JP2015126720A - Cell culture method and cell culture apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a suspension culture method in which shear power is reduced by suppressing turbulent flow generation in a culture fluid.SOLUTION: The present invention relates to a cell culture apparatus that includes: an incubator having the culture fluid into which a cell to be cultured is impregnated; support means which supports the incubator in an inclined posture to the horizontal plane; and driving means which makes the incubator revolve, wherein the incubator is made to be revolved around a vertical revolution axis or the revolution axis inclined to the horizontal plane as the center of the revolution orbit. The present invention also relates to a cell culture method using the cell culture apparatus.

Description

  The present invention relates to a cell culture method and a cell culture apparatus for performing the method. In particular, it relates to a suspension culture method.

Stem cells continue to retain the ability to differentiate into cells with specific functions, such as blood cells, mucosal epithelium, and epidermis, even after cell division. In the medical field, it is expected that stem cells of tissues or organs to be treated are cultured and transplanted to patients.
Embryonic stem cells (ES cells) that develop from fertilized eggs have pluripotency that can differentiate into any cell. Techniques for artificially producing such pluripotent stem cells have also been improved. By using arbitrary cells differentiated from pluripotent stem cells, the possibility of elucidating the cause of intractable diseases and confirming the safety of drugs is increasing at the molecular level.

Progress and practical application of the basic research and clinical research described above are premised on the mass production of undifferentiated stem cells that can be induced to differentiate into desired cells. Conventionally, there is a suspension culture method as a mass culture method of pluripotent stem cells. The suspension culture method is a cell culture method in which cells are suspended and three-dimensionally cultured by flowing a culture solution in a culture tank. The suspension culture method can culture stem cells at high density. Comparing the culture efficiency between the suspension culture method and the static culture method, for example, when animal cells having a diameter of 20 μm are generally cultured, the density of cells cultured in the suspension culture method is 1 × 10 ⁶ cells / cm ² Degree. On the other hand, the density in the static culture method is about 6 × 10 ⁴ cells / cm ² .

  In the three-dimensional culture of cells, the shearing force generated by flowing the culture solution becomes a cause of reducing the culture efficiency of undifferentiated cells. For example, cell differentiation of vascular endothelium and heart valve tissue cells is promoted by excessive shearing force. When three-dimensional culture is performed using a carrier, cells are easily detached from the carrier due to shearing force.

  In order to solve the problems exemplified above, various cell culture techniques in which the shearing force of the culture solution is suppressed have been proposed. Such techniques include improving the shape of the stirring blade or forming a laminar flow in the culture tank. Patent Document 1 discloses a device that includes a first substrate and a second substrate each having a microchannel structure, and in which a first substrate, a porous film, and a second substrate are stacked. . In the above device, the width and height of the second substrate are set so that the Reynolds number is less than 2000 in consideration of the physical properties and flow velocity of the liquid flowing through the microchannel structure of the second substrate. .

  However, since the above device has a complicated structure, it is difficult to easily mass-produce cells. In stem cell culture, particularly pluripotent stem cell culture, a large amount of cell loss occurs under the influence of a slight shear force. Therefore, a cell culture device and a cell culture method that can further suppress shearing force with a simple device configuration are desired.

JP 2011-147387 A

  The subject of this invention is providing the floating culture method which suppresses generation | occurrence | production of the turbulent flow in a culture solution, and reduces a shear force.

  The present invention relates to a cell culture method in which a cell to be cultured is immersed in a culture medium in a culture vessel to culture the cell, and revolves around a revolution axis in a state where the culture vessel is inclined with respect to a horizontal plane. Cell culture method. The cell culture method includes at least one of an inclination angle formed by the bottom surface of the incubator and a horizontal plane, a revolving radius of the incubator, and a revolving speed of the incubator. A laminar flow is formed in the culture solution by changing within a range to follow the movement. In the present invention, it is preferable to revolve the incubator with the revolution axis of the incubator inclined with respect to the horizontal plane. The present invention includes an incubator having a culture solution in which cells to be cultured are immersed, support means for supporting the incubator in an inclined posture with respect to a horizontal plane, and drive means for revolving the incubator supported by the support means. And a cell culture device characterized in that the incubator is revolved with a vertical revolution axis or a revolution axis inclined with respect to a horizontal plane as the center of the revolution trajectory.

  The present invention can suppress the generation of turbulent flow in the culture solution and improve cell culture efficiency.

It is the schematic which shows the example of the cell culture apparatus of this invention. It is the schematic which shows the example of the cell culture apparatus of this invention.

[Cell culture method]
The cell culture method of the present invention revolves around the revolution axis in a state where the incubator is inclined with respect to the horizontal plane. Thereby, the culture solution in the incubator follows the revolution movement of the incubator to form a laminar flow. As a result, the shearing force of the culture solution can be suppressed and the culture efficiency can be improved.

  In order to explain the cell culture method, an example of the cell culture apparatus used in the present invention is shown. FIG. 1 is a schematic view of a cell culture apparatus for revolving an incubator around a vertical revolution axis. In FIG. 1, 100 is a cell culture device, and 200 is a horizontal plane on which the cell culture device 100 is placed. The cell culture device 100 includes a drive source 102 in the housing 101 thereof. The driving force of the driving source 102 is transmitted to the support shaft 104 through the driving means 103.

  The support shaft 104 is inserted through the opening 105 of the housing 101 and exposed outside the housing, and supports the platform 107 on which the incubator 106 is placed. The support shaft 104 is supported by the driving means 103 and the opening 105 of the housing 101 by a structure that does not rotate the support shaft 104. As an example of such a structure, there is a structure in which the end portion of the support shaft 104 is fixed to the driving unit 103 and the insertion portion of the support shaft 104 facing the opening 105 of the housing 101 is pivotally supported in multiple axes. . The support shaft 104 and the platform 107 are examples of support means for supporting the incubator of the present invention. The incubator 106 is supported in an inclined posture with respect to the horizontal plane 200 by the support means. θ1 is an inclination angle formed by the bottom surface 106a of the incubator 106 and the horizontal plane 200.

  When the driving force of the drive source 102 is transmitted to the support shaft 104 via the drive means 103, the support shaft 104 rotates about the revolution shaft 300. As the support shaft 104 rotates, the incubator 106 on the platform 107 revolves around the revolution axis 300 in a state where the bottom surface 106a of the incubator keeps the inclination angle θ1 with respect to the horizontal plane constant. In the present invention, the revolution trajectory of the incubator means the trajectory of the contact point between the extension line of the axis of the support shaft and the bottom surface of the incubator. An arrow A1 is a trajectory of a contact point between the extension line of the axis of the support shaft 104 and the bottom surface 106a of the incubator 106. r1 is the revolution radius of the revolution orbit A1.

  In the present invention, a laminar flow can be formed by causing the culture solution to follow the revolving motion of the incubator, in other words, by causing the culture solution to flow in a multiaxial manner. Thereby, the shearing force of the culture solution can be suppressed, and the loss of cells to be cultured suspended in the culture solution can be reduced. The above laminar flow formation is based on the inclination angle formed by the bottom surface of the incubator and the horizontal plane, the revolution radius of the incubator, the revolution trajectory of the incubator determined by the attitude of the revolution axis with respect to the horizontal plane, and the revolution speed of the incubator. It is a combined effect. By combining the above elements within a predetermined range, the turbulent flow suppressing effect and the cell sedimentation suppressing effect of the present invention are exhibited.

  The present invention adjusts at least one of the inclination angle formed by the bottom surface of the incubator and the horizontal plane, the revolving radius of the incubator, and the revolving speed of the incubator in order to make the revolving motion that allows the culture medium to follow in the incubator. . It is also preferable to adjust the inclination angle of the revolution axis with respect to the horizontal plane. FIG. 1 shows an example in which the incubator 106 is revolved around a posture in which the revolution axis 300 is 90 ° with respect to a horizontal plane, that is, a vertical revolution axis. An example of the present invention in which the incubator is revolved with the revolution axis tilted will be described later with reference to FIG.

  In the cell culture apparatus 100 that revolves the incubator 106 around the vertical revolution axis 300 illustrated in FIG. 1, the inclination angle θ1 of the incubator 106 formed by the horizontal plane 200 and the bottom surface 106a of the incubator 106 is 1 The angle is preferably from 30 ° to 30 °. When the inclination angle is less than 1 °, the culture solution cannot sufficiently follow the revolution movement of the incubator. When the inclination angle exceeds 30 °, a turbulent flow of the culture solution is easily formed.

  The revolution radius r1 of the revolution track A1 of the incubator 106 is preferably 1 cm or more and 50 cm or less. When the revolution radius exceeds 50 cm, the centrifugal force increases and adverse effects such as collision of the carrier occur.

  The revolution speed of the incubator 106 is preferably 0.1 to 60 rpm, and more preferably 5 to 20 rpm. The revolution speed of the incubator can be linked to the rotation speed of the support shaft. In that case, the incubator can be revolved at a predetermined revolution speed by setting the rotation speed of the support shaft 104 within the range of the preferable revolution speed. In the present invention, the culture solution follows the revolving motion of the incubator well. Therefore, a laminar flow is formed in the culture solution from the beginning of the revolving motion. Cells and carriers can be suspended on the laminar flow. When the revolution speed is slower than 0.1 rpm, the culture solution cannot sufficiently follow the revolution movement of the incubator. When the revolution speed is higher than 60 rpm, a turbulent flow of the culture solution is easily formed.

  In the present invention, the incubator is preferably revolved around a revolution axis inclined at a predetermined inclination angle with respect to a horizontal plane. This further facilitates the formation of a laminar flow of the culture solution. FIG. 2 is a schematic diagram showing an example of a cell culture device that revolves around the revolution axis inclined with respect to the horizontal plane as the center of the revolution trajectory. In FIG. 2, reference numeral 301 denotes a revolution axis inclined with respect to the horizontal plane 200. The cell culture device illustrated in FIG. 2 has the same configuration as the cell culture device illustrated in FIG. 1 except that the revolution axis 301 is inclined. Therefore, the description of the same reference numerals as those in FIG. 1 is omitted. Arrow A2 is the revolution trajectory of the incubator 106, and r2 is the revolution radius of the revolution trajectory A2. θ2 is an inclination angle of the revolution axis formed by the revolution axis 301 and the horizontal plane 200. In FIG. 2, θ2 is represented by an angle formed between a plane parallel to the horizontal plane forming the housing and the revolution axis for the sake of simplicity of explanation, but this angle is the revolution axis formed between the revolution axis and the horizontal plane. It is the same size as the tilt angle.

  The inclination angle θ2 of the revolution axis with respect to the horizontal plane 200 is preferably 75 ° or more and less than 90 °. When the tilt angle of the revolution axis is less than 75 °, it is difficult to keep the liquid stable. The inclination angle θ2 of the revolution axis with respect to the horizontal plane can be obtained by selecting a cell culture device in which the revolution axis is previously designed to have a desired inclination angle. As another method, it can also be obtained by using a known angle adjusting member provided in the cell culture device so as to adjust to an angle obtained by adding 1 ° to 30 ° to a desired inclination angle of the revolution axis.

  When the incubator 106 is revolved around the revolution axis 301 inclined with respect to the horizontal plane, the inclined posture of the incubator 106 that revolves around the revolution trajectory A2 changes as the incubator 106 advances along the revolution trajectory A2. That is, the inclination angle θ1 formed by the horizontal plane 200 and the bottom surface 106a of the incubator 106 changes with the revolution movement of the incubator 106. The incubator 106 for revolving the incubator around the inclined revolution axis preferably has an inclination angle θ1 that changes within a range of 0 ° to 45 °, preferably within a range of 0 ° to 30 °. It is more preferable to change with.

  The preferable range of the revolution speed and the revolution radius when the incubator is revolved around the inclined revolution axis is the same as the case where the incubator is revolved around the vertical revolution axis.

  The inclination angle between the bottom of the incubator and the horizontal plane, the revolution radius of the incubator, the revolution speed of the incubator, and the inclination angle between the revolution axis and the horizontal plane are the type of cell to be cultured, cell diameter, and seeding density. It is set in consideration of the volume of the incubator and the inner radius of the incubator. The incubator is filled with a culture solution to be described later, supplied with a carrier, seeded with cells dispersed in a single cell, and then set each of the installation conditions such as the inclination angle of the incubator and the revolution radius of the incubator. At least one is filled and installed in the cell culture device. After installation of the incubator, the incubator is revolved at a revolution speed within the above preferred range.

  In the present invention, since the culture solution has high followability to the revolution movement of the incubator, the laminar flow is formed at the same time as the revolution movement starts, and turbulence hardly occurs. Therefore, cells can be three-dimensionally cultured in an environment in which the shearing force of the culture solution is suppressed from the beginning of the revolution movement of the incubator. Thereby, in this invention, the cell seed | inoculated in the culture solution is hard to be damaged. When cell culture is performed using a carrier, the cells are less likely to be detached from the carrier. That is, the present invention has low cell loss and good cell culture efficiency.

When cultivating mouse pluripotent stem cells with a diameter of 20 μm in a culture vessel with a seeding density of 1 × 10 ⁵ cells / ml and a volume of 15 ml that revolves around the vertical revolution axis and an inner radius of 5 cm, the incline angle It is preferable that θ1 is 2 ° to 10 ° and the revolution radius r1 of the incubator is 0 cm or more and 5 cm or less. In that case, the incubator is preferably revolved at a revolution speed of 10 to 60 rpm.

In addition, when the above mouse pluripotent stem cells are cultured in a 15 ml incubator with a seeding density of 1 × 10 ⁵ cells / ml and revolving around a revolution axis inclined at an angle of 85 ° with respect to a horizontal plane, It is preferable to adjust the support shaft for supporting the incubator so that the revolving radius r2 is 0 to 3 cm. In that case, it is preferable that the inclination angle θ1 of the incubator changes within a range of 0 ° or more and 10 ° or less with the revolution motion of the incubator. Thereby, a laminar flow of the culture solution can be formed.

  In the present invention, the turbulent flow suppressing effect can be further improved by rotating the incubator in addition to the above revolving motion. The rotation axis is preferably provided in the central region of the bottom of the incubator. A preferable rotation speed is 0.01 to 20 rpm, and more preferably 0.1 to 5 rpm.

  Cells and carriers floating on the formed laminar flow are less likely to settle even when the revolution speed of the incubator is slow. Even if the cells and carriers start to settle, they are rebounded and resuspended when they come into contact with the cells and carriers floating in the lower layer. Therefore, this invention can avoid that a cell settles and forms a colony.

The cell to be cultured to be seeded in the culture solution is not particularly limited, but the present invention is not limited to ES cells, artificial pluripotent stem cells (iPS cells), pluripotency derived from various mammals such as humans, mice and cows. Suitable for germ stem cells (mGS cells) and the like. Seeding density to the carrier of the stem cells, preferably ^{5~100cells / cm 2, 20~50cells / cm} 2 is more preferable.

  In the present invention, since the shearing force of the culture solution is small, there is little damage to the cells, and the cells are not easily detached from the carrier. The adhesion efficiency to the carrier is 50 to 100%. In addition, since the cells and the carrier hardly settle down to the bottom of the incubator, three-dimensional culture can be performed efficiently. The present invention is useful for mass culture of cells.

Continue culturing the cells on the carrier by the above method until confluent. As the culture conditions, conventionally known suspension culture conditions can be applied. For example, when culturing mouse ES cells with the culture apparatus of the present invention, it can be carried out in an environment of temperature conditions of 35 to 37 ° C. and 2 to 5% CO ₂ .

  When stem cells are cultured using the carrier having an appropriate shape as described above, the undifferentiated rate of stem cells is estimated to be about 95 to 99%. When culturing stem cells in a dispersed state, the contact area between the stem cells is small. Therefore, it is difficult for stem cells to form colonies. Therefore, the differentiation inhibitor can sufficiently act on all the stem cells attached to the carrier. As a result, the undifferentiated state of the stem cell can be maintained.

  Confirmation of the undifferentiated state of the cultured cells can be performed by measuring the ALP activity corresponding to the type of cell to be cultured and the expression level of a specific marker gene. As the specific marker, expression of gene products such as Oct-3 / 4 and Rex1 / Zfp42 can be used.

  An example of detection of ALP activity is that cultured ES cells are washed with PBS, fixed with a citric acid solution containing 66% acetone / 3% formalin, washed with PBS, and then washed with naphthol AS-BI phosphate alkaline staining solution for 15 minutes. It is carried out by processing and color reaction.

  In addition, antigenic molecules may be used as undifferentiated markers. This is because the differentiation of ES cells can be confirmed by the reduction or disappearance of various antigenic molecules. Examples of such antigenic molecules include SSEA-1 for mouse ES cells, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM- for human ES cells. There are 2 mags.

  The following method is an example of using an undifferentiated marker. First, RNA is prepared from cultured ES cells, and cDNA is synthesized according to a conventional method. A gene fragment of a predetermined undifferentiated marker is amplified by PCR using the cDNA as a template. The resulting PCR product is stained and detected. By significantly detecting the undifferentiated marker, it can be confirmed that the cultured ES cells are undifferentiated.

  When the culture solution becomes confluent, the present invention can pass the cells to be cultured by a conventional method. Passaging can be performed up to at least 3 to 10 passages.

  When recovering the cultured stem cells, a carrier on which the stem cells are attached in a dispersed state is recovered from the culture solution, and single cells are obtained from the carrier by a conventional method. When the revolving motion of the incubator is stopped, the carrier naturally settles to the bottom of the incubator in 3 to 4 hours. The cells are collected by washing the carrier and obtaining single cells using a protease enzyme solution such as trypsin or an EDTA solution.

[Cell culture equipment]
The cell culture device according to the present invention includes an incubator having a culture solution in which cells to be cultured are immersed, support means for supporting the incubator in an inclined posture with respect to a horizontal plane, and the incubator supported by the support means. Drive means for revolving, and the incubator is revolved with a vertical revolution axis or a revolution axis inclined with respect to a horizontal plane as a center of a revolution trajectory. In the present invention, the culture solution follows the revolving motion of the incubator well and forms a laminar flow. Thereby, cell culture efficiency can be improved.

  The cell culture device of the present invention has the device configuration illustrated in FIG. 1 or FIG. The supporting means provided in the present invention is not particularly limited as long as it has a structure in which the incubator is supported in an inclined posture having the predetermined inclination angle with respect to the horizontal plane. As a specific example, there is a configuration including a support shaft made of a single rod-like body and a platform connected to an end of the support shaft via an angle adjusting member and fixed to the support shaft. In this case, the support shaft and the platform are adjusted by the angle adjusting member so that the bottom of the incubator placed on the platform forms a desired inclination angle with respect to the horizontal plane, and then the platform is fixed to the support shaft.

  The driving means provided in the present invention is not particularly limited as long as it has a structure capable of transmitting the driving force of a driving source such as a motor to the supporting means. As a specific example, there is a configuration in which a plate is fixed to the tip of a motor shaft, and a support means of an incubator is fixed to the plate. In that case, the torque output of the motor is transmitted to the plate via the shaft, and the plate rotates. The support shaft can be rotated by transmitting the rotational movement of the plate to support means such as a support shaft. In addition, in the support shaft, a region different from the shaft fixing region is pivotally supported by two support shafts so as to be able to obtain the revolution motion of the incubator according to the present invention while suppressing the rotation of the support shaft. Can do.

  The revolution axis of the incubator according to the present invention is inclined with respect to the vertical or horizontal plane. FIG. 1 is an example of the present invention in which an incubator is revolved with a vertical revolution axis as the center of a revolution trajectory. FIG. 2 shows an example of the present invention in which the revolution axis in which the incubator is inclined with respect to the horizontal plane is revolved around the revolution trajectory. In an incubator that revolves around a revolution axis in which the incubator is inclined with respect to a horizontal plane, the inclination angle formed by the revolution axis and the horizontal plane is preferably 75 ° or more and less than 90 °.

  In the present invention, the incubator may be rotatably supported by connecting the support shaft to the driving means 103 using a flexible joint or the like. In that case, the culture solution can be stirred more uniformly by rotating the incubator in addition to the revolving motion.

  Regarding the inclining posture of the incubator with respect to the horizontal plane, in the incubator that revolves around the vertical revolution axis, the inclination angle formed by the bottom surface of the incubator and the horizontal plane is preferably 1 to 30 °. The inclination angle set within the above range is kept constant during the revolution of the incubator. On the other hand, in an incubator that revolves around an inclined revolution axis, the inclination angle is preferably adjusted so as to change within a range of 0 ° or more and 45 ° or less with the revolution motion of the incubator.

  The revolution radius of the incubator in the present invention is preferably 0 cm or more and 50 cm or less. The revolution radius of the incubator can be adjusted by adjusting the distance between the fulcrum of the indicator shaft and the incubator. The revolution speed of the incubator may be any speed that allows the culture solution to follow the revolution motion of the incubator, preferably 0.1 to 60 rpm, and more preferably 5 to 20 rpm.

  As the incubator used in the present invention, a vinyl bag, a cylindrical vessel, a petri dish or the like is preferably used. You may apply | coat the component which suppresses attachment of a cell to the inner wall of an incubator. The present invention can be applied to perfusion culture. In that case, the incubator is provided with an inlet and an outlet for the culture solution. In that case, it is preferable to attach a filter or the like to the outlet to prevent the carrier and cells from flowing out.

  In the present invention, it is preferable to use a carrier in an incubator. Undifferentiated cells can be cultured in a large amount by dispersing and culturing the cells on a carrier. The shape of the carrier is preferably a disc shape, a rod shape, or a polygonal shape of hexagon or more. In the carrier having the shape exemplified above, the cells attached to the surface of the carrier are less susceptible to friction and difficult to peel off from the surface of the carrier. Moreover, it becomes easy to float a support | carrier by selecting a shape with a slow sedimentation speed. Such a carrier is suitable for suspension culture because it easily resuspends when it comes into contact with other carriers even if it begins to settle. From the above viewpoint, it is particularly preferable to use a disk-shaped carrier.

  The maximum size of the carrier is preferably 0.1 to 6 mm, more preferably 2 to 4 mm. Further, it is preferable to select a carrier having a size of 1/10 or less of the minimum internal dimension of the incubator. When a disc-shaped carrier having a diameter of 0.1 mm is used, cells having a diameter of 20 μm are combined with the front and back of the disc to have an area of 50 cells. When 5 cells are attached to such a disk-shaped carrier, the cells can be grown 10 times depending on the type of cells.

The shape and size of the carrier are appropriately selected according to the volume of the incubator, the diameter of cells to be cultured, the seeding density, and the like. The supply amount of the carrier is preferably 10 to 100 pieces / cm ³ . As a specific example, the supply amount of the carrier with respect to 3 to 20 ml of the culture solution is preferably 3 to 100, and more preferably 6 to 50.

  As the material for the carrier, known materials used for culturing animal cells, in particular, stem cells, mouse ES cells and human ES cells can be used. From the viewpoint of moldability, polystyrene, nitrocellulose, nylon, polyurethane and the like are preferable.

  In stem cell culture, it is also preferable to coat a carrier with a stem cell adhesion substance. As examples of the stem cell adhesion substance, cell binding substances such as fibronectin and collagen are preferable, and E-cadherin, matrigel, laminin and the like are more preferably used. In the present invention, the above stem cell adhesion substance may be used alone or in combination of two or more.

  The stem cell adhesion substance may be produced according to a conventional method, or a commercially available product may be used. Stem cells that are in contact with the surface of the carrier are sufficiently adhered to the surface of the carrier because a predetermined basement membrane component expressed on the surface of the stem cell and the stem cell adhesion substance coated on the surface of the carrier are strongly bonded to each other due to their affinity. be able to.

  E-cadherin is a kind of protein expressed in undifferentiated mouse ES cells, and is known as a substance involved in the adhesion of pluripotent stem cells. Matrigel is a basement membrane component extracted and generated from mouse EHS sarcoma. Matrigel contains type IV collagen, laminin, heparan sulfate proteoglycan, entactene and the like. Laminin is one of the major proteins that make up the basement membrane, and is thought to be involved in cell adhesion, migration, and proliferation. Preferably, laminin, human laminin, human laminin α5β1γ1, human laminin α3β3γ2 are used.

The coating amount of the stem cell adhesion substance is preferably the maximum amount that can be coated on the carrier from the viewpoint of improving adhesion. Coating amount of stem cells adherent material to the support substrate surface, preferably ^{0.6~1.1μg / cm 2, 0.8~1.1μg / cm} 2 is more preferable.

  The method for coating the surface of the carrier with the stem cell adhesion substance is not particularly limited. As an example, after preparing the stem cell adhesion substance to 10 μg / ml with distilled water or Phosphate Buffered Salin (PBS), the carrier substrate is brought into contact with the filtered and sterilized solution, and left at room temperature 35 to 37 ° C. for 30 to 60 minutes. Can be mentioned.

  By bringing the stem cell adhesion substance into contact with the carrier, the stem cell adhesion substance can be adsorbed or covalently bonded to the carrier, thereby fixing the stem cell adhesion substance to the carrier. Before bringing the stem cell adhesion substance into contact with the carrier substrate, an antigenic molecule is previously bound to the stem cell adhesion substance, and an antibody molecule that specifically binds to the antigenic molecule is added to the surface of the carrier substrate. It is also preferable to keep it. As a result, the antigen in the stem cell adhesion substance specifically binds to the antibody on the surface of the carrier base material, and the stem cell adhesion substance can be well fixed on the carrier surface.

  The carrier used in the present invention may be formed with a height on its surface. By using a carrier having an uneven surface composed of a high part and a low part, the cells can be easily fitted to the carrier surface, and the high part formed on the carrier surface functions as a scaffold for supporting the cells. Therefore, the carrier can suppress cell detachment. In addition, since the surface area of the carrier can be increased, a large amount of stem cell adhesion substance can be easily held on the surface. Thereby, it has favorable stem cell adhesiveness.

The number of low portions in the carrier having the above surface roughness is about 400 to 3000000 per 1 million μm ² of the carrier surface area. The number is preferably 1000000 to 3000000, more preferably 1300,000 to 1700,000 and even more preferably 150,000 per 1000000 μm ^{2 of the} carrier surface area. The other preferable low part existence number is 400 to 14000 per carrier surface area of 1000000 μm ² , more preferably 4000 to 7000.

  In the carrier having the above surface roughness, if the distance between the apex of the adjacent high part and the lowest point of the low part is the height difference, the average value of the height difference is preferably larger than 0.5 μm. The distance between apexes between adjacent high parts is preferably about 1 to 5% of the cell diameter. As a result, a scaffold for the cells is secured, and the cultured cells are easily supported on the surface of the carrier.

  The number of low parts, the distance between vertices, and the height difference are appropriately determined depending on the diameter and flexibility of cells to be cultured, the moving speed, and the like. Cells migrate on the carrier surface and grow. The speed depends on the surface properties of the carrier. When the moving speed of the cell is high, the cell rapidly expands and contracts. Cells that rapidly shrink to a nearly spherical shape have a reduced contact area with the carrier. Therefore, it becomes difficult to obtain preferable adhesion. From the viewpoint of complementing the low adhesiveness presumed above, it is considered preferable to use a carrier that can provide a high part that serves as a scaffold when cells with high migration speed are cultured. That is, when culturing cells having a high moving speed, it is preferable to use a carrier having a relatively large number of high parts within the predetermined range of the present invention.

  In the case of a carrier used for culturing mouse pluripotent stem cells, a preferable distance between vertices is 0.1 to 2.7 μm, and more preferably 0.5 to 2.0 μm. The average height difference on the surface of the carrier is preferably 0.5 to 2.7 μm, more preferably 0.5 to 2.0 μm.

  In order to prevent colony formation in culturing highly adherent stem cells, it is preferable to use a carrier having a surface roughness appropriate for the size of the stem cells. Thereby, it can avoid that many stem cells enter a bottom part, and can prevent colony formation. As a result, undifferentiated stem cells can be cultured in large quantities.

  Cells can be attached in a dispersed state on the surface of the carrier. In the present invention, the dispersed state means a state in which stem cells are individually attached as single cells to the surface of the carrier, and further, the stem cells are attached in a state where there is little contact area so that each stem cell does not form a clear colony. Is included. Therefore, the stem cells cultured according to the present invention are unlikely to become a cell mass and easily maintain an undifferentiated state.

  Examples of a method for forming the height on the surface of the carrier include a method in which a glass plate is pressed against the carrier and an injection molding method. When performing the manufacturing method by the above-mentioned pressure welding, the pressure welding material is appropriately selected according to the hardness of the selected carrier. Preferable pressure contact materials include metals such as glass, stainless steel and iron, and ceramics.

  A conventionally known culture solution can be used as the culture solution in which the cells to be cultured are immersed. Examples of conventionally known culture solutions include Dulbecco's Modified Eagle's Medium (DMEM), Grass Minimum Essential Medium (GMEM), RPMI640, and the like.

  L-glutamine, insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid Known components such as glycine, L-proline, and L-serine can be added. Although fetal bovine serum (FBS) can be added to the culture solution, serum-free culture may be performed.

  A leukemia inhibitory factor (LIF) may be added to the culture medium as a differentiation inhibitor. When culturing human pluripotent stem cells, a known method that does not use feeder cells can be applied. Thereby, the influence of a co-culture method can be avoided and a human pluripotent stem cell can be applied.

  In the case of subjecting cells to be cultured in suspension using the predetermined cell culture apparatus of the present invention, the culture solution can follow the revolution of the incubator to form a laminar flow of the culture solution. Thereby, the shear force of a culture solution is suppressed. Therefore, cell culture efficiency can be improved.

[Example 1]
The cell culture device of the present invention having the basic structure shown in FIG. 1 was adjusted so as to be in the state shown in Table 1. A 15 ml FBS, 15% FBS, non-essential amino acids, pyruvic acid, glutamic acid, 0.1 mM mercaptoethanol, and GIF with addition of LIF are filled as a culture medium. 300 carriers were supplied. The carrier was previously coated with E-cadherin. Furthermore, mouse stem cells having a diameter of 20 μm were seeded at a seeding density of 1 × 10 ⁵ cells / ml. While the above incubator was revolved at the revolution speed described in Table 1, culture was started at a temperature condition of 36 to 38 ° C. and 5% CO ₂ .

  The culture was continued while exchanging the medium according to a conventional method, and after 72 hours, the revolving motion of the incubator was stopped. The flow of the culture broke down and the carrier settled naturally. After all the carrier was completely settled, the carrier was removed from the incubator and the cells were collected. Cells were collected by adding Accutase to a carrier and incubating at 37 ° C. for about 10 minutes, and then suspending in a culture solution to form single cells.

[Example 2]
The cell culture device of the present invention having the basic structure shown in FIG. 2 was adjusted to the state shown in Table 1. A 15 ml FBS, 15% FBS, non-essential amino acids, pyruvic acid, glutamic acid, 0.1 mM mercaptoethanol, and GIF with addition of LIF are filled as a culture medium. 300 carriers were supplied. The carrier was previously coated with E-cadherin. Furthermore, mouse stem cells having a diameter of 20 μm were seeded at a seeding density of 1 × 10 ⁵ cells / ml. While the above incubator was revolving at the revolving speed shown in Table 1, culturing of mouse stem cells was started under temperature conditions of 35 to 37 ° C. and 2 to 5% CO ₂ .

  The culture was continued while changing the medium according to a conventional method, and after 72 hours, the revolution of the incubator was stopped. The flow of the culture broke down and the carrier settled naturally. After all the carrier was completely settled, the carrier was removed from the incubator and the cells were collected. Cells were collected in the same manner as in Example 1.

[Comparative example]
Using a WAVE bioreactor, mouse stem cells were cultured under temperature conditions of 35 to 37 ° C. and 2 to 5% CO ₂ . The volume of the incubator used was 200 ml, and the seeding density of mouse stem cells was 1 × 10 ⁵ cells / ml. The peristaltic angle was 5 ° and the peristaltic speed was 30 rpm. The culture was continued while exchanging the medium according to a conventional method. After 72 hours, the incubator was stopped. The flow of the culture broke down and the carrier settled naturally. After all the carrier was completely settled, the carrier was removed from the incubator and the cells were collected. Cells were collected in the same manner as in Example 1.

[Method for measuring cell number and calculating cell density]
Cells cultured in Example 1, Example 2 and Comparative Example 1 and then dispersed with Accutase were collected, placed in an incubator, and allowed to settle. Three photographs of cells settled on the bottom of the incubator were taken from different fields of view, and the average number of cells was counted. The total number of cells was calculated as total cell number = average cell number × incubator bottom area / viewing area. The total cell number obtained was divided by the volume of the incubator to calculate the cell density. Table 1 shows the cell densities obtained in Example 1, Example 2, and Comparative Example.

100 cell culture equipment
101 housing
102 Drive source
103 Drive means
104 Support shaft
105 opening
106 Incubator
106a Bottom of the incubator
107 platform
108 medium
200 Horizontal plane (Cell culture device mounting surface)
300, 301 Revolving axis
Revolution trajectory of A1, A2 incubator
r1, r2 Revolution radius of incubator θ1 Inclination angle of incubator θ2 Inclination angle of revolution axis X Vertical direction

Claims (4)

  A cell culture method for culturing cells by immersing cells to be cultured in a culture medium in an incubator, characterized in that the incubator is revolved around a revolution axis in a state inclined with respect to a horizontal plane. Cell culture method.   Range in which at least one of the inclination angle formed by the bottom surface of the incubator and the horizontal plane, the revolution radius of the incubator, and the revolution speed of the incubator follows the revolving motion of the incubator. The cell culture method according to claim 1, wherein a laminar flow is formed in the culture solution.   The cell culture method according to claim 1 or 2, wherein the incubator is revolved in a state where the revolution axis of the incubator is inclined with respect to a horizontal plane.   An incubator having a culture solution for immersing the cells to be cultured; a supporting means for supporting the incubator in an inclined posture with respect to a horizontal plane; and a driving means for revolving the incubator supported by the supporting means, A cell culture apparatus characterized in that the incubator is revolved with a vertical revolution axis or a revolution axis inclined with respect to a horizontal plane as a center of a revolution trajectory.