JP2015223108A - Cell culture chamber and production method thereof, and method and kit for cell culture using cell culture chamber - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture chamber capable of suppressing sag of a vitrigel membrane and uniformly dispersing and culturing cells on the vitrigel membrane, in a cell culture chamber comprising the vitrigel membrane of dried material.SOLUTION: A cell culture chamber 1 comprises a cylindrical chamber body 2, a vitrigel membrane 3 of dried material, and a substrate 4. In the chamber body 2, open end faces 2a and 2b are formed at both opposite ends. The open end face 2b is fixed in a state that one surface side of the vitrigel membrane 3 of dried material is in contact, and the substrate 4 is arranged on the other surface side of the vitrigel membrane 3 of dried material.

Description

  The present invention relates to a cell culture chamber and a method for producing the same, a cell culture method using the cell culture chamber, and a cell culture kit.

  Research on drug discovery and alternative methods for animal experiments has long required the development of a culture system that can easily construct a three-dimensional tissue model that reflects the living body using various functional cells. In particular, the three-dimensional culture technique using collagen gel as a cell culture carrier is useful for reconstructing angiogenesis models, cancer invasion models, epithelial mesenchymal models, etc., but has not been widely used. .

  The reason for this is that conventional collagen gels are soft and difficult to handle because they are composed of low-density fibers, and because they are opaque, phase-contrast microscopy of cultured cells is not always easy. I came.

  In order to solve such a problem, the present inventor injected a collagen sol imparted with a salt concentration and a hydrogen ion concentration (pH) optimal for gelation at low temperature into a culture dish, After the collagen sol is gelled by keeping it at the optimum temperature, it is vitrified by gradually removing free water as well as bound water by sufficiently drying at low temperature, and then rehydrated (Rehydration) has established a technique for converting the physical properties of collagen gel into a thin film excellent in strength and transparency with good reproducibility (Patent Document 1).

  And if it is a hydrogel, gel of components other than collagen can be converted into a new stable physical state by virtue of rehydration after vitrification. A gel having a new physical property state is named vitrigel (Non-patent Document 1).

  In particular, the collagen vitrigel thin film that has been developed so far is a transparent thin membrane with a thickness of several tens of micrometers in which high-density collagen fibers comparable to connective tissues in the living body are intertwined with each other, and has excellent protein permeability. And has a characteristic of having strength. In addition, since various substances can be added to the collagen sol in the production process, the characteristics of the added substance can be reflected in the collagen vitrigel thin film. Furthermore, for example, a collagen vitrigel thin film in which a cyclic nylon membrane support is embedded can be easily handled with tweezers.

  The present inventors further develop the technology relating to the collagen vitrigel thin film, a technique for improving the transparency and production reproducibility of the collagen vitrigel thin film (Patent Document 2), and the collagen vitrigel thin film. In addition, a technique (Patent Document 3) for producing a filamentous or tubular shape, a technique for fixing or moving a collagen vitrigel using magnetism (Patent Document 4), and the like are also proposed.

  Further, the present inventor has devised a method for rapidly and mass producing a dried vitrigel membrane that can be molded into a desired shape and has excellent handleability (Patent Document 5). According to this method, the dried vitrigel membrane can be obtained in a membrane state without adhering to the culture petri dish. Can be established.

  And this inventor is applying the method of patent document 5, and has created the cell culture chamber using a vitrigel membrane dry body. Furthermore, using this cell culture chamber, a three-dimensional tissue model capable of evaluating ADMET (absorption / distribution / metabolism / excretion / toxicity) of a chemical substance has been successfully constructed (Patent Document 6).

  Furthermore, using a three-dimensional tissue model produced in this cell culture chamber, attempts have been made to apply it to peritoneal permeation analysis and eye irritation tests, and its usefulness has been clarified (Non-Patent Document 3, 4). In addition, various test methods such as liver metabolism test, hepatotoxicity test, corneal permeability test, skin sensitization test and the like have been vigorously developed using this cell culture chamber.

JP-A-8-228768 WO2005 / 014774 JP 2007-204881 A JP 2007-185107 A WO2012 / 026531 JP 2012-115262 A

Takezawa T, et al., Cell Transplant. 13: 463-473, 2004 Takezawa T, et al., J. Biotechnol. 131: 76-83, 2007 Aoki T, et al., Am. J. Physiol. Renal. Physiol. 306: F116-122, 2013 Yamaguchi H, et al., Toxicol. Sci. 135: 347-355, 2013

  However, when cells are cultured in the cell culture chamber of Patent Document 6, if a liquid such as a culture solution is injected into the chamber, the hydrated vitrigel membrane may be slackened downward, thereby supplying the liquid into the chamber. It was confirmed that the collected cells gathered in the center of the vitrigel membrane. In this case, cells that have a low aggregation property between cells and are relatively easy to handle (for example, mouse embryonic fibroblasts (NIH3T3), human colon cancer-derived cells (Caco-2), human corneal epithelial cells (HCE-T) ) Etc.) is not a big problem in the construction of tissue models, but for example, in the case of cells that tend to aggregate, such as human hepatoma-derived cells (HepG2), an aggregate is formed in the center and uniform on the Vitrigel membrane. It was found that it was difficult to construct a simple cell layer as a point to be improved.

  The present invention has been made in view of the above circumstances, and in a cell culture chamber equipped with a dried vitrigel membrane, the slack of the vitrigel membrane is suppressed and the cells are uniformly dispersed on the vitrigel membrane. It is an object of the present invention to provide a cell culture chamber that can be used. Another object of the present invention is to provide a method for producing the cell culture chamber, a cell culture method using the cell culture chamber, and a cell culture kit.

In order to solve the above problems, the present invention provides the following cell culture chamber, method for producing the cell culture chamber, cell culture method, and cell culture kit.
<1> A cell culture chamber comprising a cylindrical chamber body, a dried vitrigel membrane, and a support, wherein the chamber body has open end faces at opposite ends, of which One open end surface is fixed in a state where one surface side of the dried vitrigel membrane is in contact with the other, and the support is disposed on the other surface side of the dried vitrigel membrane. A cell culture chamber characterized by the above.
<2> The cell culture chamber according to <1>, wherein the support is a plastic thin film.
<3> The cell culture chamber according to <1>, wherein the support is a silicon-treated PET thin film.
<4> The following steps:
A step of abutting and fixing one surface side of the dried vitrigel membrane to one open end surface of a cylindrical chamber body having opposite open end surfaces formed at both ends; and covering the open end surface; and A method for producing a cell culture chamber, comprising a step of disposing a support on the other surface side of the dried vitrigel membrane.
<5> The following steps:
Supplying one or more types of cells into the inside of the chamber body of the cell culture chamber of any one of claims 1 to 3;
Culturing cells on the vitrigel membrane in a state where the vitrigel membrane obtained by rehydrating the dried vitrigel membrane is supported on the support; and removing the support from the vitrigel membrane. A cell culture method characterized.
<6> A cell culture kit including a cylindrical chamber body, a dried vitrigel membrane, and a support, wherein the chamber body has an open end surface with opposite end surfaces open, and the open end surface One of the two is fixed in a state where one surface side of the dried vitrigel membrane is in contact, and the support is disposed on the other surface side of the dried vitrigel membrane. A cell culture kit characterized by the above.

  The cell culture chamber of the present invention suppresses slackening of the vitrigel membrane, and can cultivate the cells evenly distributed on the vitrigel membrane. This reflects the tissue and organ units in the living body using the characteristics of Vitrigel (polymer permeability, sustained release of physiologically active substances such as proteins, transparency, fiber density close to the living body, stability, etc.) Various organizational models can be constructed more reliably.

It is the cross-sectional schematic diagram which illustrated one Embodiment of the cell culture chamber of this invention. It is a photograph of the cell culture chamber of the present invention. It is the photograph which showed the time-dependent change of the vitrigel membrane of the cell culture chamber of this invention. It is the photograph which showed the time-dependent change of the vitrigel membrane of the cell culture chamber of this invention. It is a photograph which shows the dispersion | distribution state at the time of adding microparticles | fine-particles to the cell culture chamber of this invention. It is a photograph which shows the mode of the HepG2 cell of the culture | cultivation 2nd day cultured by the cell culture chamber of this invention.

  FIG. 1 is a schematic cross-sectional view illustrating an embodiment of the cell culture chamber of the present invention.

  The cell culture chamber 1 includes a cylindrical chamber body 2, a dried vitrigel membrane 3, and a support 4.

  In this embodiment, the chamber body 2 has a substantially cylindrical shape having a space for holding cells therein, and has open end surfaces 2a and 2b that are open at opposite ends (upper surface and bottom surface). .

  One surface side (upper side in FIG. 1) of the dried vitrigel membrane 3 is in contact with and fixed to the open end surface 2b (bottom surface in FIG. 1), and the open end surface 2b (bottom surface) of the chamber body 2 is covered. Has been. As the material of the chamber body 1, a material suitable for cell culture can be appropriately selected. For example, an acrylic or polystyrene cylindrical tube can be preferably exemplified.

  Further, a locking portion 5 that protrudes outward is provided on the outer peripheral edge of the open end surface 2a (upper surface in FIG. 1) of the chamber body 2. The locking portion 5 can be formed into a rod shape, a flange shape, or the like, for example, with a plastic material or the like.

  Further, a support 4 is disposed on the other surface side (lower side in FIG. 1) of the dried vitrigel membrane 3. The support 4 is approximately the same as or larger than the inner diameter of the chamber body 2 and covers the lower surface of the dried vitrigel membrane 3. Therefore, even if the dried vitrigel membrane 3 is rehydrated with a culture solution or the like, and the cells are supplied to the inside of the chamber body 2 and cultured, the vitrigel membrane is supported by the support 4 and the slack is suppressed. The cells can be uniformly distributed and cultured. Therefore, the formation of cell aggregates on the vitrigel membrane is suppressed, and the characteristics of vitrigel (polymer permeability, sustained release of physiologically active substances such as proteins, transparency, fiber density close to the living body, stable A tissue model having a uniform cell layer can be more reliably constructed.

  When the cells are cultured on the rehydrated Vitrigel membrane, the support 4 can be detached and removed after a predetermined time has elapsed after the start of the culture and the cells adhere to the Vitrigel membrane. Thus, nutrient components and oxygen can be permeated through the vitrigel membrane and supplied to the cells. Therefore, it is desirable that the support 4 has both moderate adhesiveness and releasability with respect to the dried vitrigel membrane 3 (re-hydrated vitrigel membrane). For example, when the support 4 is difficult to peel from the vitrigel membrane, the vitrigel membrane may be broken or stretched when the support 4 is removed.

  Preferred examples of the material of the support 4 include plastic, rubber, glass, metal, and composite materials thereof. Specifically, siliconized PET (polyethylene terephthalate), PET, polyethylene, polycarbonate, polystyrene, PTFE (polytetrafluoroethylene), fluororesin, urethane resin, silicon resin, polypropylene, acrylic, vinyl chloride, siliconized glass, glass Examples thereof include silicon-treated stainless steel, fluororesin-treated stainless steel, and stainless steel. Of these, silicon-treated PET, PET, polyethylene, PTFE, and silicon resin are preferable, and silicon-treated PET is particularly preferable. Silicon-treated PET is excellent in water repellency and realizes appropriate adhesion and peelability to the dried vitrigel film (the vitrigel film after rehydration). “Silicon-treated PET” means one or both sides of a PET film covered with silicon. The support 4 illustrated in FIG. 1 has a form in which one surface of a PET film 41 is covered with silicon 42, and the surface on the silicon 42 side is brought into contact with and adhered to the dried vitrigel film 3.

  The form of the support 4 is not particularly limited as long as the surface in contact with the dried vitrigel membrane is smooth. Specifically, the support 4 can be exemplified by a film shape (thin film shape) or a cap shape fitted to the end of the chamber body 2.

  For example, when the support 4 is in the form of a thin film, the thickness is not particularly limited, but can be appropriately designed in the range of, for example, about 10 μm to 20000 μm depending on the type. Further, the support 4 preferably has water repellency, and specifically, the contact angle is particularly preferably 70 ° or more. When the contact angle of the support 4 is 70 ° or more, moderate adhesiveness and peelability are realized with respect to the dried vitrigel membrane (the vitrigel membrane after rehydration).

  Moreover, as a method of disposing the support 4 on the dried vitrigel membrane 3, for example, an aqueous solution is supplied into the chamber body to rehydrate the dried vitrigel membrane and convert it into a vitrigel membrane. A method of adhering the support to the dried vitrigel membrane can be exemplified by bringing the support into contact with the support and then drying it.

  Furthermore, for the treatment such as drying and rehydration of the dried vitrigel membrane in the cell culture chamber of the present invention, a method similar to the method described in WO2005 / 014774 can be appropriately employed.

  Further, as another embodiment of the cell culture chamber of the present invention, for example, one side (one chamber side or two chamber side surface) of the dried vitrigel membrane of the two-chamber cell culture chamber described in Patent Document 6 In addition, a form in which the support is disposed can be exemplified.

  Next, the cell culture method of the present invention will be described.

  The cell culture method of the present invention includes the following steps.

  Supplying one or more types of cells inside the chamber body of the cell culture chamber;

  A step of culturing cells on the vitrigel membrane in a state where the vitrigel membrane obtained by rehydrating the dried vitrigel membrane is supported by the support.

  Removing the support from the vitrigel membrane.

  In the cell culture method of the present invention, one type or two or more types of desired cells are supplied onto the dried vitrigel membrane in the cell culture chamber. By adding a cell-containing suspension or culture solution, the dried vitrigel membrane in the cell culture chamber is rehydrated and converted into a vitrigel membrane. As illustrated in FIG. 1, cells are cultured on the vitrigel membrane in a state where the vitrigel membrane converted from the dried vitrigel membrane is supported by the support. After a predetermined time has elapsed after the start of the culture, the cells can stably adhere to the vitrigel membrane, and then the support can be detached and removed to continue the culture. In this case, a tissue model or the like can be constructed by performing liquid phase-liquid phase interface culture or liquid phase-gas phase interface culture using the characteristics of the vitrigel membrane.

  Since the cell culture chamber includes a support, slackness of the vitrigel membrane is suppressed, and cells can be uniformly dispersed and cultured on the vitrigel membrane. For this reason, even if it is a cell which is easy to aggregate, formation of the aggregate is suppressed and the tissue model etc. in which the uniform cell layer was formed on the vitrigel membrane can be constructed reliably.

  Here, the “tissue model” refers to a model of a cell state, tissue, or organ in a living body. For example, a chemical substance or a physiologically active substance (drugs such as various pharmaceuticals, nutritional components, proliferation, etc.) The effects of factors, etc.) can be tested. The tissue model can be constructed by, for example, seeding and culturing cells derived from various mammals, and is preferably a human-derived cell. According to the tissue model of human-derived cells, it is possible to establish an evaluation system free from the problem of species differences when examining ADMET (absorption, distribution, metabolism, excretion, toxicity) of chemical substances for humans.

  Although the form of the tissue model is not limited, for example, an epithelial tissue model that can be constructed by culturing capped epithelial cells or glandular epithelial cells, a connective tissue model that can be constructed by culturing fibroblasts, adipocytes, etc., myoblasts Tissue model that can be constructed by culturing cells, cardiomyocytes, smooth muscle cells, etc., nerve tissue model that can be constructed by culturing nerve cells or glial cells, and other cells derived from two or more types of tissues Organ-like models that can be constructed by Here, the cells used are not limited to normal mature differentiated cells. Embryonic stem (ES) cells, somatic stem cells, induced pluripotent stems (iPS) ) Undifferentiated cells such as cells, lesion-derived cells such as cancer cells, or transformed cells into which a foreign gene has been introduced. In this way, by selecting cells to be used as appropriate, not only normal tissue models but also tissue models that are in the process of development or regeneration, tissue models of lesions such as cancer, or artificially transformed cells Forms such as structured organizational models can be created. In particular, as a tissue model that can extrapolate the action of chemical substances such as pharmaceuticals, physiologically active substances, cosmetics or detergents on the living body, it is possible to construct a tissue model that can reflect the migration path of chemical substances exposed to or administered to the living body. It becomes important. From this point of view, the “tissue sheet (one type of cell)” model composed of only epithelial cells or endothelial cells to which chemical substances are first exposed, and the interval between epithelial cells or endothelial cells that are exposed to chemical substances. An “organ-like plate (two types of cells)” model composed of two types of cells, epithelial cells including mesenchymal cells and mesenchymal cells or endothelial cells and mesenchymal cells. An “organ-like plate (three types of cells)” model composed of three types of cells, ie, epithelial cells, mesenchymal cells, and endothelial cells, can be exemplified. Specifically, such tissue models include chemical transdermal absorption models, corneal permeability models, intestinal and other gastrointestinal absorption models, lung and other airway absorption models, vascular permeability models, liver metabolism models, Including renal glomerular filtration and excretion model, mature tissue model of various organs such as skin, cornea, oral mucosa, nerve, liver, kidney, etc. useful for chemical toxicity evaluation and embryonic tissue model useful for chemical development toxicity evaluation Or an angiogenesis model or a cancer invasion model useful for drug development.

  The tissue model test method is not specifically limited. For example, a method of directly adding a chemical substance into the chamber or a method of causing a chemical substance to act on cells using the permeability of a vitrigel membrane may be exemplified. it can.

  Next, the cell culture kit of the present invention will be described.

  The cell culture kit of the present invention includes a cylindrical chamber body, a dried vitrigel membrane, and a support. The cylindrical chamber body, the dried vitrigel membrane and the support are the same as those illustrated in FIG.

  As illustrated in FIG. 1, the chamber body has an open end face where both opposite end faces are open, and one of the open end faces is in contact with one face side of the dried vitrigel membrane. It is fixed. On the other hand, the support is disposed on the other side of the dried vitrigel membrane, but is separate from the dried vitrigel membrane.

  According to the cell culture kit of the present invention, for example, when an experimenter cultures cells in a cell culture chamber, the dried vitrigel membrane is converted into a vitrigel membrane, and a support is appropriately attached to the vitrigel membrane. Cells can be cultured on the membrane.

  Next, a method for producing a dried vitrigel membrane used in the cell culture chamber of the present invention and a method for disposing the dried vitrigel membrane in the chamber body will be described.

  The dried vitrigel membrane can be produced according to the method described in Patent Document 5. Moreover, about the method of arrange | positioning a vitrigel membrane dry body in a chamber main body, it can carry out according to the method of patent document 6. FIG.

  Hereinafter, one embodiment of the dried vitrigel membrane used in the cell culture chamber of the present invention and the method for fixing the dried vitrigel membrane will be described in detail.

The dried vitrigel membrane used in the cell culture chamber of the present invention can be produced, for example, by going through the following steps (1) to (5).
(1) A film on which a dried vitrigel membrane can be peeled is laid on a substrate, a hydrogel is formed inside a wall mold placed on the film, and a part of free water in the hydrogel is removed between the substrate and the wall mold. Draining from the process,
(2) removing the wall surface mold from the substrate;
(3) Drying the hydrogel to remove residual free water and producing a vitrified hydrogel dried body,
(4) A step of rehydrating the dried hydrogel to produce a vitrigel membrane,
(5) A step of re-drying the vitrigel membrane to remove free water and producing a vitrified dried vitrigel membrane,
Here, “hydrogel” refers to a substance in which a polymer has a network structure formed by chemical bonds, and a large amount of water is retained in the network. This refers to a gel made by introducing cross-links into a molecular artificial material.

  Further, the “hydrogel dried product” refers to a product obtained by removing free water from a hydrogel and vitrifying it. Furthermore, the “vitrigel membrane” refers to a rehydrated body of this hydrogel. As described above, the “new stable gel that can be produced through the vitrification process” is named “vitrigel” by the present inventor. The “dried vitrigel membrane” refers to a vitrigel obtained by vitrification again. The dried vitrigel membrane can be converted to a vitrigel membrane by rehydration when necessary.

  Hereinafter, each step will be described.

  Step (1): A film on which a dried vitrigel membrane can be peeled is laid on a substrate, and a wall surface mold is placed on the film. And after inject | pouring sol into this wall surface mold and gelatinizing, a part of free water in hydrogel flows out from the space | interval of a board | substrate and a wall surface mold.

  For the substrate and the wall surface mold, a material that can withstand sterilization by 70% ethanol or autoclave can be used as appropriate. Specific examples include plastics such as polystyrene and acrylic, glass, stainless steel, and the like.

  Non-water-absorbing films such as parafilm, polyethylene, polypropylene, Teflon (registered trademark), silicon, saran wrap (registered trademark), vinyl, etc. can be exemplified as the film from which the dried vitrigel membrane can be peeled. Is preferred. Parafilm is a thermoplastic film that uses paraffin as a raw material, and has the characteristics that it has stretchability and adhesiveness, and is excellent in airtightness and waterproofness. Hereinafter, it is simply referred to as “film”.

  The wall surface mold can be, for example, a cylindrical frame that does not have an upper surface and a bottom surface, and the shape of the wall surface mold can be designed to be the same as the shape of the desired vitrigel film. Specifically, for example, when a circular vitrigel film is produced, a wall surface (frame) having an annular shape (cylindrical shape) can be used. Moreover, when producing a rectangular vitrigel film | membrane, a wall surface (frame) can be made into a rectangular shape (square cylinder shape).

  Then, a wall surface mold is placed on the film laid on the substrate. At this time, the area where the film is laid is larger than the cross section of the wall mold, and the film and the bottom surface of the wall mold are in contact with each other. A slight gap is formed so that it can flow out. Depending on the desired number of vitrigel films, a plurality of wall surface molds can be arranged on the film.

  Examples of the natural product-derived polymer used as a raw material for producing hydrogel include collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, Examples include basement membrane components (trade name: Matrigel) reconstituted from heparan sulfate proteoglycans, etc., gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan and the like. It is possible to prepare a hydrogel by selecting components such as salt, concentration, pH and the like that are optimal for each gelation. By combining the raw materials, it is possible to obtain a vitrigel membrane that imitates various in vivo tissues.

  In addition, examples of the synthetic polymer used for producing the hydrogel include polyacrylamide, polyvinyl alcohol, methyl cellulose, polyethylene oxide, poly (II-hydroxyethylmethacrylate) / polycaprolactone, and the like. It is also possible to produce a hydrogel using two or more of these polymers. The amount of hydrogel can be adjusted in consideration of the thickness of the vitrigel film to be produced.

  Among these, collagen is preferable as the raw material for the hydrogel. When collagen gel is used, collagen sol can be injected into a wall surface mold placed on a substrate and gelled by an incubator.

  The case where collagen sol is used will be described as an example. Collagen sol is considered to have an optimal salt concentration, including physiological saline, PBS (Phosphate Buffered Saline), HBSS (Hank's Balanced Salt Solution), basic culture solution, It can be prepared with a serum culture solution or a serum-containing culture solution. In addition, the pH of the solution during collagen gelation is preferably about 6 to 8.

  Here, it is desirable to prepare the collagen sol at 4 ° C. Thereafter, the heat retention during gelation should be lower than the collagen denaturation temperature depending on the animal species of the collagen to be used, but generally it can be gelled at a temperature of 37 ° C. or less for several minutes to several tens of minutes. It can be carried out while keeping the temperature.

  Collagen sol is too dilute when the collagen concentration is 0.2% or less and weakly gelled, and when it is 0.3% or more, it is too thick and difficult to homogenize. Therefore, the collagen concentration in the collagen sol is preferably 0.2 to 0.3%, more preferably about 0.25%.

  The collagen sol thus adjusted is injected into the wall surface mold. Since the collagen sol having the above-mentioned concentration has viscosity, if the collagen sol is injected into the wall mold and kept warm quickly, the collagen sol does not flow out of the gap between the substrate and the wall mold within a few minutes. Can be

  The formed collagen gel adheres closely to the substrate and the wall mold, but by leaving it for a predetermined time, as time passes, a part of the free water in the collagen gel passes through the gap between the substrate and the wall mold. It flows out to the outside. Here, by slightly moving the wall surface mold up and down or the like, the adhesion between the gel and the wall surface mold is released and a slight gap is generated, so that the free water can be promoted.

  Step (2): The wall surface mold is removed from the substrate.

  The wall mold is removed leaving the hydrogel on the film laid on the substrate. In the hydrogel, since the free water flows out, the shape held by the wall surface mold can be maintained without being deformed on the film.

  Step (3): The hydrogel is dried to remove residual free water, and a vitrified hydrogel dried body is produced.

  By drying, the free water in the hydrogel is completely removed and vitrified. The longer the period of this vitrification step is, the more vitrigel film excellent in transparency and strength can be obtained when rehydrated. If necessary, the vitrigel film obtained by rehydration after vitrification for a short period of time can be washed with PBS or the like and then vitrified again.

  As a drying method, for example, various methods such as air drying, drying in a sealed container (circulating air in the container and always supplying dry air), drying in an environment where silica gel is placed, and the like can be used. . For example, examples of the air drying method include a method of drying in an incubator kept sterile at 10 ° C. and 40% humidity for 2 days, or a method of drying at room temperature all day and night in a sterile clean bench.

  Step (4): A dried hydrogel is rehydrated to produce a vitrigel membrane.

  A vitrigel membrane can be prepared by rehydrating the dried hydrogel with PBS or a culture medium to be used. Here, the liquid to be rehydrated may contain various components such as a physiologically active substance. Examples of the physiologically active substance include various pharmaceuticals including antibiotics, cell growth factors, and differentiation induction. Examples of factors such as factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, or extracellular matrix components that do not gel include fibronectin, vitronectin, entactin, and osteopoietin. It is also possible to contain a plurality of these.

  Step (5): The vitrigel membrane is re-dried to remove free water to produce a vitrified dried vitrigel membrane.

  As in the step (3), there are various drying methods such as air drying, drying in a sealed container (circulating air in the container and always supplying dry air), drying in an environment where silica gel is placed, and the like. Can be used.

  By re-drying the vitrigel membrane, a vitrified dried vitrigel membrane can be produced. This dried vitrigel membrane can be converted again to a vitrigel membrane by rehydration when necessary.

  Since this dried vitrigel membrane is layered with a peelable film, the dried vitrigel membrane can be handled freely with the film, and the vitrified dried vitrigel membrane can be cut into any shape. Is possible.

  The components contained in the “hydrogel dried product” and the “vitrigel membrane dried product” are not necessarily the same. “Hydrogel dried product” contains components of hydrogel, but “Vitrigel membrane dried product” contains components remaining in the vitrigel membrane equilibrated with an aqueous solution when the hydrogel dried product is rehydrated. Contains.

  Moreover, since the vitrigel membrane obtained by rehydrating the dried vitrigel membrane in the step (5) has a longer vitrification period than the vitrigel membrane obtained in the step (4), the strength and transparency. Is excellent.

  Furthermore, the period of vitrification can be prolonged in the state of “hydrogel dry body”, but “hydrogel dry body” is a state in which all components at the time of hydrogel preparation coexist. When maintaining a dry body or when using a vitrigel membrane, unnecessary components are also mixed. On the other hand, with respect to the vitrigel membrane after the “hydrogel dried product” is rehydrated to remove unnecessary mixed components, unnecessary components are also removed from the dried product. Therefore, when it is necessary to maintain the vitrification period for a long time, it is preferable to keep the dried vitrigel membrane in this state, which is unnecessary for the vitrigel membrane obtained by rehydrating the dried vitrigel membrane. It is excellent in that no components are mixed.

  Furthermore, the dried vitrigel membrane can be prepared, for example, by mixing a physiologically active substance to be contained in a sol solution before gelation and then producing a vitrigel membrane such as gelation or vitrification. . Since the dried vitrigel membrane containing a physiologically active substance can supply factors necessary for cell adhesion, proliferation, differentiation and the like from the vitrigel membrane side, a better culture environment can be realized. Moreover, it is very useful for conducting a test for examining the effect of the contained physiologically active substance on cells. A vitrigel membrane containing a physiologically active substance can also function as a drug delivery system by being implanted into the body (Non-patent Document 2). Furthermore, the vitrigel membrane can permeate a bioactive substance having a large molecular weight, thereby allowing mutual interaction between each cell seeded on two different surfaces of the vitrigel membrane via the bioactive substance. Can greatly contribute to testing and research of action (Non-patent Document 2).

  According to the above method, the dried vitrigel membrane can be obtained in a membrane state without adhering to the culture dish. Moreover, since this dried vitrigel membrane is easy to cut, it can be used in the cell culture chamber of the present invention.

  Next, an embodiment of the method for producing a cell culture chamber of the present invention will be described.

  The manufacturing method of the cell culture chamber of the present invention includes the following steps.

  A step of covering and covering the open end surface by fixing one surface side of the dried vitrigel membrane to one open end surface of a cylindrical chamber main body on which the open end surfaces facing each other are formed.

  A step of disposing a support on the other surface side of the dried vitrigel membrane;

  Specifically, in this method for manufacturing a cell culture chamber, for example, an adhesive is applied to one open end surface of the chamber body, and one surface of the dried vitrigel membrane is brought into contact and fixed. When using the dried vitrigel membrane layered with the film, the vitrigel membrane dried body side can be adhered to the chamber body, and then the film can be peeled off. The adhesive can be appropriately selected in consideration of adhesiveness and cytotoxicity. Specifically, a urethane adhesive can be preferably exemplified. For example, rubber-based, cyan acrylate-based, and acrylic-based materials may show cytotoxicity, and a hot-melt system is not preferable because the dried vitrigel membrane may be thermally denatured. In addition, as a method of bonding and fixing the dried vitrigel membrane and the chamber body, a method of bonding and fixing a double-sided tape between the dried vitrigel membrane and the chamber body, a heat sealer, a hot plate, Examples thereof include a method of thermally welding the dried vitrigel membrane and the chamber body using sound waves, lasers, and the like.

  Furthermore, the dried vitrigel membrane bonded and fixed to one end surface of the chamber body can be cut into substantially the same shape as the end surface of the chamber body. Since the dried vitrigel membrane layered with the film is easy to cut, an excess portion protruding from the open end surface of the chamber body can be cut. Also in this case, after cutting the dried vitrigel membrane, the film can be peeled off and removed. As a result, the dried vitrigel membrane can be disposed on the open end surface of the chamber body.

  And a support body is arrange | positioned in the other surface side (lower surface side in FIG. 1) of the dried vitrigel membrane.

  When the support is disposed on the dried vitrigel membrane, for example, an aqueous solution is supplied into the chamber body to rehydrate the vitrigel membrane to form a vitrigel membrane, and the vitrigel membrane and the support are brought into contact with each other. Thereafter, by drying, the support can be bonded to the dried vitrigel membrane. In addition to this, as a method for producing a cell culture chamber, a support is first bonded to a dried vitrigel membrane, and then an adhesive is applied to one open end surface of the chamber body, and the support is bonded to the vitrigel membrane. One surface of the dry body can be brought into contact with and fixed by adhesion.

  The form and production method of the cell culture chamber of the present invention are not limited to the above forms, and can be appropriately designed in consideration of, for example, Patent Documents 5 and 6.

  Hereinafter, the cell culture chamber, the method for producing the cell culture chamber, and the cell culture method of the present invention will be described in more detail together with examples, but the present invention is not limited to the following examples.

<Example 1> Examination of silicon-treated PET thin film (Si-PET) (1) According to the method described in Patent Document 6, a vitrigel membrane dry body is disposed on the open end surface of a cylindrical chamber body, and a cell culture chamber Was made.

Furthermore, PBS was supplied to the chamber body of the cell culture chamber prepared by the same method, and the dried vitrigel membrane was rehydrated for 10 minutes to convert it to a vitrigel membrane, and the PBS used for rehydration was removed. . A silicon-treated PET thin film (Si-PET) with the silicon (Si) treated surface exposed on the petri dish is placed on the petri dish, and a cell culture chamber is placed on top of it to prevent air from entering the vitrigel membrane. Abutted. Thereafter, the Vitrigel membrane is dried in a clean bench overnight to dry the Vitrigel membrane, and Si-PET, which is a thin support (hereinafter referred to as a support thin film), is disposed on the dried Vitrigel membrane. A cell culture chamber was prepared (FIG. 2).
(2) Next, the effect of arranging Si-PET was examined. Specifically, PBS 1.0mL was added to the chamber body of a cell culture chamber with Si-PET (thickness: 25μm, 50μm, 75μm, 100μm), and slacked over time while suspended in a 12-well plate. Inspected. As a comparison, the slackness of a conventional cell culture chamber in which no supporting thin film was disposed was also examined under the same conditions.

  The results are shown in FIG.

  As shown in FIG. 3, in the case of a conventional cell culture chamber without a supporting thin film, it was confirmed that the Vitrigel membrane was slightly loosened with time (0.8 mm (day 0). ) → 1.0mm (7th day)). On the other hand, in the cell culture chamber provided with Si-PET, no slack was observed in any case. It was confirmed that the loosening of the vitrigel membrane was suppressed by disposing a support thin film that supports the vitrigel membrane of the cell culture chamber from below.

<Example 2> Examination of other supporting thin films In the same manner as in Example 1, PET (thickness 75 μm), polyethylene (thickness 80 μm), polycarbonate (thickness 1000 μm), polystyrene (thickness 400 μm), PTFE ( Regarding cell culture chambers using 100 μm, 300 μm, and 1000 μm in thickness and silicon rubber (thickness of 100 μm, 500 μm, and 1000 μm) as supporting thin films, the loosening of the vitrigel membrane was examined.

  The results are shown in FIG.

  As shown in FIG. 4, PET (thickness 75 μm), polyethylene (thickness 80 μm), polycarbonate (thickness 1000 μm), polystyrene (thickness 400 μm), silicon rubber (thickness 100 μm, 500 μm, 1000 μm) No loosening of the vitrigel membrane was observed. On the other hand, with respect to PTFE (thickness 100 μm, 300 μm, 1000 μm), the PTFE sometimes peeled off as time passed.

<Example 3> Study on dispersion of fine particles The cell culture chambers produced in Examples 1 and 2 are suspended in a 12-well plate, and spherical cellulose fine particles having a particle size of 44 to 105 μm (Kanto) Chemical Co., Ltd .: Cellulofine KANTO GH-25) was added, and the state of dispersion was observed.

  The results are shown in FIG.

  As shown in FIG. 5, in the case of a conventional cell culture chamber without a supporting thin film, it was confirmed that spherical cellulose fine particles gather at the center of the chamber due to the vitrigel membrane slackened downward. Therefore, it can be seen that when cells are cultured, an agglomerate may be formed at the center, and a uniform cell layer may not be formed.

  On the other hand, Si-PET (thickness: 25 μm, 50 μm, 75 μm, 100 μm), PET (thickness 75 μm), polyethylene (thickness 80 μm), polycarbonate (thickness 1000 μm), polystyrene (thickness 400 μm), PTFE (thickness) 100μm, 300μm, 1000μm) and silicon rubber (thickness 100μm, 500μm, 1000μm) are confirmed to be uniformly dispersed on the vitrigel membrane because the loosening of the vitrigel membrane is suppressed. It was done.

<Example 4> Measurement of contact angle The contact angle of the support thin film in the cell culture chamber was measured. Specifically, each supporting thin film was cut to a diameter of 18 mm, 50 μL of MQ water was dropped thereon, the droplet was photographed from the side, and the contact angle was measured.

  As a result, Si-PET (thickness 25 μm) is 97 °, Si-PET (thickness 50 μm) is 100 °, Si-PET (thickness 75 μm) is 100 °, and Si-PET (thickness 100 μm) is 98 °. Met.

  Furthermore, PET (thickness 75 μm) is 78 °, polyethylene (thickness 80 μm) is 91 °, polycarbonate (thickness 1000 μm) is 78 °, polystyrene (thickness 400 μm) is 80 °, PTFE (thickness 100 μm) is 105 °, PTFE (thickness 300μm) 98 °, PTFE (thickness 1000μm) 110 °, silicone rubber (thickness 100μm) 99 °, silicone rubber (thickness 500μm) 102 °, silicone rubber (thickness 1000μm) ) Was 100 °.

<Example 5> Measurement of tensile strength About the cell culture chamber of the present invention prepared in Examples 1 and 2, a commercially available tensile strength measuring instrument (manufactured by Nidec Sympo Co., Ltd .: with the chamber body sandwiched between clips). The strength when peeling the supporting thin film was measured by hooking it on the hook of the digital force gauge FGPX-0.2). For cell culture chambers, both dried Vitrigel membranes were rehydrated with PBS and converted to Vitrigel membranes (hydrated (+)) and those that were not rehydrated (hydrated (-)) It was used.

  The results are shown in Table 1.

  As shown in Table 1, it was confirmed that polycarbonate (thickness 1000 μm) and polystyrene (thickness 400 μm) had high tensile strength and were strongly adhered to the vitrigel film. Since the support thin film is peeled off after the cells adhere, the polycarbonate (thickness 1000 μm) and polystyrene (thickness 400 μm) are careful not to damage or stretch the vitrigel membrane when removing the support thin film. It is thought that it is necessary to do.

  On the other hand, other supporting thin films (Si-PET (thickness: 25 μm, 50 μm, 75 μm, 100 μm), PET (thickness 75 μm), polyethylene (thickness 80 μm), PTFE (thickness 100 μm, 300 μm, 1000 μm) silicon rubber (Thickness 100 μm, 500 μm, 1000 μm)), it was confirmed that the tensile strength was not high and the thin film could be easily removed.

<Example 6> Cell culture using cell culture chamber The cell culture chamber in which Si-PET (thickness: 75 μm) as a support thin film prepared in Example 1 was placed in a 10 cm dish. And 200 μL of the culture solution was added to the chamber body, and the state was maintained until just before seeding of the cells.

Then, immediately before the seeding of the cells, the culture medium in the chamber body was removed, and HepG2 cells were seeded at a cell density of 5.0 × 10 ⁴ cells / cm ² (culture day 0). This was cultured statically in a CO ₂ incubator (37 ° C., 5% CO ₂ /95% air).

  Next, in order to start liquid phase-liquid phase interface culture or liquid phase-gas phase interface culture, the culture solution or PBS is poured into the chamber body or on Si-PET on the second day of culture, and Si-PET is lightly added. When we tried to remove the Si-PET by moving it, the Si-PET could be peeled off easily.

  The state of HepG2 cells on the second day of culture is shown in FIG.

  Microscopic observation confirmed that HepG2 cells were uniformly dispersed and cultured on the Vitrigel membrane without forming aggregates, as shown in FIG.

DESCRIPTION OF SYMBOLS 1 Cell culture chamber 2 Chamber main body 3 Vitrigel membrane dry body 4 Support body

Claims (6)

A cell culture chamber comprising a cylindrical chamber body, a dried vitrigel membrane, and a support,
The chamber body has open end faces at opposite ends, and one of the open end faces is fixed in a state in which one surface side of the dried vitrigel membrane is in contact, and A cell culture chamber, wherein the support is disposed on the other surface side of the dried vitrigel membrane.   The cell culture chamber according to claim 1, wherein the support is a plastic thin film.   The cell culture chamber according to claim 1, wherein the support is a silicon-treated PET thin film. The following steps:
A step of abutting and fixing one surface side of the dried vitrigel membrane to one open end surface of a cylindrical chamber body having opposite open end surfaces formed at both ends; and covering the open end surface; and A method for producing a cell culture chamber, comprising a step of disposing a support on the other surface side of the dried vitrigel membrane. The following steps:
Supplying one or more types of cells into the inside of the chamber body of the cell culture chamber of any one of claims 1 to 3;
Culturing cells on the vitrigel membrane in a state where the vitrigel membrane obtained by rehydrating the dried vitrigel membrane is supported on the support; and removing the support from the vitrigel membrane. A cell culture method characterized. A cell culture kit comprising a cylindrical chamber body, a dried vitrigel membrane, and a support,
The chamber body has an open end face where both opposite end faces are open, and one of the open end faces is fixed in a state where one face side of the dried vitrigel membrane is in contact with the end face, A cell culture kit, wherein the support is disposed on the other surface side of the dried vitrigel membrane.