JP2014168394A - Cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel in which culture solution does not flow out to the outside when centrifugal force is given.SOLUTION: A cell culture vessel 10 comprises: a vessel body 11 which has an inner bottom surface 11a having a cell non-adhesive property or being able to change to have the cell non-adhesive property, and an upper opening 11b; and a lid 12 covering the upper opening 11b of the vessel body 11. The mass of the lid 12 is more than that of the vessel body 11. For this reason, when centrifugal force is given to the cell culture vessel 10, the lid 12 presses the vessel body 11, which prevents culture solution within the vessel body 11 from flowing out to the outside.

Description

  The present invention relates to a cell culture container suitable for production of a biological tissue having a multilayer structure in which a plurality of cells are laminated in the thickness direction.

  A biological tissue produced by culturing cells on a culture carrier such as collagen is not suitable as a tissue for transplantation for medical purposes because of its low cell density. A technique for producing a tissue such as a cell sheet having a high cell density suitable for transplantation is an important technique in the field of tissue engineering. However, there are several problems in the conventional method for producing a high cell density biological tissue.

  It is generally widely practiced to seed cells on a culture support to form a cell sheet. For the purpose of facilitating cell sheet peeling, a technology that promotes cell peeling by providing a layer of temperature-responsive polymer compound on the cell adhesion surface has been developed, and it is possible to collect cell sheets containing almost no foreign matter. It is. However, the cell sheet formed by this method is usually composed of a single layer or three or less cell layers. For this reason, in order to carry out multilayering, it is necessary to superimpose a plurality of cell sheets. Since cell sheets are extremely thin and difficult to handle, it is not easy to superimpose a plurality of cell sheets, which is troublesome.

  Therefore, in order to obtain a biological tissue suitable for transplantation, a cell culture container having an inner bottom surface that is non-cell-adhesive or can be changed to cell-non-adhesive has been developed. When producing a biological tissue, first, a culture solution containing cells is injected into a cell culture container. Next, the cell culture vessel is placed in a centrifuge and a centrifugal force is applied toward the inner bottom surface, and cell culture is performed under conditions where cell-cell adhesion is formed, and cells are adhered to form a tissue. Thus, biological tissue is deposited on the inner bottom surface (see Patent Documents 1 to 3).

JP 2010-161952 A JP 2010-161953 A JP 2010-161954 A

  As described above, a cell culture container for obtaining a biological tissue suitable for transplantation has been developed. Moreover, such a cell culture container is installed in a centrifuge, and a cell culture is performed by applying a centrifugal force to the cell culture container.

  However, when the cell culture container is installed in a centrifuge and a centrifugal force is applied to the cell culture container, the culture solution may flow out from the cell culture container during the step of applying the centrifugal force. There is.

  The present invention has been made in consideration of such points, and an object of the present invention is to provide a cell culture vessel capable of preventing an internal culture solution from flowing out during a step of applying a centrifugal force. And

  A cell culture container according to one embodiment of the present invention contains a culture solution containing cells, and performs cell culture under a condition in which cell-cell adhesion is formed while acting a centrifugal force toward the inner bottom surface. A cell culture container that adheres to form a tissue and deposits biological tissue on the inner bottom surface, and has an inner bottom surface that is non-cell-adhesive or can be changed to cell non-adhesive, and above A container main body provided with an opening; and a lid that covers an upper opening of the container main body, wherein the mass of the lid is larger than the mass of the container main body.

  The cell culture container according to one aspect of the present invention is characterized in that the specific gravity of the material of the lid is larger than the specific gravity of the material of the container body.

  The cell culture container according to one aspect of the present invention is characterized in that the lid has a double structure of a lid body and a metal member.

  The cell culture container according to an aspect of the present invention is characterized in that the metal member is provided leaving an observation region in the lid body.

  The cell culture container according to one aspect of the present invention is characterized in that the observation region is provided at a size of at least 1 mm square in a central portion of the lid body.

  In the cell culture container according to one aspect of the present invention, the lid and the container main body are made of the same material, and the specific gravity of the lid is larger than the specific gravity of the container main body.

  In the cell culture container according to an aspect of the present invention, the material is polystyrene, and the lid is made of polystyrene having a higher density than the container body.

  A cell culture container according to one embodiment of the present invention contains a culture solution containing cells, and performs cell culture under a condition in which cell-cell adhesion is formed while acting a centrifugal force toward the inner bottom surface. A cell culture container that adheres to form a tissue and deposits biological tissue on the inner bottom surface, and has an inner bottom surface that is non-cell-adhesive or can be changed to cell non-adhesive, and above A container body provided with an opening; and a lid that covers an upper opening of the container body. The specific gravity of the material of the lid body is greater than the specific gravity of the material of the container body.

  As described above, according to the present invention, when a centrifugal force is applied to the cell culture container after injecting the culture liquid into the cell culture container, the culture liquid flows out from the cell culture container. Can be prevented.

Sectional drawing of the cell culture container by the 1st Embodiment of this invention. The perspective view of the cell culture container by the 1st Embodiment of this invention. The figure which shows the effect | action of the cell culture using a cell culture container. The figure which shows a centrifuge. Sectional drawing of the cell culture container by the 2nd Embodiment of this invention. The perspective view of the cell culture container by the 2nd Embodiment of this invention. Sectional drawing of the cell culture container by the modification of 2nd Embodiment. The perspective view of the cell culture container by the modification of 2nd Embodiment. Sectional drawing of the cell culture container by the 3rd Embodiment of this invention. The perspective view of the cell culture container by the 3rd Embodiment of this invention. Sectional drawing of the cell culture container by the modification of 3rd Embodiment. The perspective view of the cell culture container by the modification of 3rd Embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  (First Embodiment) FIGS. 1 to 4 are views showing a first embodiment of a cell culture container 10 according to the present invention.

  As shown in FIGS. 1 and 2, the cell culture container 10 has an inner bottom surface 11a that is non-cell-adhesive or can be changed to cell-non-adhesiveness, and has an upper opening 11b. And a lid 12 that covers the upper opening 11b of the container body 11.

  Such a cell culture vessel 10 can store the culture solution 1 containing the cells 2 in a space surrounded by the inner bottom surface 11a and the inner side surface 11c, and while applying centrifugal force toward the inner bottom surface 11a, The cell culture is performed under the condition that the adhesion between the two is formed, and the tissue is formed by adhering between the cells 2 to deposit the biological tissue on the inner bottom surface 11a (FIGS. 3A and 3B). ) (C)).

  In this case, at least the inner bottom surface 11a out of the inner surface of the container portion containing the cell-containing culture solution 1 (see FIGS. 3A, 3B, and 3C) is non-cell-adhesive or non-cell-adhesive. Although it is only necessary to be able to change, in particular, of the inner surface of the container portion that contains the cell-containing culture solution, all of the surfaces that come into contact with the cells (for example, the inner bottom surface 11a and the inner surface 11c of the container) It is preferable that it is adhesive or can be changed to non-cell-adhesive.

  The cells used in the present invention are not particularly limited as long as they are adherent cells. Examples of such cells include liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation has ended. Any of the cells may be used. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.

  These cells are cultured in advance by a conventional method, the culture is treated with trypsin treatment or the like, and is stored in a culture vessel in a state of being suspended in a culture solution. Any culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art. For example, depending on the type of cell used, MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. A basal medium as described in “Tissue culture technology third edition”, page 581, edited by the Japanese Society for Tissue Culture can be used. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids and the like may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell tissue, it is preferable to use a medium that does not contain animal-derived components.

  In the present invention, the non-cell-adhesive surface includes a hydrophilic surface, specifically, a surface having a static water contact angle at 20 ° C. of 45 ° or less. Such a surface can be obtained by forming a film of an organic compound having a carbon-oxygen bond on the surface of the substrate. Or you may comprise the base material itself with the material which has hydrophilic property.

  The cell non-adhesive surface can be formed by a hydrophilic film formed of an organic compound having a carbon-oxygen bond and having a static water contact angle of 45 ° or less.

  The carbon oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon-oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

  Examples of the main raw material for the hydrophilic film include hydrophilic organic compounds such as water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surface active substances, and amphiphilic substances. These are physically or chemically cross-linked with each other and physically or chemically bonded to the substrate to form a hydrophilic film.

  The average thickness of the hydrophilic film is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. An average thickness of 0.8 nm or more is preferable because it is not easily affected by a region not covered with the hydrophilic film on the substrate surface. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

  The hydrophilic film can be formed on the substrate surface by directly adsorbing the hydrophilic organic compound on the substrate, coating the hydrophilic organic compound directly on the substrate, or coating the hydrophilic organic compound on the substrate. A method of performing a crosslinking treatment later, a method of forming a hydrophilic film in a multi-stage manner in order to increase the adhesion to the substrate, a base layer is formed on the substrate in order to increase the adhesion to the substrate, Examples thereof include a method of coating a hydrophilic organic compound, a method of forming a polymerization initiation point on the substrate surface, and then polymerizing a hydrophilic polymer brush.

  The cell contact region on the inner surface including the inner bottom surface of the culture vessel used in the present invention is preferably cell non-adhesive from the viewpoint of ease of tissue detachment. However, it may be a surface that can change to non-cell-adhesiveness upon peeling. Such a surface is formed by immobilizing a covalent bond on the surface of a substrate with at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer. Can be formed. The stimulus-responsive polymer is preferably a temperature-responsive polymer that can be easily applied with a stimulus, but is not limited thereto.

  Next, the cell culture container 10 will be further described. As described above, the cell culture container 10 includes the container body 11 having the inner bottom surface 11a and the upper opening 11b, and the lid body 12 that covers the upper opening 11b of the container body 11, and the container body 11 has a cylindrical shape. It is a petri dish type container body.

  The mass of the lid body 12 is larger than the mass of the container body 11. For example, the lid body 12 is made of high-density polystyrene, and the container body 11 is made of polystyrene having a lower density than that used for the lid body 12.

  The culture medium 1 containing the cells 2 is stored in the container main body 11 from the upper opening 11 b, and the upper opening 11 b of the container main body 11 is covered with the lid body 12, thereby sealing the upper opening 11 b of the container main body 11 with the lid body 12. can do.

  Since the mass of the lid body 12 is larger than the mass of the container body 11, the lid body 12 presses the container body 11 in the centrifugation step described later, so that the container body 11 is displaced and the culture solution 1 in the container body 11 is removed. Can be prevented from flowing out.

  In this case, the upper opening 11b of the container body 11 can be completely sealed by the lid body 12, and the inside of the container body 11 is shut off from the external atmosphere.

  However, without completely sealing the upper opening 11b of the container main body 11 with the lid 12, the inside of the container main body 11 communicates with the external atmosphere while preventing the culture medium 1 in the container main body 11 from flowing out. The carbon dioxide in the container body 11 that is lowered by cell culture may be supplemented from the external atmosphere.

  Next, the operation of the present embodiment having such a configuration will be described.

  First, as shown in FIG. 3A, the culture solution 1 containing the cells 2 is injected into the container body 11 from the upper opening 11b.

  Next, the upper opening 11 b of the container body 11 is covered with the lid body 12, and the upper opening 11 b of the container body 11 is sealed with the lid body 12.

  Next, the cell culture container 10 is installed in the centrifuge 20 shown in FIGS. 4 (a) and 4 (b).

  As shown in FIGS. 4A and 4B, the centrifuge 20 swings through a rotating shaft 21, an arm 22 fixed to the rotating shaft 21, and a tip of the arm 22 via a swing shaft 24. And has a basket 23 attached thereto.

  Then, the cell culture container 10 is placed on the basket 23 of the centrifuge 20 (see FIG. 4A), and then the rotation shaft 21 is rotated (see FIG. 4B).

  In this case, the basket 23 attached to the tip of the arm 22 via the swing shaft 24 is lifted upward, and the inner bottom surface of the container body 11 is moved with respect to the cell culture container 10 as the rotation shaft 21 rotates. Centrifugal force in the direction of 11a works.

  When the centrifugal force acts on the inner bottom surface 11a side of the container body 11, the cells 2 in the culture solution 1 adhere to form a tissue, and the sheet-like biological tissue 3 is deposited on the inner bottom surface 11a.

  In this way, the sheet-like biological tissue 3 is separated from the other culture solution 1 (see FIG. 3B).

  Next, the step of applying centrifugal force to the cell culture container 10 will be further described.

  By applying a centrifugal force in the direction of the inner bottom surface 11a to the culture solution 1 accommodated in the cell culture vessel 10, cell culture is performed under conditions where cell-cell adhesion is formed, and cells are adhered to form a tissue. To do. In a state where the cells 2 are in close contact with the inner bottom surface 11a according to the shape of the inner bottom surface 11a due to the centrifugal force, the cells 2 adhere to each other to form a tissue having a desired shape.

  The magnitude of the centrifugal force applied from the centrifuge 20 shown in FIGS. 4 (a) and 4 (b) can be appropriately selected as long as the tissue can be formed without adversely affecting the function of the cells 2. For example, 2G-1440G is preferable and 2G-720G is more preferable.

  When the centrifugal force is applied, the lid body 12 having a large mass presses the container body 11, the upper opening 11b of the container body 11 is completely sealed by the lid body 12, and the culture solution 1 flows out to the outside. Can be prevented.

  Here, “the conditions under which cell-cell adhesion is formed” refers to conditions under which cells can act and adhere to each other. Although it varies depending on the type of cells to be cultured, for example, the temperature condition is preferably 20 to 40 ° C., the atmospheric gas condition is preferably a carbon dioxide concentration of 3 to 5%, and the culture time is 0.5 to 24 hours is preferred. One of the advantageous features of the present invention is that the culture time can be shortened and the damage to the cells can be reduced. In the embodiment in which an extracellular matrix described later is separately prepared and added, the culture time can be shortened, and the culture time can be 0.5 to 3 hours, which is preferable. In embodiments where no extracellular matrix is added, the culture time can be 1 to 24 hours.

  In this embodiment, the culture vessel is left in an incubator filled with a desired atmospheric gas (carbon dioxide concentration 5%) for several minutes, and then the culture vessel is covered and weighted culture is performed so that the atmospheric gas does not change. . However, the present invention is not limited to this. For example, if it is in an incubator in which the atmospheric gas and temperature are controlled, weighted culture can be performed with the culture vessel opened.

  When cell culture is performed by applying a centrifugal force, it is sufficient that cell-cell adhesion is formed, and it is not essential to increase the number of cells by proliferation. By appropriately adjusting the number of cells in the culture solution, it is possible to control the thickness of the produced tissue (that is, the number of cells stacked in the thickness direction of the tissue). As described above, since it is not necessary to proliferate cells in the cell culture process, a tissue can be obtained in a relatively short time. The thickness and shape of the tissue can be freely adjusted.

  In addition, according to the cell culture method using the cell culture container of the present embodiment, it is possible to obtain a tissue in which cells are densified. A tissue in which cells are densified is preferable for transplantation.

  Next, after the step of applying the centrifugal force is completed, the obtained biological tissue 3 is peeled from the inner bottom surface 11a of the cell culture container 10 and collected (see FIG. 3C). For example, the cells can be detached by a physical operation such as a pipetting operation. If the inner bottom surface 11a of the cell culture vessel 10 is a non-cell-adhesive surface, this operation is easy, and tissue can be prepared and peeled and collected in a short time of culture. When the inner bottom surface 11a of the cell culture vessel 10 is a surface that changes to non-cell-adhesive properties, such as a stimulus-responsive polymer, in an environment in which cell-non-adhesive properties are obtained (for example, a temperature below the lower critical temperature). Perform peeling operation.

  As described above, according to the present embodiment, after injecting the culture medium 1 from the upper opening 11 b into the container body 11, the upper opening 11 b of the container body 11 is covered with the lid body 12 having a larger mass than the container body 11. . When the cell culture container 10 is installed on the basket 23 of the centrifuge 20 and the cell culture is performed while the centrifugal force acting toward the inner bottom surface 11a is applied to the cell culture container 10, the lid 12 attaches the container body 11 to the cell culture container 10. Pressing can prevent the culture solution 1 in the container body 11 from flowing out.

  Although it is preferable that the difference in mass between the lid 12 and the container main body 11 is large, a centrifugal force is applied in the centrifugation step, so that the lid 12 is not affected even if the mass difference between the lid 12 and the container main body 11 is small. The container body 11 can be sufficiently pressed to prevent the culture solution 1 in the container body 11 from flowing out.

  When cell culture is performed, the lid body 12 is preferably thinner because the upper body 11 of the container body 11 is covered with the lid body 12 and the cell culture state in the container is observed through the lid body 12. Also, higher transparency is preferred. Therefore, it is preferable that the lid body 12 is made of a material having a specific gravity greater than that of the container body 11 and the lid body 12 is made thin.

  (Second Embodiment) FIGS. 5 to 8 show a schematic configuration of a cell culture container according to a second embodiment. This embodiment is different from the first embodiment shown in FIGS. 1 to 4 in that the lid body 12 has a double structure of a lid body 12a and a metal member 12b. 5 to 8, the same parts as those of the first embodiment shown in FIGS.

  As shown in FIGS. 5 and 6, the metal member 12b is provided on the outer periphery of the lid body 12a. Here, FIG. 5 is a sectional view showing the container main body 11 and the lid body 12, and FIG. 6 is a perspective view of the cell culture container 10.

  For example, aluminum, iron, copper, or the like can be used for the metal member 12b. The metal member 12b may be fixed to the outer peripheral portion of the lid body 12a with an adhesive or the like, or may be detachably fitted from the lid body.

  The mass of the lid 12 provided with the metal member 12 b is larger than the mass of the container body 11. The container body 11 is made of, for example, polystyrene. The total mass of the lid body 12a and the metal member 12b only needs to be larger than the mass of the container body 11, and the material of the lid body 12a is not particularly limited. For example, polystyrene, polyethylene terephthalate, polycarbonate, polyethylene, fluororesin, or the like can be used as the material of the lid body 12a.

  Alternatively, as shown in FIGS. 7 and 8, the metal member 12b may be provided on the upper surface of the lid body 12a. In this case, the metal member 12b is preferably provided so as to exclude the observation region of the central portion 12c of the container main body 12a. For example, when the ring-shaped metal member 12b is provided on the peripheral edge of the upper surface of the lid main body 12a. Good. Thereby, the cell culture state in a container can be observed through the center part 12c of the lid body 12a. The size of the observation region of the central portion 12c of the lid body 12a is preferably at least 1 mm square. By configuring the lid body 12a with a highly transparent resin such as a fluororesin, it becomes easy to observe the cell culture state in the container.

  As a modification of the present embodiment, a magnet is disposed on the container body 11 side so as to face the metal member 12b, and the lid body 12 is fixed to the container body 11 by the mass and magnetic force of the lid body 12. Good. In this case, the metal member 12b on the lid side may be replaced with a magnet that generates a magnetic force while increasing the mass of the lid, and a metal member or the like may be provided on the container body side.

  According to the present embodiment, as in the first embodiment, after injecting the culture solution 1 from the upper opening 11 b into the container body 11, the upper opening 11 b of the container body 11 has a larger mass than the container body 11. Cover with a large lid 12. Therefore, when the cell culture container 10 is installed on the basket 23 of the centrifuge 20 and the cell culture is performed while the centrifugal force acting toward the inner bottom surface 11a is applied to the cell culture container 10, the lid 12 is the container body. 11 can be pressed to prevent the culture solution 1 in the container body 11 from flowing out.

  (Third Embodiment) A third embodiment of the present invention will be described with reference to FIGS. As shown in FIGS. 9 and 10, the cell culture container 30 is mounted on a container body 31 having a plurality of liquid storage portions 31 </ b> A and a connecting plate 31 </ b> B connecting the liquid storage portions 31 </ b> A. The container main body 31 includes a plate-shaped container main body.

  Here, FIG. 9 is a cross-sectional view showing the cell culture container 30, and FIG. 10 is a perspective view showing the cell culture container 30.

  As shown in FIGS. 9 and 10, each liquid storage portion 31A of the container body 31 has an inner bottom surface 31a and an inner side surface 31c, and an upper opening 31b is formed on the upper side. The upper opening 31 b of each liquid storage portion 31 </ b> A of the container body 31 is covered with a lid body 32.

  Further, the mass of the lid 32 is larger than the mass of the container body 31. For example, the container body 31 is made of ordinary polystyrene, and the lid body 32 is made of high-density polystyrene.

  9 and 10, the culture solution 1 containing the cells 2 is injected into each solution storage portion 31 </ b> A of the container body 31 from the upper opening 31 b (see FIG. 3A). Thereafter, the upper openings 31 b of the liquid storage portions 31 </ b> A of the container body 31 are covered with the lid body 32.

  The cell culture container 30 into which the culture solution 1 has been injected is then installed in the centrifuge 20 (see FIGS. 4A and 4B). Next, a centrifugal force is applied to the cell culture container 30 by the centrifuge 20, cell culture is performed, and the living tissue 3 is deposited on the inner bottom surface 31a of each liquid storage portion 31A (see FIG. 3B). ). At this time, since the lid 32 having a large mass presses the container main body 31, it is possible to prevent the culture solution 1 in the container main body 11 from flowing out.

  The inner bottom surface 31a of each liquid storage portion 31A is non-cell-adhesive or can be changed to cell-non-adhesive. For this reason, the biological tissue 3 deposited on the inner bottom surface 31a of each liquid storage unit 31A can be easily peeled and recovered by pipetting operation (see FIG. 3C).

  As shown in FIGS. 11 and 12, the cell culture container 30 includes a plate-shaped container body 31 having a plurality of liquid storage portions 31A and a connecting plate 31B for connecting the liquid storage portions 31A, and a container main body 31. And a lid body 32 having a double structure of a lid body 32a and a metal member 32b.

  Here, FIG. 11 is a cross-sectional view showing the cell culture container 30, and FIG. 12 is a perspective view showing the cell culture container 30.

  As shown in FIG. 11, each liquid storage portion 31A of the container body 31 has an inner bottom surface 31a and an inner side surface 31c, and an upper opening 31b is formed on the upper side. The upper opening 31 b of each liquid storage portion 31 </ b> A of the container body 31 is covered with a lid body 32.

  The lid 32 has a double structure of a lid body 32a and a metal member 32b, and the metal member 32b is provided on the outer periphery of the lid body 32a. For example, aluminum, iron, copper, or the like can be used for the metal member 32b. The metal member 32b may be fixed to the outer periphery of the lid body 32a with an adhesive or the like, or may be detachably fitted from the lid body 32a.

  The mass of the lid 32 provided with the metal member 32b is larger than the mass of the container body 31. The container body 31 is made of, for example, polystyrene. The total mass of the lid body 32a and the metal member 32b only needs to be larger than the mass of the container body 31, and the material of the lid body 32a is not particularly limited. For example, polystyrene, polyethylene terephthalate, polycarbonate, polyethylene, fluororesin, or the like can be used as the material of the lid body 32a.

  11 and 12, the culture solution 1 containing the cells 2 is injected into each solution storage portion 31A of the container body 31 from the upper opening 31b (see FIG. 3A). Thereafter, the upper openings 31 b of the liquid storage portions 31 </ b> A of the container body 31 are covered with the lid body 32.

  The cell culture container 30 into which the culture solution 1 has been injected is then installed in the centrifuge 20 (see FIGS. 4A and 4B). Next, a centrifugal force is applied to the cell culture container 30 by the centrifuge 20, cell culture is performed, and the living tissue 3 is deposited on the inner bottom surface 31a of each liquid storage portion 31A (see FIG. 3B). ). At this time, since the lid 32 having a large mass presses the container main body 31, it is possible to prevent the culture solution 1 in the container main body 11 from flowing out.

  The inner bottom surface 31a of each liquid storage portion 31A is non-cell-adhesive or can be changed to cell-non-adhesive. For this reason, the biological tissue 3 deposited on the inner bottom surface 31a of each liquid storage unit 31A can be easily peeled and recovered by pipetting operation (see FIG. 3C).

  In the configuration shown in FIGS. 11 and 12, the metal member 32b may be provided on the peripheral edge of the upper surface of the lid body 32a. However, in order to secure an observation area, when the container main body 31 is covered with the lid 32, the metal member 32b is not positioned above each liquid storage portion 31A.

  In the first to third embodiments, the container main body 11 or 31 may be provided with a container main body fixing means, and the lid bodies 12 and 32 may be provided with a lid fixing means for engaging with the container main body fixing means. For example, one of the container body fixing means and the lid fixing means is composed of an inner screw, and the other is composed of an outer screw. By engaging the outer screw and the inner screw, the container main bodies 11 and 31 are sealed by the lid body 12. This can more effectively prevent the culture medium in the container bodies 11 and 31 from flowing out.

  In addition, when the size of the lid is (almost) the same, the mass of the lid is smaller than the mass of the container main body, and the specific gravity of the material of the lid is larger than the specific gravity of the material of the container main body. Since the mass of the lid is larger than the mass of the container body and the material of the lid body is the same as the material of the container body, the effect of preventing the culture medium from flowing out in the container body can be improved. it can.

  Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage. In addition, various inventions can be formed by appropriately combining a plurality of components disclosed in the embodiment. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined.

DESCRIPTION OF SYMBOLS 10 Cell culture container 11 Container main body 11a Inner bottom face 11b Upper opening 11c Inner side surface 12 Lid 20 Centrifugal machine 30 Cell culture container 31 Container main body 31A Liquid storage part 31B Connection plate 31a Inner bottom face 31b Upper opening 31c Inner side face 32 Lid

Claims (8)

Cell culture medium is stored, and cell culture is performed under conditions where cell-to-cell adhesion is formed while acting a centrifugal force toward the inner bottom surface. In a cell culture container for depositing biological tissue,
A container body having an inner bottom surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and provided with an upper opening;
A lid covering the upper opening of the container body;
With
The cell culture container, wherein the mass of the lid is larger than the mass of the container body.   The cell culture container according to claim 1, wherein the specific gravity of the material of the lid is larger than the specific gravity of the material of the container body.   The cell culture container according to claim 1 or 2, wherein the lid has a double structure of a transparent lid body and a metal member.   The cell culture container according to claim 3, wherein the metal member is provided leaving an observation region in the lid body.   The cell culture container according to claim 4, wherein the observation region is provided at a size of at least 1 mm square in a central portion of the lid body.   The cell culture container according to claim 1, wherein the lid and the container main body are made of the same material, and the specific gravity of the lid is larger than the specific gravity of the container main body.   The cell culture container according to claim 6, wherein the material is polystyrene, and the lid is made of polystyrene having a density higher than that of the container body. Cell culture medium is stored, and cell culture is performed under conditions where cell-to-cell adhesion is formed while acting a centrifugal force toward the inner bottom surface. In a cell culture container for depositing biological tissue,
A container body having an inner bottom surface that is non-cell-adhesive or capable of changing to cell-non-adhesion, and provided with an upper opening;
A lid covering the upper opening of the container body;
With
The cell culture container, wherein the specific gravity of the material of the lid is larger than the specific gravity of the material of the container main body.

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