Patent Application
20150247118
The present disclosure relates to a method for producing a three-dimensional cell culture construct.
BACKGROUND ART
In recent years, regenerative medicine has attracted considerable attention as new medical care, and various methods have been proposed to construct a three-dimensional tissue of cells (e.g., Patent Documents 1 and 2). Patent Document 1 discloses a technology including the steps of (a) forming a cell layer; (b) bringing the cell layer into contact with a solution containing a first material and a solution containing a second material alternately; (c) repeating the step (a) and the step (b); and (d) laminating the cell layers via an extracellular matrix. Patent Document 2 discloses a technology including coating the entire surface of cultured cells with an adhesive film, and culturing the coated cells with the adjacent coated cells being bound together.
Patent Document 1: JP 2007-228921 A
Patent Document 2: JP 2012-115254 A
In the field of tissue engineering and regenerative medicine, the construction and application of a three-dimensional cell culture construct have been expected. Therefore, the present disclosure provides a method for producing a three-dimensional cell culture construct, which allows a three-dimensional cell culture construct with high activity to be produced.
In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; culturing the seeded coated cells in a culture medium; and using at least a portion of the culture medium continuously for 5 days or more.
In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; and culturing the seeded coated cells in a culture medium. The culture process of the coated cells includes culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells.
The present disclosure can provide a three-dimensional cell culture construct with high activity.
The present disclosure is based on the findings that coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component are cultured using at least a portion of a culture medium continuously for 5 days or more and/or cultured in 70 μL or more of a culture medium per 1×104 cells, thereby producing a three-dimensional cell culture construct with high activity.
In the present disclosure, the details of the mechanism of formation of a three-dimensional cell culture construct with high activity is unclear, but can be estimated as follows. Cells and a three-dimensional cell culture construct are generally produced by replacing a culture medium every day or two. It is reported that when a three-dimensional cell culture construct is produced by culturing coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component, the number of layers of the three-dimensional cell culture construct obtained is larger in the case where the culture medium is replaced after the culture for 12 hours than in the case where the culture medium is replaced after the culture for 24 hours (e.g., Adv. Mater. 2011, 23, 3506-3510). Liquid factors such as cytokine generated by the coated cells themselves are accumulated in the culture medium, in which the coated cells are being cultured. Replacing the culture medium removes the liquid factors. When the coated cells are cultured by using the culture medium continuously for at least 5 days without replacement, the liquid factors are accumulated in the culture medium. Then, the coated cells are cultured while receiving the liquid factors accumulated in the culture medium, and thus a three-dimensional cell culture construct with higher activity may be provided. Moreover, the volume of the culture medium is 70 μL or more per 1×104 cells. This can reduce damage to the three-dimensional cell culture construct due to waste products accumulated in the culture medium. Since the amount of the liquid factors accumulated in the culture medium is increased, the activity of the three-dimensional cell culture construct may be improved. However, the present disclosure is not limited to the above estimation.
In one or more embodiments, the “three-dimensional cell culture construct with high activity” in the present specification means that the cells constituting the three-dimensional cell culture construct have high activity, and/or that the three-dimensional cell culture construct is likely to maintain a three-dimensional form. In one or more embodiments, when the activity of the cells constituting the three-dimensional cell culture construct is high, e.g., the expression level of genes related to metabolism is high. In one or more embodiments, when the three-dimensional form of the three-dimensional cell culture construct is likely to be maintained, e.g., the thickness and/or the number of layers of the three-dimensional cell culture construct obtained is increased.
The “three-dimensional cell culture construct” in the present specification is the assembly of cells and an extracellular matrix, in which the cells are arranged in at least two layers via the extracellular matrix. The “three-dimensional cell culture construct including two or more laminated cell layers” in the present specification indicates that it is not a cell culture construct having a single cell layer. Moreover, the three-dimensional cell culture construct may include a structure of cells in which the cells adhere to each other and grow without being attached to a substrate. The three-dimensional cell culture construct may include one type or two or more types of cells.
The “coated cells” in the present specification include cells and a coating film containing an extracellular matrix component, in which the surface of the cells is coated with the coating film. In one or more non-limiting embodiments, the cells to be coated may be, e.g., cultured cells. The cultured cells are human cells or cells other than human cells. Examples of the cultured cells include primary cultured cells, subcultured cells, and cell line cells. In one or more embodiments, the cells may be adherent cells such as fibroblasts, cancer cells such as hepatoma cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, tissue stem cells, embryonic stem cells, and immune cells. The cells may be derived from human cells or cells other than human cells. The cells may be either one type or two or more types. In one or more embodiments, the coated cells can be prepared by a method disclosed in JP 2012-115254 A.
The “extracellular matrix component” in the present specification is a substance that is filled into a space outside of the cells in a living body and has the functions of serving as a framework, providing a scaffold, and/or holding biological factors. The extracellular matrix component may further include a substance that can have the functions of serving as a framework, providing a scaffold, and/or holding biological factors in in vitro cell culture.
In one or more embodiments, the “coating film containing an extracellular matrix component” in the present specification preferably includes a film containing a material A and a film containing a material B that interacts with the material A. In one or more embodiments, the combination of the material A and the material B may be (i) a combination of a protein or polymer having an RGD sequence (also referred to as a “material having an RGD sequence” in the following) and a protein or polymer that interacts with the protein or polymer having an RGD sequence (also referred to as an “interacting material” in the following) or (ii) a combination of a protein or polymer having a positive charge (also referred to as a “material having a positive charge” in the following) and a protein or polymer having a negative charge (also referred to as a “material having a negative charge” in the following).
[First Method for Producing Three-Dimensional Cell Culture Construct]
In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; culturing the seeded coated cells in a culture medium; and using at least a portion of the culture medium continuously for 5 days or more. This method is also referred to as a “first production method of the present disclosure” in the following. In one or more embodiments, the first production method of the present disclosure can provide a three-dimensional cell culture construct with high activity.
The first production method of the present disclosure includes culturing the coated cells by using at least a portion of the culture medium continuously for 5 days or more. In one or more embodiments, “culturing the coated cells by using at least a portion of the culture medium continuously for 5 days or more” in the present disclosure means that the coated cells are cultured while 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or substantially 100% of the culture medium, in which the coated cells are cultured, continues to be used for at least 5 days. The above culture process also includes culturing the coated cells without removing 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or substantially 100% of the culture medium, in which the coated cells are cultured, for at least 5 days. In one or more embodiments, the first production method of the present disclosure may include adding a new culture medium during the culture of the coated cells. The period in which at least a portion of the culture medium is continuously used is at least 5 days. In one or more embodiments, the period may be 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 15 days or more, 20 days or more, or 25 days or more. The upper limit of the period of continuous use is not particularly limited and may be appropriately determined, e.g., by the volume of the culture medium, the number of cells to be seeded, and the type of cells. In one or more embodiments, the upper limit may be 90 days or less, 60 days or less, or 40 days or less. In the first production method of the present disclosure, in one or more embodiments, the coated cells may be cultured without replacing the culture medium for at least 5 days, or the cycle of replacing the culture medium may be 5 days or more. In one or more embodiments, the first production method of the present disclosure may include replacing the culture medium for the first time (first culture medium replacement) after at least 5 days from the start of the culture, or replacing the culture medium for the second time after at least 6 days from the start of the culture. The “culture medium replacement” in the present disclosure means that approximately the total volume of the culture medium, in which the coated cells are cultured, is removed and replaced with a new culture medium.
In one or more embodiments, the first production method of the present disclosure may include replacing the culture medium after 16 to 36 hours from the seeding of the coated cells.
The volume of the culture medium during the culture is not particularly limited, but is preferably 70 μL or more, and more preferably 170 μL or more or 1 mL or more per 1×104 cells in terms of reducing the influence of waste products included in the culture medium during the culture. In one or more embodiments, the volume of the culture medium may be 100 mL or less or 10 mL or less per 1×104 cells.
In one or more embodiments, the first production method of the present disclosure includes using at least a portion of the culture medium continuously for 5 days or more, and culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells. In one or more embodiments, the first production method of the present disclosure includes culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells while at least a portion of the culture medium continues to be used.
The culture medium is not particularly limited and may be appropriately determined in accordance with the cells. Examples of the culture medium include Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RDMI, GlutaMAX medium, and serum-free medium.
In one or more embodiments, the coated cells may be seeded so that at least two layers of the coated cells are formed. In one or more embodiments, the density of the coated cells during seeding may be appropriately determined, e.g., by the thickness of an intended three-dimensional cell culture construct and/or the number of cells to be layered. In one or more embodiments, the density may be 1×102 cells/cm3 to 1×109 cells/cm3, 1×104 cells/cm3 to 1×108 cells/cm3, or 1×105 cells/cm3 to 1×107 cells/cm3.
In one or more embodiments, the incubation temperature may be 4 to 60° C., 20 to 40° C., or 30 to 37° C.
For ease of handling, it is preferable that the coated cells are cultured on a membrane filter. More preferably, the coated cells are cultured using a culture plate that includes a membrane filter. Further preferably, the coated cells are cultured using a culture plate that includes a housing portion and a base portion, in which the base portion serves as a membrane filter. The housing portion is preferably transparent. These culture plates may be commercial products. The commercial products include Transwell (registered trademark), Cell Culture Insert (trade name), etc.
The pore size of the membrane filter is not particularly limited as long as the cultured cells can remain on the membrane filter. In one or more embodiments, the pore size may be 0.1 μm to 2 μm or 0.4 μm to 1.0 μm. In one or more embodiments, the material of the membrane filter may be, e.g., polyethylene terephthalate (PET), polycarbonate, or polytetrafluoroethylene (PTFE).
In one or more embodiments, when the coated cells are cultured by placing an insert in a container such as a well, the ratio of the base area of the container holding the insert to the base area of the insert (base area of container/base area of insert) may be 7 or more, 30 or more, 100 or more, or 160 or more. Moreover, the ratio may be 16000 or less or 1600 or less.
[Second Method for Producing Three-Dimensional Cell Culture Construct]
In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; and culturing the seeded coated cells in a culture medium. The culture process of the coated cells includes culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells. This method is also referred to as a “second production method of the present disclosure” in the following.
In the second production method of the present disclosure, the volume of the culture medium during the culture is 70 μL or more, and preferably 170 μL or more or 1 mL or more per 1×104 cells in terms of reducing the influence of waste products included in the culture medium during the culture. Moreover, the volume of the culture medium may be 100 mL or less or 10 mL or less per 1×104 cells.
In one or more embodiments, the second production method of the present disclosure may include replacing the culture medium. The cycle of replacing the culture medium is not particularly limited. In one or more embodiments, the culture medium may be replaced every other day, every three days, every four days, or every five days. In one or more embodiments, the second production method of the present disclosure may include culturing the coated cells by using at least a portion of the culture medium continuously for 5 days or more because this can provide a three-dimensional cell culture construct having a large number of layers and/or a large thickness. In one or more embodiments, the period of continuous use may be 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 15 days or more, 20 days or more, or 25 days or more. Moreover, the period of continuous use may be 90 days or less, 60 days or less, or 40 days or less. In the second production method of the present disclosure, in one or more embodiments, the cycle of replacing the culture medium may be 5 days or more.
In one or more embodiments, the second production method of the present disclosure may include replacing the culture medium for the first time (first culture medium replacement) since the start of the culture and after 16 to 36 hours from the seeding of the coated cells.
The culture conditions such as the type of the culture medium and the incubation temperature in the second production method of the present disclosure are the same as those in the first production method of the present disclosure.
In the first and second production methods of the present disclosure, in one or more embodiments, the thickness of the coating film containing an extracellular matrix component is preferably 1 nm to 1×103 nm or 2 nm to 1×102 nm, and more preferably 3 nm to 1×102 nm because this can provide a three-dimensional cell culture construct in which the coated cells are more densely layered. The thickness of the coating film containing an extracellular matrix component can be appropriately controlled, e.g., by the number of films constituting the coating film. The coating film containing an extracellular matrix component is not particularly limited. In one or more embodiments, the coating film may be either a single layer or a multi-layer such as 3, 5, 7, 9, 11, 13, 15 layers or more. The thickness of the coating film may be determined by a method as described in the examples.
The first and second production methods of the present disclosure may further include preparing coated cells. The coated cells can be prepared by bringing cells into contact with a solution containing a material A and a solution containing a material B alternately. As described above, the combination of the material A and the material B may be a combination of the material having an RGD sequence and the interacting material or a combination of the material having a positive charge and the material having a negative charge.
(Material having RGD Sequence)
The material having an RGD sequence is a protein or polymer having an “Arg-Gly-Asp” (RGD) sequence, which is an amino acid sequence that is associated with cell adhesion activity. The material “having an RGD sequence” in the present specification may be a material that inherently has an RGD sequence or a material to which an RGD sequence is chemically bound. The material having an RGD sequence is preferably biodegradable.
In one or more embodiments, the protein having an RGD sequence may be, e.g., a conventionally known adhesive protein or a water-soluble protein having an RGD sequence. In one or more embodiments, examples of the adhesive protein include fibronectin, vitronectin, laminin, cadherin, and collagen. In one or more embodiments, examples of the water-soluble protein having an RGD sequence include collagen, gelatin, albumin, globulin, proteoglycan, enzymes, and antibodies, to each of which an RGD sequence is bound.
In one or more embodiments, the polymer having an RGD sequence may be, e.g., a naturally occurring polymer or a synthetic polymer. In one or more embodiments, examples of the naturally occurring polymer having an RGD sequence include water-soluble polypeptide, low molecular weight peptide, polyamino acid such as a-polylysine or ε-polylysine, and sugar such as chitin or chitosan. In one or more embodiments, examples of the synthetic polymer having an RGD sequence include straight-chain, graft, comb, dendritic, or star polymers or copolymers having an RGD sequence. In one or more embodiments, examples of the polymers or copolymers include the following: polyurethane, polycarbonate, polyamide, or copolymers thereof, polyester; poly(N-isopropylacrylamide-co-polyacrylic acid); polyamidoamine dendrimer; polyethylene oxide; poly(ε-caprolactam); polyacrylamide; and poly(methyl methacrylate-y-polyoxyethylene methacrylate).
Among them, the material having an RGD sequence is preferably fibronectin, vitronectin, laminin, cadherin, polylysine, elastin, collagen to which an RGD sequence is bound, gelatin to which an RGD sequence is bound, chitin, or chitosan. The material having an RGD sequence is more preferably fibronectin, vitronectin, laminin, polylysine, collagen to which an RGD sequence is bound, or gelatin to which an RGD sequence is bound.
(Interacting Material)
The interacting material is a protein or polymer that interacts with the material having an RGD sequence. In one or more embodiments, the term “interact” in the present specification means that the material having an RGD sequence and the interacting material approach each other to the extent that bonding, adhesion, adsorption, or electron transfer can occur chemically and/or physically between them, e.g., due to electrostatic interaction, hydrophobic interaction, hydrogen bond, charge transfer interaction, covalent bond formation, specific interaction between proteins, and/or Van der Waals force. The interacting material is preferably biodegradable.
In one or more embodiments, the protein that interacts with the material having an RGD sequence may be, e.g., collagen, gelatin, proteoglycan, integrin, enzymes, or antibodies. In one or more embodiments, the polymer that interacts with the material having an RGD sequence may be, e.g., a naturally occurring polymer or a synthetic polymer. In one or more embodiments, examples of the naturally occurring polymer that interacts with the material having an RGD sequence include water-soluble polypeptide, low molecular weight peptide, polyamino acid, elastin, sugar such as heparin, heparan sulfate, or dextran sulfate, and hyaluronic acid. In one or more embodiments, examples of the polyamino acid include polylysine such as α-polylysine or ε-polylysine, polyglutamic acid, and polyaspartic acid. In one or more embodiments, examples of the synthetic polymer that interacts with the material having an RGD sequence include straight-chain, graft, comb, dendritic, or star polymers or copolymers having an RGD sequence. In one or more embodiments, examples of the polymers or copolymers include the following: polyurethane, polyamide, polycarbonate, or copolymers thereof; polyester; polyacrylic acid; polymethacrylic acid; polyethylene glycol-graft-polyacrylic acid; poly(N-isopropylacrylamide-co-polyacrylic acid); polyamidoamine dendrimer; polyethylene oxide; poly(ε-caprolactam); polyacrylamide; and poly(methyl methacrylate-γ-polyoxyethylene methacrylate).
Among them, the interacting material is preferably gelatin, dextran sulfate, heparin, hyaluronic acid, globulin, albumin, polyglutamic acid, collagen, or elastin. The interacting material is more preferably gelatin, dextran sulfate, heparin, hyaluronic acid, or collagen. The interacting material is further preferably gelatin, dextran sulfate, heparin, or hyaluronic acid.
The combination of the material having an RGD sequence and the interacting material is not particularly limited and may be a combination of different materials that interact with each other. Specifically, one of the materials may be a polymer or protein having an RGD sequence, and the other may be a polymer or protein that reacts with the polymer or protein having an RGD sequence. In one or more embodiments, examples of the combination of the material having an RGD sequence and the interacting material include the following: fibronectin and gelatin; fibronectin and ε-polylysine; fibronectin and hyaluronic acid; fibronectin and dextran sulfate; fibronectin and heparin; fibronectin and collagen; laminin and gelatin; laminin and collagen; polylysine and elastin; vitronectin and collagen; and RGD-bound collagen or RGD-bound gelatin and collagen or gelatin. Among them, the combination is preferably fibronectin and gelatin, fibronectin and ε-polylysine, fibronectin and hyaluronic acid, fibronectin and dextran sulfate, fibronectin and heparin, or laminin and gelatin. The combination is more preferably fibronectin and gelatin. Each of the material having an RGD sequence and the interacting material may be either one type or two or more types as long as they interact with each other.
(Material Having Positive Charge)
The material having a positive charge is a protein or polymer having a positive charge. In one or more embodiments, the protein having a positive charge is preferably a water-soluble protein. In one or more embodiments, examples of the water-soluble protein include basic collagen, basic gelatin, lysozyme, cytochrome c, peroxidase, and myoglobin. In one or more embodiments, the polymer having a positive charge may be, e.g., a naturally occurring polymer or a synthetic polymer. In one or more embodiments, examples of the naturally occurring polymer include water-soluble polypeptide, low molecular weight peptide, polyamino acid, and sugar such as chitin or chitosan. In one or more embodiments, examples of the polyamino acid include polylysine such as poly(α-lysine) or poly(ε-lysine), polyarginine, and polyhistidine. In one ore more embodiments, examples of the synthetic polymer include straight-chain, graft, comb, dendritic, or star polymers or copolymers. In one or more embodiments, examples of the polymers or copolymers include the following: polyurethane, polyamide, polycarbonate, or copolymers thereof; polyester; polydiallyldimethylammonium chloride (PDDA); polyallylamine hydrochloride; polyethyleneimine; polyvinylamine; and polyamidoamine dendrimer.
(Material Having Negative Charge)
The material having a negative charge is a protein or polymer having a negative charge. In one or more embodiments, the protein having a negative charge is preferably a water-soluble protein. In one or more embodiments, examples of the water-soluble protein include acid collagen, acid gelatin, albumin, globulin, catalase, β-lactoglobulin, thyroglobulin, α-lactalbumin, and ovalbumin. The polymer having a negative charge may be, e.g., a naturally occurring polymer or a synthetic polymer. In one or more embodiments, examples of the naturally occurring polymer include water-soluble polypeptide, low molecular weight peptide, polyamino acid such as poly((β-lysine), and dextran sulfate. In one or more embodiments, examples of the synthetic polymer include straight-chain, graft, comb, dendritic, or star polymers or copolymers. In one or more embodiments, examples of the polymers or copolymers include the following: polyurethane, polyamide, polycarbonate, or copolymers thereof; polyester; polyacrylic acid; polymethacrylic acid; polystyrene sulfonic acid; polyacrylamide methylpropane sulfonic acid; carboxy-terminated polyethylene glycol; polydiallyldimethylammonium salt; polyallylamine salt; polyethyleneimine; polyvinylamine; and polyamidoamine dendrimer.
In one or more embodiments, examples of the combination of the material having a positive charge and the material having a negative charge include the following: ε-polylysine salt and polysulfonate; ε-polylysine and polysulfonate; chitosan and dextran sulfate; polyallylamine hydrochloride and polystyrene sulfonate; polydiallyldimethylammonium chloride and polystyrene sulfonate; and polydiallyldimethylammonium chloride and polyacrylate. The combination is preferably ε-polylysine salt and polysulfonate or polydiallyldimethylammonium chloride and polyacrylate. In one or more embodiments, the polysulfonate may be, e.g., poly(sodium sulfonate) (PSS). Each of the material having a positive charge and the material having a negative charge may be either one type or two or more types as long as they interact with each other.
Hereinafter, a preferred embodiment of a method for preparing coated cells will be described. In this embodiment, first, cells are brought into contact with a solution A containing a material having an RGD sequence, and then the cells are brought into contact with a solution B containing a material that interacts with the material having an RGD sequence, thereby preparing coated cells. However, the present disclosure should not be limited to the following embodiment.
First, cells are brought into contact with the solution A. Consequently, a film containing the material having an RGD sequence is formed on the surface of the cells, and thus the surface of the cells is coated with the film containing the material having an RGD sequence. In one or more embodiments, the contact between the cells and the solution A can be made, e.g., by applying or adding the solution A to the cells, immersing the cells in the solution A, or dropping or spraying the solution A on the cells. In particular, for ease of operation, it is preferable that the cells are brought into contact with the solution A by immersion in the solution A.
In one or more embodiments, the contact conditions may be appropriately determined, e.g., by the contact process, the type of the material having an RGD sequence and/or the type of cells, and the concentration of the solution. In one or more embodiments, the contact time is preferably 30 seconds to 24 hours, 1 minute to 60 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, or 1 minute to 5 minutes. In one or more embodiments, the ambient temperature and/or the temperature of the solution A during contact is preferably 4 to 60° C., 20 to 40° C., or 30 to 37° C.
The solution A may contain at least the material having an RGD sequence, and preferably contains the material having an RGD sequence and a solvent or a dispersion medium (also simply referred to as a “solvent” in the following). In one or more embodiments, the content of the material having an RGD sequence in the solution A is preferably 0.0001 to 1 mass %, 0.01 to 0.5 mass %, or 0.02 to 0.1 mass %. In one or more embodiments, the solvent may be, e.g., an aqueous solvent such as water, phosphate buffered saline (PBS), or buffer. In one or more embodiments, examples of the buffer include the following: Tris buffer such as Tris-HCl buffer; phosphate buffer; HEPES buffer; citrate-phosphate buffer; glycylglycine-sodium hydroxide buffer; Britton-Robinson buffer; and GTA buffer. The pH of the solvent is not particularly limited. In one or more embodiments, the pH is preferably 3 to 11, 6 to 8, or 7.2 to 7.4.
In one or more embodiments, the solution A may further contain salt, a cell growth factor, cytokine, chemokine, hormone, biologically active peptide, or a pharmaceutical composition. In one or more embodiments, examples of the pharmaceutical composition include a therapeutic agent for diseases, a preventive, an inhibitor, an antibacterial agent, and an anti-inflammatory agent. In one or more embodiments, examples of the salt include sodium chloride, calcium chloride, sodium hydrogencarbonate, sodium acetate, sodium citrate, potassium chloride, dibasic sodium phosphate, magnesium sulfate, and sodium succinate. The salt may be either one type or two or more types. Both the solution A and the solution B may contain the salt, or one of them may contain the salt. The salt concentration in the solution A is not particularly limited. In one or more embodiments, the salt concentration is, e.g., 1×10−6 M to 2 M, preferably 1×10−4M to 1 M, and more preferably 1×10−4M to 0.05 M.
Next, the material that has not been used for the formation of the film containing the material having an RGD sequence is separated. In one or more embodiments, the separation may be performed, e.g., by centrifugation or filtration. In one or more embodiments, when the material is separated by centrifugation, e.g., the solution A in which the cells are dispersed is centrifuged, and then the supernatant is removed. The centrifugation conditions may be appropriately determined, e.g., by the type of cells, the concentration of cells, and the composition of the materials contained in the solution A.
In addition to the above separation, a washing operation is preferably performed. In one or more embodiments, the washing operation may be performed, e.g., by centrifugation or filtration. In one or more embodiments, when the washing operation is performed by centrifugation, e.g., a solvent is added to the cells from which the supernatant has been removed, and this solution is centrifuged so that the supernatant is removed. It is preferable that the solvent used for washing is the same as that of the solution A.
Next, the cells coated with the film containing the material having an RGD sequence are brought into contact with the solution B. Consequently, a film containing the interacting material is formed on the surface of the film containing the material having an RGD sequence, and thus the surface of the cells, which has been coated with the film containing the material having an RGD sequence, is further coated with the film containing the interacting material. The contact between the cells and the solution B can be made in the same manner as the contact between the cells and the solution A except that the interacting material is used instead of the material having an RGD sequence.
By repeatedly bringing the cells into contact with the solution A and the solution B alternately, the film containing the material having an RGD sequence and the film containing the interacting material can be alternately laminated to form a coating film containing an extracellular matrix component on the entire surface of the cells. The number of times of the contact between the cells and the solution A or the solution B may be appropriately determined, e.g., by the thickness of the coating film containing an extracellular matrix component to be formed.
[Three-Dimensional Cell Culture Construct]
The present disclosure relates to a three-dimensional cell culture construct that includes cells arranged in at least two layers and an extracellular matrix component, and that is produced by the first or second production method of the present disclosure. In one or more embodiments, the three-dimensional cell culture construct of the present disclosure can have high activity. The cells and the extracellular matrix component of the three-dimensional cell culture construct of the present disclosure are as described above.
[Method for Culturing Coated Cells]
In one or more aspects, the present disclosure relates to a method for culturing coated cells that includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; culturing the seeded coated cells in a culture medium; and using at least a portion of the culture medium continuously for 5 days or more. In one or more aspects, the present disclosure relates to a method for culturing coated cells that includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; and culturing the seeded coated cells in a culture medium. The culture process of the coated cells includes culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells. The culture conditions or the like in the culture method of the coated cells of the present disclosure are as described above.
[Method for Culturing Three-Dimensional Cell Culture Construct]
In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; culturing the seeded coated cells in a culture medium; and using at least a portion of the culture medium continuously for 5 days or more. In one or more aspects, the present disclosure relates to a method for producing a three-dimensional cell culture construct including at least two or more laminated cell layers. The method includes the following: seeding coated cells in which a cell surface is coated with a coating film containing an extracellular matrix component; and culturing the seeded coated cells in a culture medium. The culture process of the coated cells includes culturing the coated cells in 70 μL or more of the culture medium per 1×104 cells. The culture conditions or the like in the culture method of the three-dimensional cell culture construct of the present disclosure are as described above.
Hereinafter, the present disclosure will be described in more detail by way of examples and comparative examples. However, the present disclosure is not limited to the following examples.
[Preparation of Coated Cells]
HepG2 cells were collected from a petri dish by a trypsin treatment. The collected cells were dispersed at a concentration of 1×107 cell/ml in a 50 mM Tris-HCl solution (pH=7.4) containing 0.2 mg/ml fibronectin. The dispersion state was maintained for 1 minute while this solution was gently stirred by inverting the container. Then, the solution was centrifuged at 2500 rpm for 1 minute (FN immersion operation). After the supernatant was removed, a 50 mM Tris-HCl solution (pH=7.4) was added so that the cells were dispersed. The dispersion state was maintained for 1 minute while this solution was gently stirred by inverting the container. Then, the solution was centrifuged at 2500 rpm for 1 minute (washing operation). After the supernatant was removed, the cells were dispersed in a 50 mM Tris-HCl solution (pH=7.4) containing 0.2 mg/ml gelatin. The dispersion state was maintained for 1 minute while this solution was gently stirred by inverting the container. Then, the solution was centrifuged at 2500 rpm for 1 minute (G immersion operation). Subsequently, the washing operation was performed. The FN immersion operation, the washing operation, the G immersion operation, and the washing operation were performed in this order. In this case, a set of the FN immersion operation and the washing operation was defmed as one step, and another set of the G immersion operation and the washing operation was defmed as one step. Finally, a total of 9 steps, i.e., 5 times of the FN immersion operation and 4 times of the G immersion operation were performed, so that FN-G coated cells were prepared (the thickness of the coating film containing an extracellular matrix component: 8 nm).
[Evaluation of Culture Medium Consumption/Analysis of Gene Expression Level]
First, 7×105 HepG2 coated cells were seeded on a membrane filter of Transwell (manufactured by Corning Incorporated; pore size: 0.4 μm, surface area: 0.33 cm2) placed on a 24-well culture plate. Then, 2.5 mL of Eagle's MEM medium containing 10 wt % fetal bovine serum was added, and the coated cells were cultured at 37° C. for 1 day. The Transwell in which the coated cells had been seeded was placed on a 6-well culture plate, and 12 mL of Eagle's MEM medium containing 10 wt % fetal bovine serum was added. The coated cells were cultured at 37° C. for 8 days without replacing the culture medium, and thus a three-dimensional cell culture construct was produced (the volume of the culture medium during the culture: 170 μL per 1×104 cells).
A three-dimensional cell culture construct was produced in the same manner as Example 1 except that the coated cells were cultured while the culture medium was replaced every three days (i.e., the period in which the culture medium was not replaced was 2 days) after the Transwell was placed on the 6-well culture plate. In each replacement, almost all of the culture medium included in the well was removed and replaced with a new culture medium (the volume of the culture medium during the culture: 170 μL per 1×104 cells).
A three-dimensional cell culture construct was produced in the same manner as Example 1 except that the coated cells were cultured with the Transwell being placed on the 24-well culture plate while the culture medium was replaced after 1 day from the seeding of the coated cells, and then the culture medium was replaced every three days. In each replacement, almost all of the culture medium included in the well was removed and replaced with a new culture medium (the volume of the culture medium during the culture: 36 μL per 1×104 cells).
Evaluation of Culture Medium Consumption
The absorbances (560 nm) of the solution in the inside and the outside of the insert were measured before the replacement of the culture medium.
Analysis of Gene Expression Level
RNA was extracted from each of the samples (three-dimensional cell culture constructs) after the culture for 9 days, and the gene expression levels of CYP1A1, CYP1A2, ALB (albumin), and CYP3A4 were measured by real-time PCR.
[Relationship between Volume of Culture Medium and Gene Expression Level]
In Example 3, the coated cells were cultured under the same conditions as Example 1 except that a 100 mm dish was used instead of the 6-well culture plate (the volume of the culture medium during the culture: 1 mL per 1×104 cells). In Example 4, the coated cells were cultured under the same conditions as Example 1. In Example 5, the coated cells were cultured under the same conditions as Example 1 except that a 24-well culture plate was used instead of the 6-well culture plate (the volume of the culture medium during the culture: 36 μL per 1×104 cells). The gene expression levels of the three-dimensional cell culture constructs in Examples 3 to 5 were evaluated.
As shown in
[Relationship between Number of Cells and Gene Expression Level]
In Example 6, the coated cells were cultured under the same conditions as Example 1. In Reference Example, the coated cells were cultured under the same conditions as Example 1 except that 7×105 cells were directly seeded in a well without using an insert. The gene expression levels of the three-dimensional cell culture construct in Example 6 and the cells in Reference Example were evaluated.
As shown in
[Evaluation of Three-Dimensional Cell Culture Construct]
The three-dimensional cell culture constructs produced in Examples 3 to 5 were evaluated. As shown in
[Relationship between Period of Continuous Use of Culture Medium and Gene Expression Level]
In Example 7, the coated cells were cultured for 27 days under the same conditions (the volume of the culture medium during the culture: 1 mL per 1×104 cells, the culture medium was not replaced from the second day of the culture) as Example 3. With respect to the samples on the 9th day, the 18th day, and the 27th day of the culture, the gene expression levels of CYP1A1 and ALB were measured by real-time PCR.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2012-203154 | Sep 2012 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2013/074743 | 9/12/2013 | WO | 00 |
