JP2017046649A - Cell cryopreservation container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cell cryopreservation containers where adherence of cells to the inner wall surface of the container can be suppressed and the cells preserved in a preservation part can be tested readily.SOLUTION: The present invention provides a cell cryopreservation container comprising a preservation part capable of preserving a preservation liquid containing cells, and an inner wall surface which composes the preservation part, comprising an uneven surface having cell adhesion reduction properties on a part of the inner wall surface, and an observation part capable of observing, on the wall part, the cells which is preserved in the preservation part corresponding to at least a partial region of the inner wall surface except the uneven surface having the cell adhesion reduction properties. The inner wall surface of the observation part has a smooth surface, and the light transmittance of the observation part at 450 nm in water is 55% or more.SELECTED DRAWING: Figure 8

Description

  The present invention relates to a cell cryopreservation container for freezing and storing cells.

  In the field of regenerative medicine, cells collected from biological samples or cultured cells are used for various applications such as tissue reconstruction. The cells are cryopreserved in a state of being stored in a storage container until they are collected from a biological sample or cultured and used for medical treatment.

  Currently, polystyrene (PS) containers, polypropylene (PP) containers, or glass containers are generally used as containers for storing cells.

  However, when cells having adhesiveness are stored using these containers, there is a problem that the cells adhere to the inner wall surface of the container. There is currently no container that can suppress cell adhesion, and development is required. Moreover, in order to inspect the state of cells after storage, the cell cryopreservation container is required to be able to inspect cells in a state in which a cell-containing preservation solution is contained in the container.

  Therefore, an object of the present invention is to provide a cell cryopreservation container that can suppress the adhesion of cells to the inner wall surface of the container and can easily inspect the cells stored in the storage unit.

(1) A cell cryopreservation container comprising a storage unit capable of storing a preservation solution containing cells, and an inner wall surface constituting the storage unit,
An uneven surface having a cell adhesion reducing property on a part of the inner wall surface;
An observation unit capable of observing the cells stored in the storage unit on a wall corresponding to at least a partial region of the inner wall surface other than the uneven surface having the cell adhesion reducing property;
Have
The cell cryopreservation container, wherein an inner wall surface of the observation part is a smooth surface, and light transmittance at 450 nm in water of the observation part is 55% or more.
(2) The cell cryopreservation container according to (1), wherein light transmittance at 450 nm in water of the observation unit is 80% or more.
(3) The cell cryopreservation container according to (1) or (2), wherein the concavo-convex surface having the cell adhesion reducing property is formed on an inner wall surface located in a downward direction of the housing portion.
(4) The cell cryopreservation container is a self-supporting container, and the concavo-convex surface having the cell adhesion reducing property is formed on an inner wall surface of a bottom wall portion positioned in a downward direction of the housing portion. ) Cell cryopreservation container.
(5) including a container body including a side wall portion and a bottom wall portion positioned in a horizontal direction of the housing portion, and a lid body that is connected to the container body and seals the housing portion,
The cell cryopreservation container according to (4), wherein the observation unit is provided on at least a part of the side wall or at least a part of the lid.
(6) The cell cryopreservation container according to (5), wherein the outer wall surface of the observation unit is also a smooth surface, and the observation unit is flat.
(7) The cell cryopreservation container according to any one of (1) to (3), wherein the cell cryopreservation container is non-independent.
(8) The cell cryopreservation container according to any one of (1) to (7), wherein the uneven surface includes a plurality of fine protrusions having cell non-adhesiveness.
(9) The arrangement pitch of the plurality of fine protrusions is 60 nm or more and not more than the average diameter of cells to be accommodated,
The height of the plurality of fine protrusions is an average diameter of the cells to be accommodated × 1/2 or more,
The cell cryopreservation container according to (8), wherein the length of the short sides of the tops of the plurality of fine protrusions is equal to or less than the average diameter of the cells to be accommodated x 1/40.
(10) A method for preserving cells by suppressing cell adhesion using the cell cryopreservation container according to any one of (1) to (9).

  ADVANTAGE OF THE INVENTION According to this invention, the cell cryopreservation container which can suppress that a cell adheres to the inner wall face of a container and can test | inspect the cell preserve | saved at an accommodating part can be provided easily.

It is front view sectional drawing of a self-supporting container. It is a front view of a non-independent container. It is side surface sectional drawing which shows the fine protrusion formed in the inner surface of a container. It is a top view of the fine protrusion of FIG. It is a top view which shows the top surface of the fine protrusion of FIG. It is a schematic diagram which shows the state by which the cell was arrange | positioned on the microprotrusion of FIG. It is a schematic diagram which shows the state by which the cell was arrange | positioned on the microprotrusion as a reference example. It is a typical sectional view of the self-supporting cell cryopreservation container concerning this embodiment. It is typical sectional drawing of the non-independent type cell cryopreservation container which concerns on this embodiment.

  The present invention relates to a cell cryopreservation container for cryopreserving cells. The present invention also relates to a container filled with cells and a preservation solution. The cells and the preservation solution filled in the container may be in a cryopreserved state or may be in a state that is not cryopreserved.

[Container shape]
The shape of the cell cryopreservation container according to the present invention is not particularly limited as long as it can hold cells and a preservation solution (hereinafter, a preservation solution containing cells is also referred to as a “cell-containing preservation solution”).

  In one embodiment, the cell cryopreservation container is a free-standing container. In the present specification, the “self-supporting container” refers to a container having a structure in which the container itself can stand on its own without requiring any other device. Examples of the self-standing container include a tube shape, a vial shape, and a petri dish shape. Further, even a container that is generally considered not to be self-supporting, such as a bag shape or a container having a bag shape, may be a self-supporting container by having a base. The self-standing container is, for example, a container 10 having a bottom wall 11 and a side wall 12 standing from the periphery of the bottom wall and a lid (not shown) as shown in FIG. Although there is, it is not limited to this.

In one embodiment, the cell cryopreservation container is a non-self-supporting container. In the present specification, the term “non-self-supporting container” refers to a container that cannot stand on its own due to its own structure, as opposed to a self-supporting type. Examples of the non-self-standing container include a bag-shaped container and a bag-shaped container. A typical non-self-supporting container includes, for example, a pair of opposed film portions 21 shown in FIG. 2, a pair of side sealing portions 22 and a bottom sealing portion 23 along the outer edges of the pair of film portions, However, the present invention is not limited to this. In the container 20 shown in FIG. 2, after the cell-containing preservation solution is filled into the internal space (container) formed by the pair of film parts 21,
The upper part of the film part 21 is sealed.

  The shape of the accommodating part in the container is not particularly limited, and generally has a shape corresponding to the shape of the container. For example, the shape of the storage part of the self-supporting container includes a columnar shape or a prismatic shape.

[Uneven surface with reduced cell adhesion]
In the cell cryopreservation container according to the present invention, an uneven surface having a cell adhesion reducing property is provided on a part of the inner wall surface constituting the accommodating portion in the container.

  The concavo-convex surface having cell adhesion-reducing property means an concavo-convex surface in which cell adhesion is reduced as compared with a smooth surface formed of the same material by forming an uneven shape. The present inventors have found that cell adhesion can be reduced by imparting an uneven shape to the wall surface. It is preferable that the uneven surface includes a plurality of fine protrusions. In addition, the description regarding the specific shape of the uneven | corrugated surface which has cell adhesion reduction property is mentioned later.

  The uneven surface having cell adhesion reducing property is preferably an uneven surface having cell non-adhesiveness. Cell non-adhesive means the property that cells do not adhere or cells do not substantially adhere. The uneven surface having cell non-adhesiveness means an uneven surface to which cell non-adhesiveness is imparted by an uneven shape. The adhesion of the cells on the uneven surface can be easily confirmed by a party by microscopic observation.

  Since the cells contained in the cell-containing preservation solution tend to settle by gravity and gather below the housing part, the uneven surface having cell adhesion reducing properties is formed on the inner wall surface located below the housing part. It is preferable.

  In the present specification, the “downward direction” means the direction of gravity when the intended arrangement state of the container at the time of storage or use of the cell-containing preservation solution is used as a reference. For example, when the container is a self-supporting type, the downward direction (further upward, horizontal direction) is determined based on the state where the self-supporting container is arranged on a horizontal plane. Specifically, in the case of a self-supporting container as shown in FIG. 8, the inner wall surface located in the downward direction of the housing portion means the inner wall surface (104) of the bottom wall portion (101b). Further, when the container is non-self-supporting, a person skilled in the art can easily grasp the arrangement state intended at the time of storage or use by a sticker, a barcode or the like attached to the container. For example, when the container has a bag shape as shown in FIG. 9, since a seal with a trade name is generally attached to the upper surface, the wall (film) opposite to the wall (film) to which the seal is attached ( The film is placed on the lower side. Therefore, in FIG. 9, the inner wall surface of the wall portion (second sheet 202) arranged facing downward is the inner wall surface positioned below the housing portion. Some containers having a bag shape are used by being suspended. In the case of this suspended bag container, the gravity direction may be determined as the downward direction based on the suspended state.

[Observation part]
The cell cryopreservation container according to the present invention has an observation part capable of observing cells contained in the accommodation part on a wall part corresponding to at least a partial region of the inner wall surface other than the uneven surface having cell adhesion reduction properties. . Regarding the observation part, the inner wall surface is a smooth surface, and the light transmittance at 450 nm in water is 55% or more.

“Smooth surface” means a surface having no unevenness or substantially no unevenness, and is a concept including a flat surface, a spherical surface, and a curved surface. The phrase “substantially free of unevenness” means that the depth of the concave portion and the height of the convex portion are, for example, 1 μm or less, preferably 0.5 μm or less, even if there are concave portions and / or convex portions. Yes, more preferably 0.1 μm or less. The depth of the concave portion and the height of the convex portion can be measured using an atomic force microscope or a scanning electron microscope, as in the method for measuring fine protrusions described later.

  The observation part is not particularly limited in its shape and arrangement position as long as its inner wall surface is a smooth surface and its light transmittance is 55% or more. For example, in the case of a self-supporting container, the observation part can be provided on at least a part of the side wall part and / or at least a part of the lid. Moreover, the observation part can form the whole side wall part as an observation part, or can also form an observation part in a part of side wall part. The observation unit may be formed of the same material as that of the other container part, or may be formed of a material different from that of the other container part. The observation part is preferably formed from the same material as the other container part (wall part having an uneven surface), and is preferably formed integrally with the other container part.

  In the smooth surface, light scattering is suppressed and the light transmittance is higher than in the non-smooth surface. Therefore, it becomes possible to easily observe the filled cell-containing preservation solution from the outside of the container through a smooth surface.

  The higher the light transmittance in the observation part, the easier it is to observe the filled cell-containing preservation solution from the outside of the container. Therefore, the higher the light transmittance, the better. The light transmittance in the observation part is 55% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and particularly preferably 90% or more. Preferably it is 95% or more. When the light transmittance in the observation part is 80% or more, it is particularly preferable because the cell-containing preservation solution accommodated in the accommodation part can be observed using an optical microscope.

  The outer wall surface of the observation part is also preferably a smooth surface. Further, from the viewpoint of facilitating more precise observation, the observation part is preferably flat.

  The “light transmittance” in the present specification is a transmittance (wavelength: 450 nm) measured according to the first method of transparency test in the container / packaging material test method of the 16th revision Japanese Pharmacopoeia.

  The light transmittance varies depending on the type of material constituting the observation part, crystallinity, and the like. Therefore, desired light transmittance can be obtained by appropriately selecting the type of material, the manufacturing method of the container, and the like. In addition, since plastic has a tendency to decrease light transmittance when crystallized, the light transmittance can be improved by increasing the cooling rate during resin molding so as to prevent crystallization.

[Container material]
The material of the container is not particularly limited and is, for example, plastic, and can be appropriately selected in consideration of use for cryopreservation. Examples of the material for the container include polystyrene, polypropylene, polyethylene, polyethylene terephthalate (PET), and the like. Examples of the container material include cyclic olefin copolymers and cycloolefin copolymers. Moreover, glass can also be used as a material for the container. In addition, fluorine-based polymers can also be used, for example, ethylene-perfluoroethylene propene copolymer (EFEP), perfluoroethylene propene copolymer (FEP), perfluoroalkoxyalkane (PFA), ethylene-tetrafluoroethylene. Copolymer (ETFE), polychlorotrifluoroethylene (PCTFE), polyvinyl fluoride (PVF) and the like can be mentioned.

[Cell-containing preservation solution]
The type of cells to be cryopreserved is not particularly limited. For example, cancer cells (for example, liver cancer cells,
Glioma cells, colon cancer cells, kidney cancer cells, pancreatic cancer cells, prostate cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, ovarian cancer cells), hepatocytes, Kupffer cells, endothelial cells (eg, vascular endothelial cells, corneal endothelium) Cells), fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermis cells (eg epidermal keratinocytes), epithelial cells (eg tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells) , Corneal epithelial cells), mammary cells, pericytes, muscle cells (eg, smooth muscle cells, cardiomyocytes), kidney cells, pancreatic islets of Langerhans, nerve cells (eg, peripheral nerve cells, optic nerve cells), chondrocytes, bone Pluripotent stem cells such as cells, egg cells, fertilized eggs, ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), embryonic stem cells that are undifferentiated cells, mesenchymal stem cells having multipotency, Blood with monopotency Unipotent stem cells, such as endothelial progenitor cells, cells differentiated is completed, it may be any of such regenerative medicine cells. Moreover, a cell may be used individually by 1 type and may be used in combination of 2 or more type.

  The preservation solution for cryopreserving the cells is not particularly limited, and for example, a known cell preservation solution can be used. In addition, as the preservation solution, for example, water or a mixed solution of water and dimethyl sulfoxide (DMSO) can be used.

  The average cell diameter means the average particle diameter of the cells contained in the preservation solution, measured as a sphere equivalent diameter by a laser diffraction / scattering method using a measuring device according to JIS Z8825. The average cell diameter varies depending on the cell type, but is usually in the range of 10 μm to 100 μm. Examples of the cells contained in the preservation solution include dendritic cells.

[Embodiment of uneven surface having cell adhesion reducing property]
Hereinafter, the uneven surface for reducing cell adhesion will be described. The concavo-convex surface having cell adhesion reducing properties can be composed of a plurality of fine protrusions. The concavo-convex surface having a plurality of fine protrusions has a smaller area of adhesion with cells and can suppress cell adhesion.

  The uneven surface having cell adhesion reducing property preferably has cell non-adhesive properties. Hereinafter, the uneven surface having cell non-adhesiveness will be described with reference to the drawings. FIG. 3 is a side sectional view of the fine protrusion 1. FIG. 4 is a plan view of the fine protrusion 1.

  A microprotrusion is a non-cell-adhesive protrusion. In order for the fine protrusions to exhibit a cell non-adhesive function, preferably, the arrangement pitch of the fine protrusions is 60 nm or more and not more than the average diameter of the cells to be filled, and the height of the fine protrusions is the average of the cells to be filled. It is diameter x 1/2 or more, and the length of the short side of the top of the fine projection is average diameter of cells to be filled x 1/40 or less.

  The average cell diameter means the average particle diameter of the cells contained in the cell-containing preservation solution, measured as a sphere-equivalent diameter by a laser diffraction / scattering method using a measuring device according to JIS Z8825. The average cell diameter varies depending on the cell type, but is usually in the range of 10 μm to 100 μm. Therefore, more specifically, the arrangement pitch of the fine protrusions is 60 nm or more and the average cell diameter (10 μm to 100 μm) or less, and the height of the fine protrusions is the average cell diameter (10 μm to 100 μm) × 1/2 or more. The length of the short side of the top part of the fine protrusion is an average cell diameter (10 μm to 100 μm) × 1/40 or less.

  The arrangement pitch of the fine protrusions refers to the distance L between the centers of two adjacent fine protrusions 1 in FIG. Here, “center” means a point of symmetry of the shape when the shape of the fine protrusion 1 in FIG. 4 is point-symmetric, and in this case, coincides with the center of gravity of the shape. On the other hand, when the shape of the fine protrusion 1 in FIG. 4 is not point-symmetric, it means the center of gravity of the shape.

  The height of the fine protrusion means the distance H between the bottom 2 and the top 3 of the fine protrusion 1 in the shape of the fine protrusion 1 in FIG.

  The length of the short side of the top of the fine protrusion means that when the fine protrusion 1 has a square shape as shown in FIG. It refers to the length of one side, and when it has a rectangular shape, it refers to the length of its short side. In addition, when the top of the fine protrusion 1 has a circular shape as shown in FIG. 5 (b) when the fine protrusion 1 is viewed in plan as shown in FIG. When it has, it means the length of the minor axis. Further, when the fine protrusion 1 is viewed in plan as shown in FIG. 4, when the top of the fine protrusion 1 is viewed globally and globally as shown in FIG. (C) the vertical direction) and the short direction (that is, the horizontal direction in FIG. 5C), the length in the short direction is expected to reflect the overall tendency of the variation. Measured at a plurality of locations, and specified as an average value of the measured lengths in the short direction. Specifically, for example, four intermediate points that are located on the virtual line segment along the longitudinal direction and that divides the virtual line segment into five equal parts are identified, and the four intermediate points that are specified are identified. For each, measure the length in the short direction at each midpoint. And the value which averaged the length of a total of four short directions is made into the length of the short side of the top part of a microprotrusion. Further, when the fine protrusion 1 is viewed from the side as shown in FIG. 3, when the top of the fine protrusion 1 is sharply pointed, the length of the short side of the top is zero.

  Various dimensions and shapes relating to the fine protrusions can be specified using an atomic force microscope (AFM) or a scanning electron microscope (SEM). However, it is not necessary to specify the dimensions of the fine protrusions by examining the entire area of the target inner surface and calculating the average value. , Height, length of the short side of the top surface, etc.) in a section with an area that can be expected to reflect the overall tendency) It can be specified by examining the number obtained and calculating the average value. For example, by measuring an average of 30 points included in a 30 mm × 30 mm region with an electron microscope, each dimension related to the fine protrusions (for example, the arrangement pitch of the fine protrusions, the height, the short side of the top surface) Length etc.) can be specified.

  In the illustrated example, the fine protrusions are formed in a regular pattern, but may be formed irregularly.

  The method for forming the fine protrusions is not particularly limited. For example, the fine protrusions can be formed by injection molding using a mold having a concavo-convex shape formed on the surface by cutting, sandblasting, or dispersion plating. Further, the fine protrusions can be directly formed by cutting or etching the inner surface of the container.

  An example of a method for forming fine protrusions in a regular pattern on a film will be described. First, the uneven | corrugated shape corresponding to the negative shape of a fine protrusion is formed in the surface of metal sheets, such as copper, aluminum, and stainless steel, by etching or cutting, and the said metal sheet is wound around the surface of a heating roll. Then, while transporting the film so as to pass between the heating roll and the pressure roll rotating in opposite directions, the heating roll and the pressure roll are brought close to each other at a predetermined pressure, and the surface of the film is melted appropriately. The uneven shape on the sheet surface is transferred. Thereby, fine protrusions are formed in a regular pattern on the film.

An example of a method for irregularly forming fine protrusions on a film will be described. First, an irregular concavo-convex shape (satin texture) is formed on the surface of a sheet made of a material constituting the film by sandblasting, and the sheet is wound around the surface of a rotatable heating roll. Then, while conveying the film so as to pass between the heating roll and the pressure roll rotating in opposite directions, the heating roll and the pressure roll are brought close to each other with a predetermined pressure, and the unevenness of the surface of the sheet melted appropriately The shape is thermally transferred to the film. That is, the material of the top part of the uneven | corrugated shape of the surface of the sheet | seat melt | dissolved moderately is adhered to a film. Thereby, fine protrusions are irregularly formed on the film.

  Another example of a method for irregularly forming fine protrusions on a film will be described. First, paper (for example, high-quality paper) having irregular irregular shapes on the surface is wound around the surface of a rotatable heating roll. Then, while conveying the film so as to pass between the heating roll and the pressure roll rotating in opposite directions, the heating roll and the pressure roll are brought close to each other with a predetermined pressure, and the surface of the paper is appropriately dissolved in the film. Transfer (shape) the uneven shape. Thereby, fine protrusions are irregularly formed on the film. Here, instead of paper, a metal sheet having irregular irregular shapes formed on the surface by dispersion plating or sand blasting, or a synthetic resin sheet having irregular irregular shapes formed on the surface by sand blasting may be used.

  As shown in FIG. 6, when the arrangement pitch of the fine protrusions 1 is 60 nm or more and not more than the average diameter of the cells 4, the cells 4 contact only the tops of the fine protrusions 1 and contact the side surfaces of the fine protrusions 1. It becomes difficult. Thereby, the scaffold of the cell 4 becomes small and the adhesion of the cell 4 is suppressed.

  On the other hand, as shown in FIG. 7A, when the arrangement pitch of the fine protrusions 1 is larger than the average diameter of the cells 4, the cells 4 come into contact with the side surfaces of the fine protrusions 1, and the cells 4 adhere to each other using the side surfaces as scaffolds. there is a possibility.

  Further, as shown in FIG. 7B, when the arrangement pitch of the fine protrusions 1 is less than 60 nm, the fine protrusions 1 are recognized as a plane for the cell, and the non-adhesiveness of the cell may not be obtained. In addition, it is reported in Reference 1 that cell non-adhesiveness may not be obtained if the interval between microprojections is too narrow (Reference 1: Elisabetta Ada Cavalcanti-Adam, et al., “Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands ”, Biophysical Journal, Volume 92, Issue 8, April 2007, p.2964-2974). However, Document 1 only confirms that the non-adhesiveness of the cells can be obtained by the fine protrusions, and there is no description or suggestion until it is applied to a container filled with the cell-containing preservation solution.

  In addition, as shown in FIG. 7C, when the height of the fine protrusions 1 is less than the average diameter of the cells 4 × ½, the arrangement pitch of the fine protrusions 1 is equal to or less than the average diameter of the cells 4. In addition, there is a possibility that the cells 4 come into contact with the side surfaces of the microprojections 1 and the cells 4 adhere to each other using the side surfaces as a scaffold. Therefore, the non-adhesiveness with respect to the cells is more reliably ensured by setting the height of the fine protrusions to be equal to or larger than the average cell diameter × 1/2.

  Further, as a rule of thumb, as shown in FIG. 7 (d), when the length of the short side of the top of the fine protrusion 1 is larger than the average diameter of the cell 4 × 1/40, the cell 4 moves the top of the fine protrusion 1. There is a possibility of bonding as a scaffold. Reference 2 describes that cells can be adsorbed to microprojections having a width greater than 250 nm, but cells cannot be adsorbed to microprojections having a width of 250 nm or less (Reference 2: Takehiko Kitamori, “Nanostructures”). Cell pattern control ", [online], [July 28, 2015 search], Internet (URL: http://park.itc.u-tokyo.ac.jp/kitamori/research/nanopattern/nanopattern. html)). When the value of 250 nm is converted into the smallest average cell diameter of 10 μm, the average cell diameter × 1/40 is obtained.

Therefore, the non-adhesiveness with respect to a cell is more reliably ensured by making the length of the short side of the top part of a microprotrusion into the average diameter of a cell x 1/40 or less. Here, if the length of the short side of the top portion of the fine protrusion is equal to or less than the average cell diameter × 1/40, the planar shape of the top portion is not particularly limited. For example, as shown to Fig.5 (a), it is square, The one side may become the average diameter of a cell x 1/40 or less. Moreover, as shown in FIG.5 (b), it is circular and the diameter may be below the average diameter of a cell x 1/40. In addition, as shown in FIG. 5 (c), a rectangular portion is connected to each of the upper end and the lower end of the circular portion, and the length in the short direction (left-right direction) is equal to or less than the average cell diameter × 1/40. It may be.

(Embodiment 1)
An embodiment of the present invention will be described below with reference to the drawings.

  FIG. 8A is a schematic cross-sectional view showing a self-supporting cell storage container 100 according to this embodiment, and FIG. 8B is a schematic cross-section showing a container body 101 as a constituent member of the self-supporting container 100. FIG. 8C is a schematic cross-sectional view showing a lid 102 as a constituent member of the self-supporting container 100.

  The self-supporting container 100 includes a container main body (for example, a bottomed cylindrical main body) 101 having a storage portion 103 for storing a cell-containing preservation solution, and a lid portion 102 capable of sealing the storage portion 103 by being connected to the container main body 101. . The lid 102 can be connected to the container main body 101 by, for example, a screw-in type. The container main body 101 includes a side wall portion 101a positioned in the horizontal direction of the storage portion 103, a bottom wall portion 101b positioned in the downward direction of the storage portion 103, and a base 101c. The side wall 101a is erected from the periphery of the bottom wall 101b. On the inner wall surface of the bottom wall portion 101b, an uneven surface 104 having a cell adhesion reducing property is formed, and cell adhesion during storage or use can be suppressed by the uneven surface. Moreover, the inner wall surface of the side wall part 101a is formed as a smooth surface, and the light transmittance in water in the side wall part 101a is 55% or more.

  In this embodiment, the side wall part 101a becomes an observation part. The inner wall surface of the side wall part 101a is a smooth surface, and the light transmittance in water in the side wall part 101a is 55% or more, so that the state of the cells in the cell-containing preservation solution stored in the storage part 103 is good. Can be observed. The light transmittance in water in the side wall portion 101a is preferably 80% or more, and more preferably 90% or more.

  The outer wall portion of the side wall portion 101a is also preferably a smooth surface. Moreover, when the side wall part 101a is comprised from a flat plate-shaped wall part, the distortion of light can decrease and the precision of observation can be improved.

  Moreover, the wall part corresponding to the inner wall surface of the cover body 102 may be used as the observation part by making the inner wall surface in contact with the housing part 103 of the cover body 102 a smooth surface and having a light transmittance of 55% or more.

  Further, FIG. 8 shows a case where the entire side wall 101a is formed as an observation part, but an observation part may be provided on at least a part of the side wall or at least a part of the lid. For example, the form which provided the flat observation part in a part of side wall part of the container main body which has a substantially cylindrical shape is also assumed.

  In the container main body 101, the constituent material of the side wall portion 101a and the constituent material of the bottom wall portion 101b may be the same or different. The side wall material is preferably polyethylene, polypropylene, fluorine-based polymer, cyclic olefin copolymer, cycloolefin copolymer, or polystyrene, and the bottom wall material is polyethylene, fluorine-based polymer, cyclic olefin copolymer, cycloolefin copolymer, or the like. preferable.

  The bottom wall portion 101b is not limited to a plate shape, but is a pyramid or conical shape (V-shaped when viewed from the side), a test tube-like hemisphere, a mortar shape, or a tapered shape such as a Spitz tube. Also good.

(Embodiment 2)
An embodiment of the present invention will be described below with reference to the drawings.

  FIG. 9A is a schematic cross-sectional view showing a non-self-supporting (specifically bag-shaped) cell storage container 200 according to the present embodiment.

  The non-self-supporting container 200 is formed by welding the peripheral portions of two resin sheet-like members, that is, the first sheet 201 and the second sheet 202. In the present embodiment, it is assumed that the second sheet 202 is stored while being directed downward. A storage portion 203 for storing cells is formed by a space surrounded by the first sheet 201 and the second sheet 202. Then, on the inner wall surface of the second sheet 202, a concavo-convex surface 204 having a plurality of fine protrusions and having a cell adhesion reducing property is formed. The inner wall surface of the first sheet 201 is a smooth surface, and the light transmittance of the first sheet 201 in water is 55% or more.

  The constituent materials of the first sheet 201 and the second sheet 202 may be the same or different. The first sheet 201 is preferably polyethylene, polypropylene, fluorine-based polymer, cyclic olefin copolymer, cycloolefin copolymer, or polystyrene, and the second sheet 202 is polyethylene, fluorine-based polymer, cyclic olefin copolymer, cycloolefin copolymer, or the like. Is preferred.

  The first sheet 201 as the observation part is preferably manufactured by an inflation method, particularly a water-cooled inflation method, to improve the light transmittance. The light transmittance of the first sheet 201 in water is preferably 80% or more, and more preferably 90% or more.

DESCRIPTION OF SYMBOLS 1 Fine protrusion 2 Fine protrusion bottom part 3 Fine protrusion top part 4 Cell 10 Container 11 Bottom wall part 12 Side wall part 20 Container 21 A pair of film part 22 A pair of side sealing part 23 A bottom sealing part 100 A self-supporting cell cryopreservation container DESCRIPTION OF SYMBOLS 101 Container main body 101a Side wall part 101b Bottom wall part 101c Base 102 Cover body 103 Accommodating part 104 Uneven surface 200 having cell adhesion reducing property Non-self-supporting cell cryopreservation container 201 First sheet 202 Second sheet 203 Accommodating part 204 Uneven surface with reduced cell adhesion

Claims (10)

A cell cryopreservation container comprising a storage unit capable of storing a storage solution containing cells, and an inner wall surface constituting the storage unit,
An uneven surface having a cell adhesion reducing property on a part of the inner wall surface;
An observation unit capable of observing the cells stored in the storage unit on a wall corresponding to at least a partial region of the inner wall surface other than the uneven surface having the cell adhesion reducing property;
Have
The cell cryopreservation container, wherein an inner wall surface of the observation part is a smooth surface, and light transmittance at 450 nm in water of the observation part is 55% or more.   The cell cryopreservation container according to claim 1, wherein the light transmittance at 450 nm in water of the observation unit is 80% or more.   The cell cryopreservation container according to claim 1 or 2, wherein the uneven surface having cell adhesion reducing property is formed on an inner wall surface located in a downward direction of the housing portion.   The said cell cryopreservation container is a self-supporting container, The uneven surface which has the said cell adhesion reduction property is formed in the inner wall surface of the bottom wall part located in the downward direction of the said accommodating part. Cell cryopreservation container. A container body including a side wall portion and a bottom wall portion positioned in a horizontal direction of the housing portion, and a lid body that is connected to the container body and seals the housing portion,
The cell cryopreservation container according to claim 4, wherein the observation part is provided on at least a part of the side wall part or at least a part of the lid.   The cell cryopreservation container according to claim 5, wherein an outer wall surface of the observation unit is also a smooth surface, and the observation unit has a flat plate shape.   The cell cryopreservation container according to any one of claims 1 to 3, wherein the cell cryopreservation container is non-independent.   The cell cryopreservation container according to any one of claims 1 to 7, wherein the uneven surface includes a plurality of fine protrusions having cell non-adhesiveness. The arrangement pitch of the plurality of fine protrusions is 60 nm or more and less than the average diameter of cells to be accommodated,
The height of the plurality of fine protrusions is an average diameter of the cells to be accommodated × 1/2 or more,
The cell cryopreservation container according to claim 8, wherein the length of the short sides of the tops of the plurality of microprojections is an average diameter of the cells to be accommodated x 1/40 or less.   A method for preserving cells by suppressing cell adhesion using the cell cryopreservation container according to any one of claims 1 to 9.

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