monkey kidney, HeLa cells derived from human uterine cancer, Caco-2 cells derived from human colon cancer, Huh7 cells and HepG2 cells derived from human liver cancer.

  The cell culture carrier of the present invention has a maximum length of 50 μm to 2000 μm in the vertical direction. This size is used to efficiently introduce adherent cells into the cell culture carrier of the present invention and efficiently use the space. The size is suitable for. That is, when the size is 50 μm or less, the number of cells introduced into individual carriers is limited, and the space utilization efficiency during culture is reduced. In addition, separation from the medium becomes difficult at the time of culture, and the efficiency is lowered. In addition, those having a size of 2000 μm or more are accompanied by difficulty in uniform cell introduction, and the space utilization efficiency is lowered depending on the internal shape. The size is preferably a small piece having a size of 50 μm to 1000 μm, and most preferably 100 μm to 500 μm.

  The cell culture carrier of the present invention has an opening of 5 to 40%, but this property partially suppresses the circulation of the culture solution between the hollow part and the outer space of the carrier, and improves the self-produced cytokine concentration, In addition, this is a necessary structure for introducing cells into the carrier. When the opening of the cell culture carrier is less than 5%, since the opening is too small, the introduction efficiency at the time of cell seeding is reduced, and when the opening of the cell culture carrier exceeds 40%, This is not preferable because efficiency is lowered and an increase in the concentration of self-produced cytokines cannot be expected.

  Various shapes can be considered for the external shape of the cell culture carrier of the present invention. However, when a micro hollow body having a spherical external shape is considered, when a spherical opening is present at the top of the spherical hollow body, the radius of the sphere If there is an opening having a half radius, the ratio of the surface area of the opening is about 6.6%, and a cell culture carrier having an opening much smaller than this significantly reduces the efficiency at the time of cell introduction. There is a case. Also, when considering a hemispherical cell culture support in which a spherical hollow body is cut out in a plane at the center, the opening is about 33% if the thickness of the support is not considered, and the opening is wider than this shape. Although the efficiency at the time of cell seeding can be kept high depending on the shape, it may be difficult to obtain the effect of increasing the local concentration of the self-produced cytokine during cell culture. Therefore, in the cell culture carrier of the present invention, the opening needs to be 5 to 40% with respect to the external shape area. However, when considering the shape of the cell culture carrier, it is preferably 5 to 35%, more preferably 10-30%.

  Regarding the constituent material constituting the cell culture carrier of the present invention, any material that can be molded into the shape of the cell culture carrier of the present invention can be used, and there are no restrictions on the material adjustment method, average molecular weight, etc. Alternatively, a polymer substance can be used. Here, the polymer includes rubber, polysaccharide, protein, water-soluble synthetic polymer, organic solvent-soluble polymer, and specific examples include cellulose, gum arabic, tragagant gum, carrageenan, agar, guar gum, karaya gum, locust bean. Gum, pectin, galactan, mannan, pullulan, xanthan gum, gelatin, casein, chondroitin sulfate, collagen, elastin, fibroin, chitosan, chitin, hyaluron, carboxymethyl starch, methylhydroxypropyl starch, alginate, alginate, polyvinyl alcohol, polyvinyl Pyrrolidone, polyvinyl ethyl ether, carboxyvinyl polymer, polyacrylic acid, polyacrylamide, polystyrene, polyvinyl chloride, polyvinyl acetate, polyacrylic Nitrile, polysulfone, polyurethane, acrylic methacrylic acid, fluorine-based polymers, polyamides, polypiperazine amides, polyureas, crosslinked polyether, and the like polyacrylonitrile. Preferred are polysulfone and acrylic methacrylate, and more preferred is polysulfone used in the examples of the present invention.

  Furthermore, the cell culture carrier of the present invention may be composed of a single constituent material, or may be composed of a plurality of materials such as a cell adhesive surface composed of another constituent material. For example, in order to improve adhesion of adherent cells, it is preferable to coat the entire surface of the carrier or only the surface inside the carrier with another constituent material that improves cell adhesion. The coating material is not particularly limited, but is coated with collagen, gelatin, fibronectin, introduction of positively or negatively charged functional groups, various cell adhesion factors such as RGD peptide (Arg-Gly-Asp) Modification is preferred.

  The cell culture carrier of the present invention may have a plurality of inner voids each having an opening. However, when the hollow portion is continuous in the inner space, that is, when only one hollow portion is present. Since cell introduction efficiency and efficient use of the internal space can be realized, high performance can be exhibited, which is preferable. However, in the process of producing a large number of fine carriers such as the cell culture carrier of the present invention, some carriers may have internal spaces that are not continuous due to individual differences that occur during production. When the average number of hollow portions of the culture carrier is 1.5 or less, the same effect as when all the cell culture carriers have a continuous hollow portion can be obtained, and more preferably, the average hollow portion The number is 1.1 or less.

  The cell culture carrier of the present invention preferably has one or two openings that connect the hollow portion and the outer space. The number of openings is related to the movement of cells passing through the hollow part of the cell culture carrier and the cell culture medium. If there are one or two openings, it is effective to increase the efficiency during cell introduction. In addition, the concentration of the self-produced cytokine inside the carrier can be kept high. Even if there are two openings, it is possible to introduce a cell with high efficiency by creating a state in which the opening on one side is closed at the time of cell introduction. However, among a plurality of produced carriers, even when some of the carriers have two or more openings due to variations in production, it is preferable because the same function can be obtained if the overall average number is 2.5 or less. Preferably, the average is 2.2 or less. In addition, although a structure does not exist among the parts which connect a hollow part and external space, presence of the fine pore structure which does not have a size which can permeate | transmit a cell is not restrict | limited. Depending on the type of cell and the culture method, pores that cannot be permeated by these cells may be desirable because they facilitate the flow of the culture solution.

  The cell culture carrier of the present invention is preferably a columnar body. In order to efficiently seed cells on the cell culture carrier of the present invention, it is preferable to use a state where the openings are aligned in a dense state as described later, but the columnar body is suitable for this application. More preferably, it is a cell culture carrier having the columnar outer shape shown in FIG. However, the presence of a fine exceptional portion at the end portion is allowed as long as the main body portion is a columnar body. The diameter of the columnar body is preferably 100 to 1000 μm, and more preferably 200 to 500 μm.

  Examples of the method for producing a cell culture carrier of the present invention include production using a template, production by lithography, processing of a natural structure having a similar structure, and production by cutting a precursor structure. Among them, the cell culture carrier having the columnar shape, which is a preferred embodiment of the cell culture carrier of the present invention, is preferably produced by cutting a hollow fiber. For example, when a hollow fiber having a length of 100000 μm is cut, 2000 cell culture carriers of the present invention having a length of 50 μm can be produced. In consideration of production efficiency, it is preferable that the length of the hollow fiber is long. Preferably, the hollow fiber having a length of 100 times or more of the maximum length in the direction perpendicular to the longest diameter of the finally obtained cell culture carrier is cut. More preferably, it is a method of cutting and producing a hollow fiber having a length of 1000 times or more.

  Moreover, in the step of producing the cell culture carrier from the hollow fiber, one of the preferable production methods is to cut the hollow fiber in a bundled state. It can be produced efficiently. After being produced by bundling and cutting the hollow fiber, it can be used as it is as a cell culture carrier, or it can be used as a cell culture carrier after being subjected to treatment such as surface modification and closing one of the openings.

  The present invention also relates to a cell culture method for culturing adherent cells using the cell culture carrier. Cell culture as used herein refers to maintaining the survival of adherent cells held inside the carrier via a culture solution containing oxygen, nutrients, and the like. In addition, as a culture form, a culture tank generally used for cell culture can be used. Specifically, a Petri dish, a plastic plate, a plastic tube, a glass tube, a hollow fiber, a spiral film, a column, Examples include, but are not limited to, multiwell plates, multi-sheet plates, agitation culture chambers used to produce cell growth products, and cell holding parts of various cell assay equipment. If it is good. The medium used for cell culture is not particularly limited as long as it is a medium suitable for cell survival.

  In the cell culture method of the present invention, the cell culture carrier can be arranged in a plane in the medium by taking advantage of the shape characteristics of the cell culture carrier. Here, “arranging in a plane in the medium” means that most of the cell culture carriers are aligned without overlapping, and includes, for example, a state where they are aligned on the outer wall of a cylindrical culture tank. When the cells are seeded with the columnar cell culture support closely packed and the direction of the openings aligned, it is possible to adhere the cells to the cell culture support with high efficiency in the process of seeding the adherent cells. It is preferable because the cell culture carrier of the present invention can be used efficiently. As a method for assembling the cell culture carrier in a state where the openings are aligned and dense, a method for producing the cell culture carrier as a collection in which the openings are aligned and dense, or a method for assembling the prepared cell culture carrier later can be considered. Preferably, it is a method of producing as a dense assembly.

  The method for introducing cells into the cell culture carrier of the present invention is not particularly limited, and a method in which a cell suspension is dropped into the opening of the cell culture carrier and introduced by gravity, or the cell suspension is added to the cell culture carrier. However, when cell culture carriers are arranged in a flat plane in the medium, the openings are aligned in one direction opposite to the fixed surface. By dropping the cell suspension, the cells can be efficiently introduced into the cell culture carrier by gravity.

  There is no particular limitation on the method of arranging the cell culture carrier in a flat plane in the culture medium, which is a preferred embodiment of the cell culture method of the present invention, such as a method of temporarily fixing it on the plane of the culture tank, magnetism, gravity, centrifugal force, etc. Examples of the method include aligning without fixing to a culture tank, and a method of temporarily fixing to a culture tank plane is preferable.

  In the cell culture method of the present invention, the case where the adherent cells adhered to the cell culture carrier of the present invention are cryopreserved for cell culture is also part of the cell culture. The method of freezing is not limited, but is preferably freezing at −80 ° C. or lower, more preferably freezing at −150 ° C. or lower.

  Examples are given below to describe the details of the present invention, but the present invention is not limited to the Examples.

Example 1 Preparation of Cell Culture Carrier <Method>
After bundling polysulfone hollow fiber (for Toray, “Trevino”) with an outer diameter of 400 μm and an inner diameter of 300 μm so as not to leave a gap, it is immersed in a frozen section mounting agent (Tissuetech OCT Compound (Sakura Finetech)) It was allowed to stand for several hours until the compound sufficiently penetrated. While maintaining the bundled state, the frozen block was frozen at −150 ° C. and the frozen block was sufficiently frozen at −20 ° C. with a frozen section preparation machine (OTF5000 (Bright UK)), carbon blade (replacement blade C35 for microtome) (Feather)) and cut to a thickness of 20 μm. The cut hollow fiber assembly is fixed on a slide glass (Silane coated slide S3003 (Dako)) thinly coated with a water-soluble adhesive (silylated urethane resin adhesive (“Bond New Hitech” (Konishi))), Dried.

  The cell culture carrier sufficiently dried on the slide glass was immersed in pure water for several hours to remove the frozen section mounting agent used at the time of cutting. Thereafter, the cell culture carrier was sterilized by treatment at 120 ° C. for 20 minutes in an autoclave together with the fixed slide glass, and stored at room temperature until use.

<Result>
An electron micrograph (Hitachi S4800) of the prepared cell culture carrier is shown in FIG. 3A, and a photograph taken with a stereomicroscope is shown in FIG. 3B. This cell culture carrier has an outer diameter of 400 μm, an inner diameter of 300 μm, a height of about 300 μm, and an opening ratio of about 29%. The cell culture carrier was fixed on the slide glass in a dense state with the opening facing up and down (FIG. 3C), and no disturbance due to cutting or autoclaving occurred. In addition, the cell culture carrier fixed with a water-soluble adhesive on the slide glass was easily released from the slide glass by a weak water flow after being immersed in the cell culture medium for several hours and became dispersed. (Fig. 3D)
Example 2 Seeding of Caco-2 cells on cell culture carrier Caco-2 cells (derived from human colon) cultured in a culture medium (DMEM / 10% FBS (Gibco)) on a 10 cm cell culture plate (Falcon) The cell line was treated with a cell detachment solution (0.25% trypsin / EDTA (Sigma)) and released from the plate. The remaining cell detachment solution was removed by washing twice with the culture medium. Ten million human colon cancer-derived Caco-2 cells were suspended in 10 ml of culture medium.

The slide glass with the cell culture carrier closely aligned is placed in the center of a 10 cm cell culture plate (Falcon), and a 0.3% type I collagen solution for cell culture (“Selgen” (Koken)) is added dropwise at room temperature. The cell culture carrier surface was coated with collagen. The added collagen solution was removed, and the cell culture carrier was washed with a sterilized phosphate buffer. A small amount of Caco-2 cell dispersion was dropped from the top of the cell culture carrier. After the addition, the mixture is allowed to stand for 30 minutes, and Caco-2 cells settle on the bottom and is introduced into the cell culture carrier. The slide glass on which the cell culture carrier is fixed is transferred to a 50 ml tube (Corning), and the culture medium. In a state in which the slide glass was satisfied, the slide glass was maintained in a vertical state, and the cells were cultured for 24 hours in a culture incubator (NAPCO CO26000) at 37 ° C. and 5% CO ₂ concentration.

  After 24 hours, the lid of the 50 ml tube was opened, and the cell culture carrier was released from the slide glass by gently suspending the solution. The slide glass from which the cell culture carrier was removed was removed, and the inside of the 50 ml tube was centrifuged at 3000 rpm for 5 minutes to precipitate the cell culture carrier, and the operation of removing the culture solution was performed three times.

<Result>
FIG. 4B shows a frozen section of a cell culture carrier to which Caco-2 cells are adhered, and FIG. 4A shows an image observed with a scanning electron microscope of the cell culture carrier to which cells are adhered.
Some cells were introduced into the cell culture carrier by dropping the Caco-2 cell suspension from the top of the cell culture carrier. Thereafter, the Caco-2 cells introduced inside can be engrafted inside the cell culture carrier by keeping the glass slide on which the cell culture carrier is fixed in order to adhere to the inner wall of the cell culture carrier. confirmed.

Example 3 Suspension culture of adherent cells using cell culture carrier <Method>
The transition of cell viability during stirring suspension culture of a cell culture carrier having Caco-2 cells adhered to the inner wall and Caco-2 cells alone was compared and evaluated.

  Caco-2 cells cultured in a 10 cm cell culture plate in the same manner as in Example 1 and the cell culture carrier prepared in Example 2 and recovered in the cell detachment solution were centrifuged at 180 G for 5 minutes each 3 times. After the cells were washed, each cell was divided into 6 tubes, and suspension culture was performed at a rate of 30 rotations / minute with an inversion mixing rotator installed in a culture incubator. After 0, 1, 3, 6, 24, and 48 hours after the start of culture, the cells and the cell culture carrier were precipitated by centrifugation at 180 G for 5 minutes, and then the culture solution was removed and stored frozen at -30 ° C. . 48 hours after all samples were collected, the samples were thawed and used using a cell viability measurement kit (Cell Counting Kit (WST-8) (Dojindo)) and a plate absorptiometer (Multiscan JX (Thermo Laboratories)). Cell viability was measured.

<Result>
The change of each cell viability is shown in FIG. The survival rate of Caco-2 cells without using a cell culture carrier decreased rapidly from immediately after the start of the assay, and became 20% or less after 24 hours. On the other hand, the survival rate of Caco-2 cells adhered to the cell culture carrier shows a certain decrease after the start of the culture, but thereafter, it tends to increase, and the survival in the cell culture carrier is maintained and the cell proliferation is not increased. It had occurred.

An example of a shape satisfying the requirements of the cell culture carrier of the present invention. Two examples of A and B are shown. In the three-dimensional conceptual diagram in the upper diagram, the hatched portion indicates the inner wall surface. The figure below is a schematic cross-sectional view of the above figure, cells Examples of shapes that satisfy the requirements of the cell culture carrier of the present invention, A, B, C, D, E, and F, are shown. In the three-dimensional conceptual diagram in the figure, the hatched portion indicates the inner wall surface. Scanning electron micrograph (A), stereomicrograph (B), cell culture carrier (C) aligned on a glass slide, cell culture carrier (D) dispersed in an aqueous solution of the cell culture carrier prepared in Example 1 ). 2 is a scanning electron micrograph (A) of a cell culture carrier to which Caco-2 cells are adhered according to Example 2, and a visible light observation image (B) of a frozen section. It is a change result of the Caco-2 cell survival rate at the time of the stir culture of the cell culture carrier of this invention by Example 3 and a cell alone.

Claims (8)

  A solid body having a spherical shape, an ellipsoid shape, a polyhedron shape, a columnar shape, or a pyramid shape, the longest diameter and the outside of a micro hollow body having a maximum length in a direction perpendicular to the longest diameter of 50 to 2000 μm, and having a hollow portion A cell culture carrier having an inner wall to which adherent cells can adhere, and an opening connecting the outer space and the hollow portion of 5 to 40% on the outer shape surface.   The cell culture carrier according to claim 1, wherein the hollow portion is continuous.   The cell culture carrier according to claim 1 or 2, which has one or two openings.   The cell culture carrier according to any one of claims 1 to 3, wherein the external shape is a columnar body.   The cell culture carrier according to any one of claims 1 to 4, which is produced by cutting a hollow fiber.   A cell culture method for culturing adhesive cells using the cell culture carrier according to claim 1.   The cell culture method according to claim 6, wherein the cell culture carriers are arranged in a plane in the medium.   The cell culture method according to claim 7, wherein the cell culture carriers are arranged in a plane on a medium by bundling and cutting the hollow fibers.