JP6599310B2 - Quality evaluation method for sheet cell culture - Google Patents

Description

  The present invention relates to a method, kit, system, and the like for quality evaluation of sheet-shaped cell culture or determination of completion of sheeting.

  In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. have been tried to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. (Non-Patent Document 1).

As part of such attempts, cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed (Patent Document 1).
For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies such as the use of cell cultures are ongoing.

  When such cell cultures are applied clinically, in order to ensure their applicability, effectiveness, safety, storage stability, transportability, etc., setting of indicators to optimize the manufacturing method and quality control As part of this, it is necessary to evaluate the quality of the sheet-like cell culture such as strength. Regarding the evaluation of the strength of the sheet-shaped cell culture, Patent Document 1 describes that the strength was estimated qualitatively and empirically from the ease of desorption of the cell culture from the culture substrate. . Patent Document 2 discloses a method in which a sheet-shaped cell culture is subjected to a dissociation treatment, and the strength of the sheet-shaped cell culture is measured from the time of dissociation treatment and the dissociation state. A method for determining the quality of a sheet-shaped cell culture based on the roundness of the outline of the sheet-shaped cell culture is disclosed in Patent Document 4, which measures the concentration of non-adherent cells in a liquid medium in sheet culture or a change thereof. The method for determining the sheet-like state of the sheet-shaped cell culture by calculating the adhesion rate between the sheet-forming cells and the culture container in Patent Document 5, and forming the sheet-shaped cell culture based on this Patent Document 6 discloses a determination method in which a fragile sample such as a sheet-shaped cell culture is fixed to a fixed surface connected to a load measuring device in a state where the sample is located in a liquid or a liquid surface. Measure tensile properties In Patent Document 7, a method of forming a sheet containing magnetic particles at least in part and applying a tensile load by applying a magnetic force to the sheet to evaluate the tensile properties of the sheet, Each is listed.

Special table 2007-528755 gazette JP 2012-029598 A JP 2012-039944 A JP 2012-152188 A JP2012-152189A JP 2012-159408 A JP 2013-198438 A

Haraguchi et al., Stem Cells Transl Med. 2012 Feb; 1 (2): 136-41

  In addition to the strength of the sheet-like cell culture, the present invention includes cell density, presence or absence of minute tears or holes that cannot be detected with the naked eye, adhesion between cells, presence or absence of peeling from the culture substrate during sheeting, The purpose is to provide a method for evaluating the viability of cells, the vitality of cells, a method for determining the completion of sheeting of a sheet-shaped cell culture, a method for detecting abnormal sheeting of a sheet-shaped cell culture, etc. To do.

  While studying the quality evaluation method of the sheet-shaped cell culture, the present inventor, in addition to the strength of the sheet-shaped cell culture, the uniformity of the cell density in the sheet-shaped cell culture, and the sheet-shaped cell culture Compatibility with transplantation includes micro-breaks and holes that cannot be detected with the naked eye, adhesion between cells, separation from culture substrate during sheeting, cell viability, and cell vitality. As a result of diligent research to develop a method for evaluating these items, the cells cannot pass cells but have a semi-permeable part that allows molecules of a given size to pass through. The sheet-like cell culture is formed so as to cover the semipermeable portion, the semipermeable portion of the culture vessel is accommodated in a liquid-impermeable recovery vessel, and a label is added to the culture vessel. Left for a predetermined period , By detecting the amount of label has moved to the collection container from the culture vessel, it found that it is possible to evaluate the above items at a time. As a result of further research, the amount of label moving to the collection container decreased with the progress of sheeting, and when the sheeting was completed, the amount of label moving to the collection container became constant. Completed the invention.

That is, the present invention relates to the following.
<1> (1) a step of seeding cells in a first container having a semipermeable portion that passes through a label but does not pass cells, and forms a sheet so as to cover the semipermeable portion;
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) Simultaneously with step (1), or after step (1), quality evaluation of sheet-shaped cell culture, determination of completion of sheet formation, evaluation of cell sheet-forming ability, or sheet-shaped cell culture Method for detection of sheet peeling in the manufacturing process.

<2> (1) a step of seeding cells in a first container having a semipermeable portion that passes through the label but does not pass cells, and cultivating the sheet so as to cover the semipermeable portion;
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
(5) Sheet culture when the amount or increase of the label becomes almost constant in a plurality of consecutive measurements, or when the absolute value of the rate of change over time in the amount of label is below the reference value And the step of recovering the sheet-shaped cell culture from the first container, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes steps (1) and A method for producing a sheet-like cell culture for transplantation, which may be performed before step (1) or after step (1) at the same time.

<3> The method according to <1> or <2>, wherein the label has a molecular weight of 100 to 150,000.
<4> The method according to any one of <1> to <3>, wherein the label is selected from the group consisting of a fluorescent label, an enzyme label, a luminescent label, a radioactive label, a magnetic label, and a colorimetric label.
<5> The method according to any one of <1> to <4>, wherein the semipermeable portion has pores having a pore diameter of about 0.01 μm to about 50.0 μm.
<6> The quality of the sheet-shaped cell culture is selected from the group consisting of the strength of the sheet-shaped cell culture, the degree of sheeting, the cell density, the viability of the cells, the adhesion state between the cells, and the vitality of the cells. The method according to any one of <1> to <5>.
<7> The method according to any one of <1> to <6>, wherein the cells are selected from the group consisting of myoblasts and mesenchymal stem cells.

<8> (1) A label that inhibits passage between cells by adhesion between cells,
(2) a first container having a semi-permeable portion through which the label passes but cells do not pass, and (3) a liquid-impermeable second container capable of containing the semi-permeable portion. For production of sheet cell culture, quality evaluation of sheet cell culture, determination of completion of sheet formation, evaluation of cell sheet forming ability, and / or detection of detachment in the production process of sheet cell culture kit.
<9> (1) A label that inhibits passage between cells by adhesion between cells,
(2) a first container having a semipermeable portion through which the label passes but cells do not pass;
(3) Production of sheet-shaped cell culture, sheet-shaped cell culture, comprising a liquid-impermeable second container capable of accommodating the semi-permeable portion, and (4) a detector for detecting a label A system for quality evaluation, determination of completion of sheet formation of a sheet-shaped cell culture, evaluation of sheet-forming ability of cells, and / or detection of detachment in the manufacturing process of a sheet-shaped cell culture.

  According to the quality evaluation method of the present invention, not only the strength of the sheet-shaped cell culture but also the presence or absence of minute tears or holes that cannot be detected with the naked eye, the adhesion state between cells, and from the culture substrate during the sheeting process It is possible to evaluate the quality such as detachment, cell viability, and cell vitality at the same time, and without damaging the sheet cell culture. it can. In addition, cells are cultured until cell adhesion is completed, but the donor cells have the time until sheet formation is completed and a sheet-shaped cell culture suitable for transplantation is completed. Depending on the nature, it was impossible to detect completion of sheeting in the past, but the sheeting completion determination method of the present invention makes it possible to accurately detect completion of sheeting and has a high therapeutic effect suitable for transplantation. Sheet cell cultures can be reliably produced without time loss. Furthermore, according to the sheeting abnormality detection method of the present invention, it is possible to quickly detect an abnormality that has occurred in the sheeting process, and it is possible to prevent time loss by quickly taking necessary measures.

FIG. 1 is a diagram showing an algorithm (algorithm 1) relating to a specific embodiment of the method for producing a sheet-shaped cell culture according to the present invention. FIG. 2 is a diagram showing a modification of algorithm 1. In FIG. FIG. 3 is a diagram showing an algorithm (algorithm 2) relating to a specific embodiment of the method for producing a sheet-shaped cell culture according to the present invention. FIG. 4 is a diagram showing an algorithm (algorithm 3) relating to a specific embodiment of the method for producing a sheet-shaped cell culture according to the present invention. FIG. 5 is a graph showing that the amount of label transferred to the second container changes depending on the cell density. FIG. 6 is a graph showing that the amount of the label transferred to the second container changes according to the survival rate of the cells constituting the sheet-shaped cell culture. FIG. 7 is a graph showing the transition of the amount of label transferred to the second container as the sheet-shaped cell culture is made into a sheet. FIG. 8 is a graph showing that the amount of label transferred to the second container changes due to abnormal sheet formation. FIG. 9 is a graph showing that the amount of label transferred to the second container changes depending on the difference in cell purity.

  Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety.

One aspect of the present invention is as follows: (1) seeding cells in a first container having a semi-permeable portion through which a label passes but cells do not pass, and forming the sheet so as to cover the semi-permeable portion. ,
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) At the same time, it relates to a method for evaluating the quality of a sheet-shaped cell culture (hereinafter sometimes abbreviated as “quality evaluation method”), which may be performed before step (1) or after step (1).

  In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other by intercellular adhesion or the like, and is typically composed of one cell layer. In addition, those composed of a stack of cell layers are also included. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly derived from cells constituting the sheet-shaped cell culture. The cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected.

  The cells that can form a sheet-like cell culture are not limited and include, for example, adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal cells, gingival cells, periosteum cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) Etc. Somatic cells may be differentiated from stem cells, particularly iPS cells. Non-limiting examples of cells that can form a sheet-like cell culture include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots) , Muscle tissue, endometrium, placenta, cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelium) Cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, liver parenchymal cells), pancreatic cells (eg, islet cells), kidney cells, adrenal glands Examples include cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells and the like.

  The label used in the present invention includes any label in which passage between cells is inhibited by cell-cell adhesion. In a sheet-like cell culture, cells are connected to each other by cell-cell adhesion, and the cell gap is narrow. Therefore, if the molecule is small in size, the sheet is passed through the cell gap (ie, the cell-cell pathway). Can pass freely through cell-shaped cell cultures, but when a certain size is exceeded, cell-cell adhesion devices (eg, tight junctions, adhesive junctions, desmosome junctions, gap junctions, etc.) responsible for cell-cell adhesion The passage of is now inhibited. On the other hand, if the cell gap in the sheet-shaped cell culture is expanded for some reason, the passage of molecules that are normally inhibited from passing between the cells becomes easy, and the sheet-shaped cell culture can be removed within a predetermined time. The amount of molecules passing through increases. Therefore, by using a label that inhibits passage between cells due to cell-cell adhesion, the amount of label that passes through the cell gap of the sheet-like cell culture can be used as an index of the size of the cell gap.

  A non-limiting example of a label that inhibits passage between cells by intercellular adhesion is, for example, a label having a molecular weight in a predetermined range. The molecular weight range is not limited, and is, for example, about 100 to about 1500,000, about 300 to about 1,000,000, about 1000 to about 500,000, about 5,000 to about 200,000, about 10,000 to about 100,000, about 30,000 to about 90,000, Examples include about 40,000 to about 80,000. The label may also have a positive charge or a negative charge. Non-limiting examples of such labels include, for example, fluorescein sodium (Na-F).

  Whether or not a certain label is one that inhibits passage between cells due to cell-cell adhesion can be determined, for example, as follows. A sheet cell culture is usually formed by seeding cells that can form a sheet cell culture in a culture vessel and allowing the cells to interact with each other for a predetermined period of time (this specification) In this document, this process is sometimes referred to as “sheeting”), and as shown in the results of Example 5, the cell gap at the time of seeding is gradually reduced by cell-to-cell adhesion in this sheeting process. However, it is considered that the sheet is stabilized at a certain size by completing the sheet formation. Therefore, if the amount of label that passes through the sheet-shaped cell culture that has been formed into a sheet is less than the amount of label that passes through the sheet-shaped cell culture that is not sufficiently sheeted, the label will adhere between cells by cell-cell adhesion. It can be said that the passage of is inhibited. Moreover, as the result of Example 4 shows, when the viability of the cell which comprises this sheet-like cell culture falls, the connection between cells will become weak and a cell gap | interval will be expanded. Thus, if the amount of label that passes through the sheet cell culture that does not reduce cell viability is less than the amount of label that passes through the sheet cell culture with reduced cell viability, then the label is It can be said that cell-cell adhesion is inhibited by cell-cell adhesion.

  The label used in the present invention includes any one that can be quantified by a known measuring method, and non-limiting examples include, for example, fluorescent labels, enzyme labels, luminescent labels, radioactive labels, magnetic labels, and colorimetric labels. It is done. The label may be a known detectable labeling substance, such as a fluorescent substance, an enzyme, a luminescent substance, a radioisotope, a magnetic particle, a dye, etc., and the labeling substance may be combined with another substance (for example, a carrier such as dextran). It may be a labeling substance that binds and inhibits passage between cells by cell-cell adhesion. In addition, substances that absorb electromagnetic waves of a specific wavelength, such as nucleic acids and proteins, antibodies and antigen-binding fragments thereof, antigens recognized by antibodies (eg, DIG), solubilized receptors, ligands for receptors, lectins, Sugar chains recognized by lectins, biotin and derivatives thereof, avidin and derivatives thereof, and the like may be used as labels. When using antibodies, antigen-binding fragments, solubilized receptors, lectins, biotin and their derivatives as labels, detect them with antigens, ligands, sugar chains, avidin and their derivatives bound to the labeling substances. When an antigen recognized by an antibody, a ligand for a receptor, a sugar chain recognized by a lectin, avidin or a derivative thereof is used as a label, the antibody bound to the labeling substance or its antigen-binding fragment, solubilized receptor Body, lectin, biotin and derivatives thereof.

  Further examples of the label used in the present invention include substances that color or emit light by an enzymatic reaction.

Examples of fluorescent substances that can be used for labeling include the Cy ^™ series (eg, Cy ^™ 2, 3, 5, 5.5, 7, etc.), the DyLight ^™ series (eg, DyLight ^™ 405, 488, 549, 594). 633, 649, 680, 750, 800, etc.), Alexa Fluor ^(R) series (eg Alexa Fluor ^(R) 405, 488, 549, 594, 633, 647, 680, 750 etc.), HiLyte Fluor ^TM series ( For example, HiLyte Fluor ^™ 488, 555, 647, 680, 750, etc.), ATTO series (eg, ATTO 488, 550, 633, 647N, 655, 740, etc.), FAM, FITC, Texas Red, Rhodamine, GFP, RFP, Qdot , Lucifer Yellow, Fluorescein, DRAQ7 ^™, etc., as enzymes, HRP, β-galactosidase, etc., as luminescent substances, luminol, luciferin, lucigenin, aequorin, etc., as radioisotopes, technetium-99m ( ^99m Tc), indium ^{-111 (111} In), iodine -123 ⁽¹²³ I), iodine -124 ⁽¹²⁴ I), iodine -125 ⁽¹²⁵ I), iodine--131 ⁽¹³¹ I), thallium ^{-201 (201} Tl ), carbon ^{-11 (11} C), nitrogen ^{-13 (13} N), oxygen ^{-15 (15} O), fluorine ^{-18 (18} F), copper ^-64 (64 Cu), gallium ^-67 (67 Ga), krypton ^{-81m (81m} Kr), xenon ^{-133 (133} Xe), strontium ^-89 (89 Sr), yttrium -90 ⁽⁹⁰ Y), etc., as the dye, Nile Blue, neutral red, janus green, calcein, propidium Examples thereof include dyes for vital dyes such as iodide.

  Examples of further fluorescent substances include fluorescent proteins such as YFP, CFP, OFP. Examples of further enzymes include luciferase, alkaline phosphatase, glucuronidase, peroxidase and the like. Examples of the substance that colors or emits light by an enzymatic reaction include, but are not limited to, a luciferase substrate, an alkaline phosphatase substrate, a β-galactosidase substrate, a glucuronidase substrate, a peroxidase substrate, and the like.

  The label is preferably less toxic and less toxic and less toxic. Examples of such a label include a cell membrane impermeable label. In one aspect, the label is biocompatible. By using a biocompatible label, even if the label is attached to the sheet culture, it can be safely used for transplantation or the like. In one embodiment, the label includes a carrier coupled to a detectable substance. Examples of the carrier include, but are not limited to, for example, dextran, biotin, albumin, agarose, polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polyethyleneimine (PEI), polyethylene Polymers such as glycol (PEG) and polylysine (for example, poly-L-lysine (PLL)), polystyrene, silica, acrylic, quartz, chitosan, gold, silver, albumin, agarose, polylactic acid, polyethyleneimine, platinum, oxidation Examples thereof include fine particles composed of aluminum, borosilicate glass, soda lime glass, PLA, PLGA, iron oxide, palladium, and sugar alcohols such as mannitol and erythritol.

In certain embodiments, the label comprises dextran conjugated with a fluorescent substance. The molecular weight of dextran may be about 1000 to about 1 million, about 5000 to about 500,000, about 10,000 to about 200000, about 20000 to about 100,000, about 30000 to about 90,000, about 40000 to about 80000, and the like. In another specific embodiment, the label includes [ ³ H] mannitol, [ ¹⁴ C] mannitol, sodium fluorescein (Na—F), lucifer yellow CH (eg, dilithium salt, dipotassium salt, etc.) and the like.

  The first container is not particularly limited as long as cells can form a sheet-shaped cell culture therein, and includes, for example, cell culture containers made of various materials. The first container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution except for the semipermeable portion. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polycarbonate, polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, Examples thereof include polydimethylacrylamide, metal (for example, iron, stainless steel, aluminum, copper, brass). The first container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. The first container may be made using the above materials, or a commercially available one may be used. The first container may be in the form of a dish or a multi-well plate with a plurality of wells, and may be opaque, translucent or transparent. Moreover, when detecting a label | marker in a 1st container, it is preferable that a 1st container is comprised with the material suitable for the detection of a label | marker, or has a part comprised with such a material at least. For example, when the label is a fluorescent label, the first container is preferably colorless and transparent or has at least a colorless and transparent portion suitable for fluorescence detection.

The first container includes a sheet-shaped cell culture forming surface for forming a sheet-shaped cell culture. The surface may be an adhesive surface suitable for the formation of a sheet-like cell culture, such as, but not limited to, a hydrophilic surface, such as a surface coated with serum, corona discharge treated polystyrene, gelatin, Collagen gel, Matrigel-coated surface, surfaces coated with hydrophilic compounds such as hydrophilic polymers, and extracellular substrates such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, cadherin family, selectin It may be a surface coated with a cell adhesion factor such as family or integrin family. Cell culture vessel having such surface are commercially available ^{(e.g., Corning (R) TC-Treated} Culture Dish, etc. manufactured by Corning), it can be prepared according to known methods.

  The sheet-like cell culture forming surface may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amides), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine), or a vinyl ether derivative (for example, methyl vinyl ether) homopolymer or copolymer, a temperature-responsive material, a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By giving a predetermined stimulus to these materials, the physical properties, for example, hydrophilicity and hydrophobicity can be changed, and peeling of the cell culture adhered on the materials can be promoted. In addition, a component such as serum can be further coated on the coating with the stimulus-responsive material as described above.

The first container has a semi-permeable portion through which the label passes but no cells pass. The semipermeable portion is not particularly limited as long as it has a property that the label can pass but the cell does not pass through it, and may be composed of, for example, a culture substrate having pores of a predetermined size. . The size (pore diameter) of the pore is not particularly limited as long as the label can pass through but the cell does not pass through. For example, about 0.01 μm to about 50.0 μm, about 0.01 μm to about 20.0 μm, About 0.01 μm to about 10.0 μm, about 0.02 μm to about 5.0 μm, about 0.03 μm to about 4.0 μm, about 0.05 μm to about 3.5 μm, about 0.1 μm to about 3.0 μm, etc. It may be. A person skilled in the art can appropriately select pores suitable for the label to be used and the cell size. The density of the pores may vary depending on the pore diameter. For example, when the pore diameter is 3.0 μm, the density is about 1 × 10 ³ pieces / cm ² to about 1 × 10 ⁸ pieces / cm ² , about 1 × 10 ⁴ pieces / cm ² to about 1 × 10 ⁷ pieces / cm ² , about 2.5 × 10 ⁴ pieces / cm ² to about 8 × 10 ⁶ pieces / cm ² , about 5 × 10 ⁵ pieces / cm ² to It may be about 5 × 10 ⁶ pieces / cm ² , about 8 × 10 ⁵ pieces / cm ² to about 2.5 × 10 ⁶ pieces / cm ^2, etc. In addition, when the pore density is expressed in terms of an open area ratio, for example, the area of the semipermeable portion is about 0.1% to about 95%, about 0.5% to about 90%, about 1% to about 90%, about 2% to about 90%, about 3% to about 85%, etc.

  The semipermeable portion may constitute part or all of the sheet-like cell culture forming surface of the first container. When constituting part of the sheet-like cell culture-forming surface, the proportion can be any proportion ranging from about 1% to about 99% of the surface area of the sheet-like cell culture-forming surface, such as about 90%, about It may be 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, etc. Moreover, the semipermeable part may exist in one place on the sheet-like cell culture formation surface, or may exist separately in a plurality of places. The material and thickness of the semipermeable part may be the same as or different from other parts of the first container. Specifically, for example, the first container may be one in which part or all of the surface of the sheet-shaped cell culture forming surface is subjected to pore treatment, or a culture substrate having pores at the bottom. It may be attached.

  The culture substrate constituting the semipermeable portion is not particularly limited as long as cells can form a sheet-like cell culture thereon, and includes, for example, plates and films of various materials. The culture substrate may be made of a material that does not allow liquid to pass through, such as a culture solution. Non-limiting examples of such materials include those described for the first container, urethane, polyethylene terephthalate (PET), non-woven fabric, polytetrafluoroethylene (PTFE), cellulose mixed esters (eg, cellulose acetate and nitrocellulose) And the like suitable for pore treatment. The culture substrate may be produced using these materials, or commercially available ones may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures. Non-limiting examples of such surfaces are as described above for sheet cell culture formation. Moreover, a porous structure can also be used as a culture substrate. For example, if a sheet-like cell culture is formed on a culture substrate of a porous structure composed of a biological material such as a collagen sponge, the culture substrate is not peeled off from the culture substrate. The whole material can be used for transplantation as it is. In this case, even if the semipermeable portion is composed of only the porous structure, the semipermeable portion is composed of the porous structure and the support that is fixed to the first container and holds the porous structure in a separable manner. May be.

  The second container has a form capable of accommodating the semi-permeable portion of the first container and is liquid impermeable. Therefore, the second container can be made of any material that is liquid impermeable. The second container may be opaque, translucent or transparent. When the label is detected in the second container, it is preferable that the second container is made of a material suitable for detection of the sign or at least has a portion made of the material. For example, when the label is a fluorescent label, the second container is preferably colorless and transparent or has at least a colorless and transparent portion suitable for fluorescence detection. The second container may be configured to be separable from the first container or may be integrated. When detecting a label in the second container, the second container may include a label detection unit configured so that the detection of the label is not inhibited by the first container. The sign detection unit is made of a material suitable for detection of the sign, or has at least a part made of such a material. The second container may have a form of a dish or a plate in accordance with the form of the first container. When the label is transferred to the second container, the second container may include a member for preventing discoloration or discoloration of the labeling substance, for example, a detachable cover or the like. For example, when a photodegradable substance such as a fluorescent substance or a dye is used as the labeling substance, a light shielding cover or the like may be provided. Further, regardless of whether the label is transferred to the first container or the label is transferred to the second container, the member for preventing discoloration or discoloration of the labeling substance is not limited, for example, a removable cover, etc. Thus, the entire first container and the second container that accommodates the first container can be covered.

  The second container may include a port for collecting the liquid in the container. Such a configuration is advantageous when a label that can be contained in the liquid in the second container is detected outside the container. The port is not particularly limited as long as it enables collection of the liquid from the second container, and the liquid contained in the second container provided between the first container and the second container. Even the opening that allows access to the liquid is a conduit provided at a position where one opening contacts the liquid in the second container, allowing the liquid in the second container to be aspirated. May be. The port is preferably one that allows collection of liquid from the second container without separating the first container and the second container.

  Cell seeding in the present invention can be performed by any known technique and conditions. The seeding of the cells may be performed, for example, by injecting a cell suspension in which the cells are suspended in the sheeting medium into the first container. For the injection of the cell suspension, an apparatus suitable for the operation of injecting the cell suspension, such as a dropper or a pipette, can be used.

  The step of forming the seeded cells into a sheet can also be performed by any known technique and condition. Non-limiting examples of such methods include, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-172925, and the like. It is described in. The step of forming a sheet can be achieved, for example, by culturing cells under conditions that form cell-cell adhesion. Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions. A person skilled in the art can select optimal conditions according to the type of cells to be seeded. In the present specification, the culture for forming the seeded cells into a sheet may be referred to as “sheet culture”.

  Sheeting can be performed to cover all or part of the semi-permeable portion. When uniform experimental conditions are required, such as comparison between a plurality of samples, it may be preferable to form a sheet so as to cover all of the semi-permeable portion. On the other hand, when evaluating changes over time in the same sheet-shaped cell culture (for example, a method for determining completion of sheet formation described later, a method for producing a sheet-shaped cell culture for transplantation, a process for producing a sheet-shaped cell culture) A good evaluation is possible even if the sheet is formed so as to cover a part of the semi-permeable portion.

  Examples of the sheeting medium used for sheeting include, but are not limited to, water, physiological saline, various physiological buffers (for example, PBS, HBSS, etc.), and various basal media for cell culture. Etc. may be used. Such a basal medium is not limited, for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like are included. Many of these basal media are commercially available, and their compositions are also known. The basal medium may be used in a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes one in which one or more components are added, removed, increased or decreased.

  The sheeting medium may contain additives such as serum (eg, bovine serum such as fetal bovine serum, horse serum, human serum, etc.), various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).

The step of adding a label can also be performed by any known technique and conditions.
The label may be added to the first container, for example, by adding the label directly to the sheeting medium contained in the first container, or by replacing the sheeting medium with another medium to which the label is added. Alternatively, the sheet-forming medium may be replaced with another medium, and a label may be added to the medium after replacement. When the sheet-forming medium is replaced with, for example, a low-nutrition medium (for example, HBSS), the state of the sheet-shaped cell culture can be maintained as it is, which is suitable for confirming the state of the completed sheet-shaped cell culture. ing. In addition, by placing the sheet-shaped cell culture under hypoxic conditions (for example, an oxygen concentration of about 0.1 to about 10%), cell growth can be stopped and the state of the sheet-shaped cell culture can be maintained as it is. it can.

For example, the label may be added to the second container by directly adding the label to the second container that is in fluid communication with the first container through the semipermeable portion. The first container may be in fluid communication with the second container containing the prepared medium through the semi-permeable portion.
The label may be added to the first container or the second container simultaneously with the seeding of the cells in the first container, before the seeding, and / or after the seeding. The label may be added only once or a plurality of times.
The amount of the label to be added may vary depending on the label to be used, the amount of the medium contained in the container, the transfer period, etc., but those skilled in the art can find an appropriate amount to be added by routine experimentation.

  The liquid communication between the first container and the second container via the semi-permeable part is, for example, that the first container is completely immersed in the liquid medium contained in the second container. As described above, it can be performed by arranging the second container. By allowing the first container and the second container to communicate with each other through the semi-permeable portion, the label added to the first container or the second container is diffused through the semi-permeable portion. This makes it possible to move to the second container or the first container. The medium in the second container may be the same as or different from the sheeted medium. If the medium in the second container is different from the sheeting medium, remove the sheeting medium contained in the first container before immersing the semi-permeable portion in the medium in the second container, or The same medium as that in the two containers may be substituted. Further, when the medium in the second container is different from the sheeted medium, it is preferable to use a medium having a small osmotic pressure difference from the sheeted medium. The medium contained in the container to which the label is transferred (hereinafter sometimes referred to as a label detection medium) is preferably one that does not adversely affect the detection of the label. For example, when the label is a fluorescent label or a colorimetric label, the medium preferably has no dye or autofluorescence.

  When a labeling substance is used as the label, the labeling is performed except that the labeling substance is not contained in the second container or the first container to which the label is transferred when the label is added to the first container or the second container. By adding the same unlabeled substance as the substance (for example, when using fluorescently labeled dextran as a label, unlabeled dextran), the influence of osmotic pressure can be eliminated and more accurate evaluation can be performed.

  For example, the step of transferring the label to the first container and the second container to which the label is not added through the semipermeable portion includes adding the label to the first container or the second container. Thereafter, the first container and the second container can be incubated for a predetermined period in a state where the first container and the second container are in fluid communication via the semipermeable portion. Liquid communication between the first container and the second container may be performed before the label is added, after the label is added, or at the same time as the label is added. The transfer of the label is caused by the diffusion of the label. The transition period is not particularly limited, and may be, for example, 1 minute to 72 hours, 5 minutes to 48 hours, 10 minutes to 36 hours, 15 minutes to 24 hours, and the like. The incubation temperature is not particularly limited as long as it does not inhibit the diffusion of the label. For example, the incubation temperature is about 2 ° C to about 45 ° C, about 4 ° C to about 40 ° C, about 8 ° C to about 38 ° C, about 10 ° C to about It may be 38 ° C, about 15 ° C to about 30 ° C, and the like.

  The transition step is performed in a state where the level difference between the level of the liquid stored in the first container and the level of the liquid stored in the second container is small. From the viewpoint of the above. The situation in which there is a difference in level between the liquid level of the liquid stored in the first container and the liquid level of the liquid stored in the second container is the liquid level of the liquid stored in the first container Is higher than the liquid level of the liquid stored in the second container, and the liquid level of the liquid stored in the first container is lower than the liquid level of the liquid stored in the second container. The height difference is preferably as small as possible, and is not limited. For example, 20 mm or less, 15 mm or less, 12 mm or less, 10 mm or less, 8 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less. , 0.5 mm or less. It is preferable that the height difference does not substantially exist, and it is most preferable that the height difference does not exist at all (that is, the height difference is 0 mm). In one aspect | mode, the said height difference exists in the range of 0-10 mm. The height difference can be adjusted, for example, by adjusting the amount of liquid added to the first container and / or the second container.

  The step of detecting the label transferred to the first container or the second container to which no label is added can be performed by any known technique capable of detecting the label. For example, if the label is a fluorescent label, the first or second container that may contain the transferred label is irradiated with light of a predetermined wavelength, and the intensity of the generated fluorescence is measured to It can be quantified. In the detection of the label, as described above, the transferred label that can be contained in the first container or the second container may be detected in the container, or the medium contained in the container is taken out and the label is detected. You may detect out of a container. When the label is detected in the first container, the semi-permeable portion may be sealed with a suitable member, such as a film or a cap, to prevent leakage of the medium.

  When the label is detected with another substance (detection agent) (for example, when a substance that is colored or luminescent by an enzymatic reaction is used as a label and this is detected by reacting with the enzyme, the enzyme is used as the label, The enzyme substrate (for example, when detecting by reacting with a substance that is colored or luminescent by an enzymatic reaction), the detection agent may be reacted with the label in the step of detecting the transferred label, A detection agent is put in a container to be transferred in advance before the detection step, and in the detection step, a signal (for example, coloration, luminescence, etc.) generated by the reaction between the label and the detection agent may be detected. .

  By the method of the present invention, the quality of the formed sheet-shaped cell culture can be evaluated from the measured amount of label. Here, the quality of the sheet-shaped cell culture includes the strength of the sheet-shaped cell culture, the degree of sheet formation, the cell density, the viability of the cells, the vitality of the cells, the adhesion state between the cells, and the like. The greater the cell gap present in the sheet cell culture, the greater the amount of label that migrates, but the greater the cell gap, the lower the strength of the sheet cell culture. Moreover, as the following examples show, the less the sheet is formed, the lower the cell density, and the lower the viability and vitality of the cells, the greater the amount of label transferred.

  The quality can be evaluated by, for example, comparing the amount of the detected label with a predetermined reference value. More specifically, for example, when the amount of the detected label is equal to or less than a predetermined reference value, the quality is evaluated as good, and when the amount exceeds the reference value, the quality is evaluated as poor. Can do. The reference value can be obtained by experiments or the like. In addition to the mass and concentration of the label, the amount of the label includes numerical values related thereto, for example, numerical values such as absorbance, fluorescence intensity, emission intensity, RFU, and radiation dose according to the property of the label.

  The quality evaluation method of the present invention can be applied to a sheet-like cell culture actually used for transplantation or the like, but a sheet-like cell culture representing a lot consisting of a plurality of sheet-like cell cultures produced under the same conditions You may apply to. In the latter case, the result of quality evaluation by this method can be applied to the whole lot to which the culture belongs.

According to another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion that allows a label to pass but cells do not pass, and is formed into a sheet so as to cover the semipermeable portion. Step,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
And the label is inhibited from passing between cells by cell-cell adhesion, and step (2) may be performed simultaneously with step (1), before step (1) or after step (1). The present invention relates to a method for determining completion of sheeting (hereinafter, sometimes referred to as “sheeting completion determination method”).

  Steps (1) to (4) in the sheeting completion determination method of the present invention are as described above for the quality evaluation method of the present invention. As Example 5 shows, the amount of transition label per unit time gradually decreases with the progress of sheeting, and is maintained at a substantially constant amount when sheeting is completed. Therefore, the completion of sheeting can be detected by periodically measuring or monitoring the amount of label transferred. For measurement of the amount of transfer label, etc., only step (4) is repeated regularly, or steps (2) to (4) are repeated periodically, or steps (3) to (4) are repeated periodically. May be. When only step (4) is repeated periodically, the amount of the label increases with the passage of time because the label continuously moves to and accumulates in a container to which no label has been added. The amount of increase per unit time gradually decreases with the progress, and becomes almost constant when the sheeting is completed. Therefore, for example, the amount of increase in the label is calculated from the amount of the label measured periodically, and when the increase becomes substantially constant in a plurality of consecutive measurements, it can be determined that the sheeting is completed. . When steps (2) to (4) are periodically repeated, for example, if the transferred label is removed after step (4), the label is newly added in step (2) of the next cycle, and step (3 ), The amount of the detected label gradually decreases with time, and becomes almost constant when the sheeting is completed. Accordingly, it can be determined that the sheeting is completed when the amount of the marker measured periodically becomes substantially constant in a plurality of consecutive measurements. Further, when steps (3) to (4) are periodically repeated, for example, if the transferred label is removed after step (4), the label is newly transferred in step (3) of the next cycle. Therefore, the amount of the detected label gradually decreases with time, and becomes almost constant when the sheeting is completed. Accordingly, it can be determined that the sheeting is completed when the amount of the marker measured periodically becomes substantially constant in a plurality of consecutive measurements.

  “When the amount or increase of the label becomes almost constant in a plurality of consecutive measurements”, the rapid decrease in the amount or increase of the label transferred to the first container or the second container at the beginning of the sheet culture Means when it has ended and it has not changed significantly over time. The fluctuation range of the label amount or the increase amount in a plurality of consecutive measurements at this time is not limited, for example, about ± 20% / hour, within about ± 10% / hour, within about ± 8% / hour, It may be within about ± 6% / hour, within about ± 5% / hour, within about ± 4% / hour, within about ± 3% / hour, within about ± 2% / hour, and the like.

  The determination of completion of sheeting can also be performed based on the average rate of change or the differential coefficient. Since the absolute value of the rate of change over time of the amount of label detected (for example, the total amount of transition and the amount of transition per unit time) becomes smaller as the sheeting progresses, The amount is expressed as a function of time, and when the change rate or the absolute value of the differential coefficient becomes smaller than a predetermined value, it can be determined that the sheeting is completed. When the completion of sheeting is determined based on the differential coefficient in the function of the amount of transition of the marker per unit time and the time, the absolute value of the differential coefficient is not limited, for example, about 1.0 or less, about 0 9.9 or less, about 0.8 or less, about 0.7 or less, about 0.6 or less, about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 It may be the following.

  Therefore, the method for determining completion of sheeting according to the present invention includes the step of periodically measuring or monitoring the amount of the transferred label and / or when the amount or increase of the label becomes substantially constant in a plurality of consecutive measurements. Alternatively, the method may further include a step of determining that the sheet formation is completed when the absolute value of the change rate of the amount of the label with time becomes equal to or less than the reference value.

  The sheet formation completion judging method of the present invention is a sheet-like cell representing a lot consisting of a plurality of sheet-like cell cultures produced under the same conditions even when applied to a sheet-like cell culture actually used for transplantation or the like. It may be applied to cultures. In the latter case, the determination result by the present method can be applied to the entire lot to which the culture belongs, and the timing at which the sheet-shaped cell culture is peeled from the culture substrate can be optimized.

In another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion through which a label passes but cells do not pass, and the sheet culture is performed so as to cover the semipermeable portion. Step to do,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
(5) Sheet culture when the amount or increase of the label becomes almost constant in a plurality of consecutive measurements, or when the absolute value of the rate of change over time in the amount of label is below the reference value And the step of recovering the sheet-shaped cell culture from the first container, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes steps (1) and At the same time, the present invention relates to a method for producing a transplanted sheet-like cell culture (hereinafter sometimes abbreviated as “manufacturing method”), which may be performed before step (1) or after step (1).

  Steps (1) to (4) in the sheeting completion determination method of the present invention are as described above for the quality evaluation method of the present invention. Also, in step (5), “when the amount or increase of the label becomes substantially constant in a plurality of consecutive measurements” and “when the absolute value of the rate of change of the amount of the label over time is below the reference value "Is as described above for the sheet formation completion determination method of the present invention.

  The recovery of the sheet-shaped cell culture from the first container in step (5) is free (detachment) from the culture substrate serving as a scaffold while the sheet-shaped cell culture is at least partially maintained in the sheet structure. For example, it can be carried out by enzymatic treatment with a proteolytic enzyme (such as trypsin) and / or mechanical treatment such as pipetting. In addition, when a cell culture is formed by culturing a cell on a culture surface coated with a material that changes its physical properties in response to stimulation, for example, temperature or light, a predetermined stimulation can be applied. It can also be released non-enzymatically.

  The production method of the present invention may further include a step of washing the sheet-shaped cell culture during, before or after step (5). By this step, the label attached to the sheet-shaped cell culture can be removed.

In another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion through which a label passes but cells do not pass, and the sheet culture is performed so as to cover the semipermeable portion. Step to do,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
And the label is inhibited from passing between cells by cell-cell adhesion, and step (2) may be performed simultaneously with step (1), before step (1) or after step (1). The present invention relates to a good method for evaluating the sheet-forming ability of cells (hereinafter sometimes abbreviated as “sheet-forming ability evaluation method”).

  Steps (1) to (4) in the sheet forming ability evaluation method of the present invention are as described above for the quality evaluation method of the present invention. As shown in Example 6, it was revealed that cells that cause abnormalities in sheet formation, such as peeling during sheet formation, have a large amount of label transfer after sheet formation. Accordingly, by performing steps (1) to (4), whether or not the cells of a certain subject (for example, a patient) have a sheet forming ability suitable for producing a clinical sheet-like cell culture used for transplantation or the like. Can be assessed in advance based on the amount of label detected. Thereby, it is possible to avoid the start of production of an incomplete sheet-like cell culture. The sheet forming ability means the ability of cells to form a sheet. Although not wishing to be bound by a specific theory, for the formation of a sheet, the ability of cells to adhere to the culture substrate, the ability to adhere to each other, the secretion ability of extracellular matrix, the ability to spread, the ability to move, etc. It is considered that various abilities of cells and cell vitality are involved. The evaluation of the sheet forming ability can be performed, for example, by comparing the amount of the detected label with a predetermined reference value. More specifically, for example, when the amount of the detected label is less than or equal to a predetermined reference value, the cell sheet forming ability is evaluated as sufficient, and when the amount exceeds the reference value, the cell sheet forming ability is evaluated. Can be evaluated as insufficient. The reference value can be obtained by experiments or the like. Moreover, the evaluation result that the sheet forming ability is insufficient by the sheet forming ability evaluation method of the present invention is used as a material for determining whether a certain object is suitable for the treatment using the sheet-like cell culture. Can do.

In another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion through which a label passes but cells do not pass, and the sheet culture is performed so as to cover the semipermeable portion. Step to do,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
And the label is inhibited from passing between cells by cell-cell adhesion, and step (2) may be performed simultaneously with step (1), before step (1) or after step (1). The present invention relates to a method for detecting sheet peeling in the production process of a sheet-shaped cell culture (hereinafter, sometimes abbreviated as “peeling detection method”).

  Steps (1) to (4) in the peeling detection method of the present invention are as described above for the quality evaluation method of the present invention. In the manufacturing process of the sheet-shaped cell culture, if peeling occurs during sheet formation, normal sheeting is not performed and a sheet-shaped cell culture with low quality is obtained. Therefore, when the amount of label transfer is measured at multiple points in the manufacturing process of the sheet-shaped cell culture, and the amount of transfer gradually decreases as the sheeting progresses, the amount of transfer increases (or the rate of decrease decreases) It can be determined that peeling has occurred. For detached sheet-like cell cultures, it is possible to stop manufacturing as soon as detachment is detected and take measures such as re-manufacturing with new cells, and to perform quality checks at the final stage of manufacturing. In comparison, it is possible to avoid loss of time.

Another aspect of the present invention is:
(1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semi-permeable portion through which the label passes but cells do not pass, and (3) a liquid-impermeable second container capable of containing the semi-permeable portion. Production of sheet cell culture, quality evaluation of sheet cell culture, determination of completion of sheet formation of sheet cell culture, evaluation of cell sheet forming ability, and / or in production process of sheet cell culture The present invention relates to a kit for detection of peeling.

  The components (1) to (3) of the kit of the present invention are as described above for the quality evaluation method of the present invention. In addition, the kit of the present invention has an arbitrary configuration suitable for one or more of the above-mentioned applications, for example, a medium added to each container, a coloring reagent, a light shielding cover, an instruction manual for each application, and information on a usage method. May be further included, for example, a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, and the like.

Another aspect of the present invention is:
(1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semipermeable portion through which the label passes but cells do not pass;
(3) Production of sheet-shaped cell culture, sheet-shaped cell culture, comprising a liquid-impermeable second container capable of accommodating the semi-permeable portion, and (4) a detector for detecting a label The present invention relates to a system for quality evaluation, determination of completion of sheet formation of a sheet-shaped cell culture, evaluation of the sheet-forming ability of cells, and / or detection of detachment in the manufacturing process of the sheet-shaped cell culture.

  The components (1) to (3) of the system of the present invention are as described above for the quality evaluation method of the present invention. The detector of the component (4) is not particularly limited as long as it can detect the label. For example, a photometer such as a spectrophotometer, an absorptiometer, a turbidimeter, a colorimeter, a scintillation counter or the like Including detectors. One skilled in the art can select a detector suitable for the label used. The system of the present invention may include only one type of detector or a plurality of types of detectors. The system of the present invention has an arbitrary configuration suitable for one or more of the above applications, for example, a processor for processing a signal from a detector and controlling an actuator, an interface such as a display device or an input device, a sign May be further provided with an injector for adding the above to the container, a manipulator for moving each container, an incubator for cell culture, a stirrer, and the like.

In another aspect of the present invention, (1) a first container having a semi-permeable portion through which a label passes but cells do not pass is coated with the isolated sheet-shaped cell culture on the semi-permeable portion. Step to arrange,
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) And a method for evaluating the quality of an isolated sheet-shaped cell culture (hereinafter referred to as “quality evaluation method for isolated sheet-shaped cell culture”, which may be performed before or after step (1). "May be abbreviated as").

  Each step of the quality evaluation method of the isolated sheet-shaped cell culture of the present invention consists in that the cells are seeded in a first container and instead of sheeting so as to cover the semipermeable part, the isolated sheet The cell-like cell culture is the same as the quality evaluation method of the present invention except that the cell-like culture is arranged so as to cover the semipermeable portion on the first container, and the quality of the same sheet-like cell culture, for example, In addition, the strength of the sheet-shaped cell culture, the degree of sheet formation, the cell density, the viability of the cells, the vitality of the cells, the adhesion state between the cells, and the like can be evaluated. An “isolated sheet cell culture” typically refers to a sheet cell culture that has been released (peeled) from a culture substrate after sheeting.

  The arrangement of the isolated sheet-shaped cell culture can be performed over a predetermined period so that the isolated sheet-shaped cell culture adheres to the semipermeable portion. Adhesion of the isolated sheet-shaped cell culture to the semipermeable portion can be achieved, for example, by culturing the isolated sheet-shaped cell culture in a state where it is in contact with the semipermeable portion for a predetermined period. . The arrangement period (for example, the culture period) is not particularly limited as long as it enables adhesion to the semipermeable portion of the isolated sheet-shaped cell culture, and for example, 5 minutes to 24 hours, 10 minutes to 12 The time may be 15 minutes to 6 hours, 30 minutes to 3 hours, or the like.

  As described above for the quality evaluation method of the present invention, the isolated sheet-shaped cell culture is composed of two or more cell layers, even if it is composed of one cell layer (single-layer sheet-shaped cell culture). (Layered sheet cell culture) may be used. A laminated sheet-shaped cell culture is produced, for example, by superposing two or more sheet-shaped cell cultures directly or via an intervening substance to form a single sheet-shaped cell culture. Examples of the intervening substance include, but are not limited to, a substance that promotes and / or strengthens adhesion between sheet-like cell cultures, and non-limiting examples thereof include, for example, an extracellular matrix component or the like. Examples thereof include compositions (eg, collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, hydrogel, gelatin, etc.), adhesion proteins (eg, cadherin family, selectin family, integrin family). In the case where the isolated sheet-shaped cell culture to be evaluated is a laminated sheet-shaped cell culture, in addition to the above-described quality items, the number of sheet-shaped cell cultures stacked and defects in the stacking (for example, the sheet shape at the time of stacking) It is also possible to evaluate cell culture bending, wrinkle formation, tearing, and the like.

In another aspect of the present invention, (1) a plurality of sheet-like cell cultures are coated on a semi-permeable portion in a first container having a semi-permeable portion through which a label passes but a cell does not pass. Step of laminating,
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) Simultaneously with step (1) or after step (1), the method for quality evaluation of the laminated sheet-like cell culture (hereinafter referred to as “the quality evaluation method of laminated sheet-like cell culture”) May be abbreviated).

Steps (2) to (4) in the quality evaluation method for a laminated sheet-shaped cell culture of the present invention are as described above for the quality evaluation method of the present invention.
In the step (1), as in the quality evaluation method of the present invention, the stacking of a plurality of sheet-like cell cultures is performed by first seeding cells in a first container and forming a sheet so as to cover the semipermeable portion. From the above, even if the isolated sheet-shaped cell culture prepared separately is stacked on the sheet-shaped cell culture formed into a sheet, the first isolated sheet-shaped cell culture is first placed in the first container. The second isolated sheet-shaped cell culture prepared separately may be placed on the semi-permeable portion and then laminated on the first isolated sheet-shaped cell culture. Good. The isolated sheet-shaped cell culture may be a single layer or a layered one, and one or two or more single-layer cell cultures are formed on the isolated sheet-shaped cell culture stacked in the first container. A release sheet-shaped cell culture may be further laminated. The number of sheet-like cell cultures to be laminated is not particularly limited, and may be, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or 11 or more.

  The lamination of the sheet-shaped cell culture is not limited, and can be achieved, for example, by placing the isolated sheet-shaped cell culture on the sheet-shaped cell culture in the first container. The arrangement is not limited, and can be achieved, for example, by superimposing two or more sheet-like cell cultures directly or via an intervening substance. About the intervening substance, it is as having mentioned above about the quality evaluation method of the isolated sheet-like cell culture of this invention. In order to adhere the sheet-like cell cultures to each other, after the isolated sheet-like cell cultures are arranged, the overlapping sheet-like cell cultures may be cultured for a predetermined period. The culture period is not particularly limited as long as it allows adhesion between the overlapping sheet-shaped cell cultures, and for example, 5 minutes to 24 hours, 10 minutes to 12 hours, 15 minutes to 6 hours, 30 minutes to 30 minutes to It may be 3 hours. The culturing may be performed every time one isolated sheet-shaped cell culture is stacked, or after all the desired number of isolated sheet-shaped cell cultures are stacked. The laminated sheet-like cell culture after evaluation may be peeled off from the first container and used for transplantation or the like.

  According to the quality evaluation method of the laminated sheet-shaped cell culture of the present invention, the strength of the laminated sheet-shaped cell culture, the degree of sheeting, the cell density, the viability of the cells, the vitality of the cells, the adhesion state between the cells, the sheet-like cells The number of cultures stacked, stacking defects (for example, bending, wrinkle formation, tearing, etc. of the sheet-shaped cell culture during stacking) can be evaluated.

According to another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion that allows a label to pass but cells do not pass, and is formed into a sheet so as to cover the semipermeable portion. Or placing an isolated sheet cell culture in the first container so as to cover the semipermeable part;
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
And the label is inhibited from passing between cells by cell-cell adhesion, and step (2) may be performed simultaneously with step (1), before step (1) or after step (1). The present invention relates to a method for evaluating the cell purity or cell composition ratio of a sheet-shaped cell culture (hereinafter, sometimes abbreviated as “cell purity evaluation method”).

Steps (1) to (4) in the cell purity evaluation method of the present invention are as described above for the quality evaluation method of the present invention and the quality evaluation method of the isolated sheet cell culture.
As shown in Example 8, it has now been clarified that the amount of transfer label per unit time varies depending on the type of cells constituting the sheet-shaped cell culture. Therefore, the amount of transfer label in a sheet-shaped cell culture whose cell purity or cell composition ratio is unknown is detected, and this is the same as the amount of transfer label in a sheet-like cell culture whose cell purity or cell composition ratio is measured under the same conditions. By comparing, it is possible to estimate the cell purity or cell constituent ratio of the sheet-like cell culture to be evaluated, or to detect an abnormality in the cell purity or cell constituent ratio.

  In the cell purity evaluation method of the present invention, the cells to be seeded may contain a plurality of cell types, and typically the cell purity and / or cell composition ratio is unknown. The cell purity evaluation method of the present invention may further include a step of comparing the detected amount of transferred label with a reference value. The reference value is not limited, and may be, for example, a transfer label amount in a sheet-shaped cell culture having a known cell purity or cell composition ratio measured under the same conditions as the sheet-shaped cell culture to be evaluated. There may be one or more reference values to be compared. For example, the reference value may be a transfer label amount for a sheet-shaped cell culture having one kind of desired cell purity, or two kinds of sheet-shaped cell cultures having an upper limit and a lower limit cell purity of an allowable range. The amount of transfer label for each of the plurality of sheet-like cell cultures having different known cell purity to the extent that a calibration curve can be created. Also good. As a result of comparison, when the deviation between the detected transfer marker amount and the reference value is larger than a predetermined level, or the detected transfer marker amount is not included in the predetermined range composed of a plurality of reference values It can be evaluated that the cell purity or the cell composition ratio of the sheet-like cell culture to be evaluated is abnormal. In addition, when the deviation between the detected amount of the transition marker and the reference value is smaller than a predetermined level, or when the detected amount of the transition marker is included in a predetermined range composed of a plurality of reference values, the evaluation target It can be evaluated that the cell purity or cell composition ratio of the sheet-like cell culture is normal. Furthermore, based on a plurality of reference values, a calibration curve showing the relationship between the cell purity or cell composition ratio and the amount of transfer label is prepared, and from the detected amount of transfer label, the sheet-like cell culture to be evaluated The cell purity or cell composition ratio can be estimated.

  In one embodiment, the cell purity is that of skeletal myoblasts. In one embodiment, the cell purity is that of fibroblasts. In one embodiment, the cell component ratio is a component ratio between skeletal myoblasts and fibroblasts.

In another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion through which a label passes but cells do not pass, and the sheet culture is performed so as to cover the semipermeable portion. Step to do,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the amount of the label transferred to the container to which the label is not added,
(5) comparing the detected amount of transfer label with a reference value;
(6) The method includes a step of detecting an abnormality in the manufacturing process of the sheet-shaped cell culture based on the comparison result, and the label is inhibited from passing between cells due to cell-cell adhesion. A method for detecting an abnormality in the production process of a sheet-shaped cell culture that may be performed prior to step (1) or after step (1) at the same time as (1) (hereinafter sometimes abbreviated as “abnormality detection method”). Is).

Steps (1) to (4) in the abnormality detection method of the present invention are as described above for the quality evaluation method of the present invention.
Reference values in step (5) are abnormalities in various parameters of the sheet-shaped cell culture being produced, for example, the strength of the sheet-shaped cell culture, the degree of sheeting, the cell density, the viability of the cells, and the vitality of the cells Including those related to abnormalities such as the state of adhesion between cells. Non-limiting examples of reference values include, for example, the amount of migration label in a standard sheet-shaped cell culture that has been sheeted (for example, the average value of the amount of migration label in a plurality of sheet-shaped cell cultures that have been sheeted) Etc.), the amount of migration label in a standard sheet-like cell culture after the lapse of a predetermined period from the start of sheeting (for example, the average value of the amount of migration label in a plurality of sheet-like cell cultures after the lapse of a predetermined period from the start of sheeting) Etc.), differential coefficient with respect to the time of the amount of migration label in the standard sheet-shaped cell culture after the lapse of a predetermined period from the start of sheeting (for example, transition in a plurality of sheet-shaped cell cultures after the lapse of the predetermined period from the start of sheeting) The average value of the derivative of the labeled amount with respect to the time of the transferred label amount, etc.), the amount of change in the transition label over time and the rate of change (for example, the average Rate, etc.), the amount of change in transition labeled amount between two different time points of the post-starting sheeting, the rate of change (e.g., the average rate of change, etc.) and the like. The reference value can be determined as appropriate by, for example, experiments described in Examples 3 to 6. The reference value may be a single numerical value or a numerical value range.

  In step (5), the detected amount of transferred label is compared with a reference value. For example, if the reference value is a single numerical value, whether the detected amount of transferred label is larger or smaller than the reference value. Or if they are the same, or if the reference value is in the numerical range, output whether the detected transition marker amount is included in the reference value, greater than the upper limit of the reference value, or less than the lower limit of the reference value To do.

  In step (6), in accordance with the comparison result in step (5), an abnormality in the manufacturing process of the sheet-shaped cell culture (for example, presence or absence of abnormality, presence or absence of possibility of abnormality) is detected. For example, as the result of Example 3 shows, the lower the cell density, the greater the amount of transfer label after the elapse of the predetermined period. Therefore, if the amount of transfer label after the elapse of the predetermined period from the start of sheeting is greater than the reference value, the cell density It is possible to detect the presence of anomaly or the possibility of being low. In addition, as Example 4 shows, the lower the viability of cells, the greater the amount of migration label after the lapse of the predetermined period. Therefore, if the amount of migration label after the lapse of the predetermined period from the start of sheeting is larger than the reference value, It is possible to detect an anomaly or a possibility that the viability is low. Furthermore, as Example 5 shows, as the sheet-like cell culture progresses to sheeting, the amount of transfer label per unit time decreases and gradually approaches 0, and the integrated amount of transfer label gradually increases to a constant value. In addition, the amount of change or rate of change per unit time of the amount of transition marker is the largest at the beginning of sheeting and gradually decreases with time. Therefore, the differential coefficient with respect to the time of the transition marker amount after the lapse of a predetermined period from the start of sheeting, the change amount and rate of change of the transition marker over time within the predetermined period after the start of sheeting, between two different time points after the start of sheeting When the amount of change and the rate of change in the amount of transition marker in the are larger than the reference value, it is possible to detect the presence of abnormality or the possibility of sheet formation failure. In addition, as shown in Example 6, since the amount of transfer label increases when cell detachment occurs during sheet formation, if the amount of transfer label after the elapse of a predetermined period from the start of sheet formation is greater than the reference value, It is possible to detect the presence of an abnormality or the possibility that peeling has occurred.

  By detecting an abnormality in the manufacturing process of the sheet-shaped cell culture or the possibility thereof, it becomes possible to control the manufacturing process of the sheet-shaped cell culture, improving the manufacturing efficiency (for example, shortening the manufacturing time, etc.) It is possible to prevent defects, improve quality, effectively use cells, improve yield, and the like. Detection of the amount of transfer label is relatively early in the production process (without limitation, for example, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours after cell seeding) , Within 8 hours, within 9 hours or within 10 hours, etc.), it becomes possible to detect abnormalities at an early stage and take necessary measures.

In another aspect of the present invention, (1) cells are seeded in a first container having a semipermeable portion through which a label passes but cells do not pass, and the sheet culture is performed so as to cover the semipermeable portion. Step to do,
(2) adding a label to the first container or a liquid-impermeable second container capable of being in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the amount of the label transferred to the container to which the label is not added,
(5) comparing the detected amount of transfer label with a reference value;
(6) The method includes the step of controlling the production process of the sheet-shaped cell culture based on the comparison result, and the label is inhibited from passing between cells by cell-cell adhesion. Step (2) includes step (1). ) At the same time as step (1) or after step (1), the present invention relates to a method for producing a sheet-shaped cell culture (hereinafter sometimes abbreviated as “controlled production method”).

Steps (1) to (5) in the control manufacturing method of the present invention are as described above for the quality evaluation method and abnormality detection method of the present invention.
In step (6), the manufacturing process of the sheet-shaped cell culture is variously controlled based on the comparison result in step (5). For example, based on the comparison result of step (5), as in step (5) of the abnormality detection method of the present invention, an abnormality in the manufacturing process of the sheet-shaped cell culture or the presence or absence of the abnormality is detected. If the possibility is not detected, continue the production. If the abnormality or the possibility is detected, stop the production, stop the production, investigate the abnormality, and take measures to resolve it. Can do. Non-limiting examples of measures for eliminating abnormalities include, for example, if the abnormalities are insufficient in cell density, discontinuing production, seeding more cells and re-staging, etc. If it is abnormal, replace the medium, etc. If the abnormality is cell detachment, stop manufacturing, re-sheeting, etc. If the abnormality is poor sheeting, extend the sheeting period And stop manufacturing and redo the sheet.

Non-limiting examples of specific aspects of the controlled manufacturing method of the present invention are described in more detail with reference to the algorithms shown in FIGS.
In the algorithm shown in FIG. 1 (algorithm 1), after the start of the treatment (Start), cells are first seeded (Plating), and sheet culture (Incubation 1) is performed so as to cover the semipermeable portion. ). After a predetermined period of time, the first transition label amount X is detected (Detection 1), and X is compared with the first reference value (Reference value 1). If X is larger than the first reference value (in the case of “No”), it is determined that there has been a problem in cell seeding (for example, a shortage of the number of cells), and seeding is performed again. The reseeding of seeding is not limited. For example, even if the seeding culture is performed by seeding additional cells in a container containing the cell culture in sheet culture, the cell culture in sheet culture is treated with a single cell by enzyme treatment or the like. After adjusting the number of cells by adding additional cells from the suspension, discard the cell culture in the sheet culture and seed with new cells even if it is performed by seeding in a container. May be performed. By detecting the amount of transfer label relatively early in the sheet culture, it is possible to quickly detect cell abnormalities and avoid time loss due to continuing the sheet culture as it is. If X is not more than the first reference value (“Yes”), the sheet culture is continued (Incubation 2). After elapse of a predetermined period, the second transition marker amount Y is detected (Detection 2), and Y is compared with the second reference value (Reference value 2). If Y is larger than the second reference value (in the case of “No”), it is determined that the sheet culture time is insufficient, and sheet culture is performed for a predetermined period. After a predetermined period, the second transfer label amount Y is detected again and compared with the second reference value. This process is repeated until Y becomes equal to or smaller than the second reference value. If Y is less than or equal to the second reference value (“Yes”), it is determined that sheeting has been completed, and an appearance inspection is performed. If there is no abnormality in the appearance inspection (in the case of “Yes”), the sheet-like cell culture is detached from the culture substrate (Detachment), and the processing is ended (End). If an abnormality is observed in the appearance inspection (in the case of “No”), the medium is replaced (Medium replacement), and the sheet culture is resumed. If abnormalities are observed in the appearance inspection even in the sheet culture after the medium replacement, the processing may be terminated without further medium replacement.

  In addition, as shown in FIG. 2, when the value of X and / or Y is larger than the reference value, whether or not the sheet-like cell culture is detached from the culture substrate (Detached) by visual inspection is investigated. It is also possible to proceed in the case of partial peeling (in the case of “Partially”), and to advance to end (End) in the case of complete peeling (in the case of “Fully”). The determination of the state of detachment of the sheet-shaped cell culture from the culture substrate can be made only if a part of the sheet-shaped cell culture is detached from the culture substrate while maintaining the integrity of the sheet-shaped cell culture. Partially peeled (Fully) if the entire sheet-shaped cell culture is peeled from the culture substrate while maintaining the integrity of the sheet-shaped cell culture, otherwise peeled otherwise No (No) can be made. In addition, when the sheet-like cell culture is agglomerated after a certain period of time after peeling, the sheet-like cell culture may be discarded without being used.

  By processing according to the above algorithm, completion of sheeting can be detected quickly, and unnecessary culture time can be cut. In addition, if abnormalities are detected, the cells can be used effectively by correcting the abnormalities by replacing the medium or adding cells instead of discarding the cells. It can be reduced and the yield can be increased.

  In the algorithm shown in FIG. 2 (algorithm 2), after the start of the treatment (Start), cells are first seeded (Plating), and sheet-cultured (Incubation) so as to cover the semipermeable portion. To start. After a predetermined period of time, the transition label amount X is detected (Detection), and X is compared with a reference value. If X is larger than the reference value (“No”), the number of cells is examined from the appearance of the cell culture (the presence of voids and detached cells that can be confirmed with the naked eye), and if there is an abnormality (“No”) ) Sow seeding again. If there are no abnormalities (in the case of “Yes”), the state of the medium is inspected, and if abnormalities (for example, discoloration, turbidity, abnormal pH, etc.) are observed (in the case of “No”), the medium is replaced (Medium replacement), restart the sheet culture. If X is below the reference value (if “Yes”), perform visual inspection, and if there are no abnormalities (if “Yes”), peel off the cell culture from the culture substrate (Detachment) and treat End (End). When visual inspection reveals abnormalities (for example, the presence of voids or exfoliated cells that can be confirmed with the naked eye, contamination with foreign matter, etc.) (in the case of “No”), the sheet culture is stopped (Stop incubation) and the process is terminated. (End). In this algorithm, when the amount of transfer label X is larger than the reference value, the abnormality in the number of cells and the abnormality in the medium are distinguished, and appropriate measures are taken for each. Effective use, the number of cells used can be reduced as a whole, and the yield can be increased.

  In the algorithm shown in FIG. 3 (algorithm 3), after start of the treatment (Start), cells are first seeded (Plating), and sheet culture (Incubation 1) is performed so as to cover the semipermeable portion. ). After a predetermined period of time, the first transition label amount X is detected (Detection 1), and X is compared with the first reference value (Reference value 1). If X is larger than the first reference value (in the case of “No”), it is determined that the culture period is insufficient, and sheet culture (Incubation 1) is repeated. If X is not more than the first reference value (“Yes”), the sheet culture is continued (Incubation 2). After a predetermined period of time, detect the amount of transition marker (Detection 2), calculate the absolute value Y of the amount of change or rate of change with the amount of transition marker detected immediately before (Detection 1 or Detection 2), and set Y as the second Compare with the reference value of 2. If Y is greater than the second reference value (in the case of “No”), the total sheet culture period (ie, elapsed time from the start of sheet culture (Incubation 1)) “t” is within the reference range A to B If t is smaller than the lower limit A (when “t <A”), it is determined that the culture period is insufficient, and sheet culture (Incubation 2) is repeated, and t is within the reference range. If it is within A to B ("A ≤ t ≤ B"), it is determined that there is an abnormality in the medium, and after performing medium replacement, sheet culture (Incubation 2) is repeated, and t is If it is larger than the upper limit B (when “t> B”), it is determined that there is an abnormality in sheet formation, the culture is stopped (Stop incubation), and the processing is ended (End). On the other hand, if Y is less than or equal to the second reference value (in the case of “Yes”), the sheet-shaped cell culture is detached from the culture substrate (Detachment), and the process is ended (End). The reference ranges A to B are not particularly limited, and may be, for example, 24 hours to 72 hours. Appearance inspection can be performed before peeling to further detect appearance abnormalities. In this algorithm, when the completion of sheeting is late, it is possible to take appropriate measures according to the length of the total sheeting culture period, and it is possible to improve the production efficiency.

  The above algorithm represents a specific aspect of the controlled manufacturing method of the present invention, and it goes without saying that the controlled manufacturing method of the present invention includes not only these aspects but also various other aspects. . Further, the above or other algorithms relating to the controlled production method of the present invention may be implemented in a system for controlled production of a sheet-shaped cell culture described below.

Another aspect of the present invention is:
(1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semi-permeable portion through which the label passes but cells do not pass, and (3) a liquid-impermeable second container capable of containing the semi-permeable portion. Quality evaluation of isolated sheet cell culture, quality evaluation of laminated sheet cell culture, evaluation of cell purity or cell composition ratio of sheet cell culture, detection of abnormalities in manufacturing process of sheet cell culture, and And / or a kit for the controlled production of sheet cell cultures.

  The components (1) to (3) of the kit of the present invention are as described above for the quality evaluation method of the present invention. In addition, the kit of the present invention has an arbitrary configuration suitable for one or more of the above-mentioned applications, for example, a medium added to each container, a coloring reagent, a light shielding cover, an instruction manual for each application, and information on a usage method. May be further included, for example, a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, and the like.

Another aspect of the present invention is:
(1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semipermeable portion through which the label passes but cells do not pass;
(3) Quality evaluation of an isolated sheet-like cell culture, laminated sheet-like, comprising a liquid-impermeable second container capable of accommodating the semi-permeable portion, and (4) a detector for detecting a label For quality evaluation of cell culture, evaluation of cell purity or cell composition ratio of sheet-shaped cell culture, detection of abnormality in manufacturing process of sheet-shaped cell culture, and / or control production of sheet-shaped cell culture About the system.

  The components (1) to (3) of the system of the present invention are as described above for the quality evaluation method of the present invention. The detector of the component (4) is not particularly limited as long as it can detect the label of the present invention. For example, a photometer such as a spectrophotometer, an absorptiometer, a turbidimeter, a colorimeter, a scintillation counter Including radiation detectors. One skilled in the art can select a detector suitable for the label used. The system of the present invention may include only one type of detector or a plurality of types of detectors. The system of the present invention has an arbitrary configuration suitable for one or more of the above applications, for example, a processor for processing a signal from a detector and controlling an actuator, an interface such as a display device or an input device, a sign May be further provided with an injector for adding the above to the container, a manipulator for moving each container, an incubator for cell culture, a stirrer, and the like. In the system for controlled production of a sheet-shaped cell culture, the processor may be implemented with the above or other algorithms relating to the controlled production method of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, this is a specific example of the present invention, and the present invention is not limited thereto.

Example 1 Examination of pore density of semipermeable part As a first container, a pore diameter of 3 μm, a pore density of 8.0 ± 2 × 10 ⁵ pieces / cm ² (low density) or 2.0 ± 0.2 × 10 ^A transparent / transparent 6-well culture insert (BD) made of PET having a density of ⁵ / cm ² (high density) was used as a second container. The culture insert was inserted into a plate well containing 2.7 ml of DMEM-F12 medium (Life Technologies) containing 20% human serum, and 4.4 × 10 ⁶ skeletal myoblasts prepared from human muscle tissue by a conventional method were used. The culture insert was suspended and seeded in 1.5 ml of the same medium as in the plate well at the density of the culture insert. Sheet culture was performed at 37 ° C. and 5% CO _{2 for} about 17 hours. After the sheet culture, the medium in the insert was changed to an HBSS (+) solution containing 50 μg / ml FITC-labeled dextran (SIGMA, average molecular weight 3000 to 5000), and the medium in the plate well was changed to 50 μg / ml unlabeled dextran. Labels were added by replacing each with an HBSS (+) solution containing (SIGMA, molecular weight ˜6000) and incubated at 25 ° C. for 1 hour. Next, the insert was taken out, and the absorbance of FITC contained in the well on the plate side was read with a plate reader (485 nm / 538 nm). The results are shown in Table 1.

As described above, it can be seen that the amount of FITC-labeled dextran migrating to the plate side is significantly reduced by the presence of the sheet-shaped cell culture. Moreover, although the difference by the difference in the pore density of an insert was hardly seen, the tendency for a better sensitivity to be obtained was seen that the density was high.

Example 2 Examination of label diffusion time A 6-well culture insert (BD) having a pore size of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ cells / cm ² was used as the first container, and incubation after label addition was performed. The same experiment as in Example 1 was performed except that the time was 20 minutes, 6 hours, or 23 hours. The results are shown in Table 2.

  As in Example 1, it was confirmed that the amount of FITC-labeled dextran transferred to the plate side was significantly reduced by the presence of the sheet-shaped cell culture. From this, by measuring the amount of FITC-labeled dextran that migrates to the plate side, how much the sheet-like cell culture permeates FITC-labeled dextran, that is, how much intercellular space within the sheet-like cell culture. Can be evaluated. Further, since the label continues to move to the plate side for 23 hours after the addition, it can be seen that the change in permeability can be monitored for a relatively long time by this method.

Example 3 Examination of influence by cell density A 6-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ cells / cm ² was used as a first container, and the cell density to be seeded was determined. 4.4 × 10 ⁶ cells / well (Normal), 2.2 × 10 ⁶ cells / well (1/2 density), 8.8 × 10 ⁵ cells / well (1/5 density) or 4.4 × 10 The same experiment as in Example 1 was performed except that the number of cells was ⁵ / well (1/10 density) and the incubation time after addition of the label was 3 hours. Measurement with only the insert was taken as Control. The results are shown in relative fluorescence units (RFU) with Control as 100. From the results shown in FIG. 1, it can be seen that the amount of FITC-labeled dextran transferred increases as the cell density decreases. Therefore, according to this method, the cell density of the sheet-like cell culture can also be evaluated.

Example 4 Examination of influence of cell viability As a first container, a 6-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ cells / cm ² was used. The cell viability is increased after seeding skeletal myoblasts in the same manner as in Example 1 except that the number of cells is 2.2 × 10 ⁶ cells / well, performing sheet culture, and forming a sheet-like cell culture. Served under conditions (room temperature storage: HBSS (+), stored at 27 ° C. for 24 hours) or conditions that do not decrease (refrigerated storage: HBSS (+), stored at about 4 ° C. (2-8 ° C.) for 24 hours) did. Next, FITC-labeled dextran was added in the same manner as in Example 1, and after 2 hours of incubation, the amount transferred to the plate side was measured. Measurement with only the insert was taken as Control. The results are shown in relative fluorescence units (RFU) with Control as 100. From the results shown in FIG. 2, it can be seen that the amount of FITC-labeled dextran transferred to the plate side increases as the viability of the cells decreases. Therefore, the viability of the cells constituting the sheet-like cell culture can be evaluated by this method.

In another experiment, it was confirmed how the viability of cells changes with room temperature storage or refrigerated storage. Skeletal myoblasts are seeded in a 3.5 cm temperature-responsive culture dish (UpCell ^(R) , 3.5 cm dish, CellSeed) with the same cell density and medium combination as in Example 1, and sheet culture is performed. After formation of dendritic cell cultures, the medium was replaced with HBSS (+) and stored at 27 ° C. or 2 ° C. for 24 or 48 hours. After the storage period, the cells were enzymatically separated, and viability was measured by cell count after trypan blue staining. As a control, the viability of the sheet-shaped cell culture without storage (unstored group) was also measured. The experiment was performed on four different lots of cells. The results are shown in Table 3.

Each numerical value represents the amount of change ± standard error (% point) relative to the viability (%) of the unstored group. The difference in viability due to storage temperature increases with time, but is relatively small at 24 hours after storage. Nevertheless, in the quality evaluation method of the present invention, there is a large difference in the amount of migration between room temperature storage and refrigerated storage at 24 hours after storage. It can be seen that the difference can be evaluated with higher sensitivity.

Example 5 Determination of Completion of Sheeting The following experiment was conducted to verify how the amount of label transferred to the second container changes as the sheeting progresses. As a first container, a 6-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ cells / cm ² was used. Seeding was performed at a density of 10 ⁶ cells / well. Cells were seeded at the same time in 4 wells, and sheet culture was performed. After 3 hours, 6 hours, 21 hours and 28 hours after seeding, a label was added to each different well in the same manner as in Example 1. After incubating at 25 ° C. for 2 hours, the absorbance of FITC contained in the plate-side well was read with a plate reader (485 nm / 538 nm). The results are shown in relative fluorescence units (RFU) with the insert alone measurement taken as 100. From the results shown in FIG. 3, it can be seen that RFU (that is, the amount of FITC-labeled dextran transferred to the plate side) rapidly decreases with the passage of time for sheet culture and is maintained at a certain level thereafter. Therefore, it can be determined that the sheeting is completed when the rapid decrease of the RFU stops and is maintained at a substantially constant level. Therefore, according to the method of the present invention, it is possible to determine completion of sheet formation of a sheet-shaped cell culture, which has been difficult to determine conventionally.

Example 6 Detection of Abnormality of Sheet Formation In order to examine whether an abnormality in sheet formation can be detected by the method of the present invention, the following experiment was performed. A 6-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ pieces / cm ² was used as the first container, and the sheet culture time was 21 to 27 hours. The amount of label transferred to the second container was measured in the same manner as in Example 1 except that the incubation time was 2 hours. At the same time as the labeled transition experiments, the cells of the same lot, the temperature-responsive culture vessels at the same density ^{(UpCell (R),} 3.5cm dish, CellSeed) were seeded in performs sheeting culture, sheets in the process It was observed whether peeling occurred. Cells were run on 5 different lots. From the results shown in FIG. 4, the amount of label transferred in the sheet-like cell culture prepared on the culture insert with the same lot of cells that were detached during the process of sheet culture in the temperature-responsive culture vessel (Detached) However, it can be seen that it is significantly more than that of the lot where no peeling occurred (Normal). Sheet exfoliation in sheet culture is considered to be due to the vitality of the cells constituting the sheet and insufficient sheet-forming ability, so that the sheet-forming ability of cells is evaluated by the method of the present invention, Whether or not it is suitable for use in producing a sheet-shaped cell culture can be determined before actually producing the sheet-shaped cell culture.

Example 7 Detection of the number of laminated sheet-like cell cultures The following experiment was conducted to examine whether the number of laminated sheet-like cell cultures can be detected by the method of the present invention.
Translucent PET 24-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ / cm ² as the first container, and colorless and transparent polystyrene as the second container A 24-well plate (BD) was used.
Temperature responsive culture vessel ^{(UpCell (R),} 48-well multi-well, CellSeed), the seeded skeletal myoblasts were prepared by a conventional method from human muscle tissue at a density of 1.3 × 10 ⁶ cells / well, 20% Sheet culture was performed in DMEM-F12 medium (Life Technologies) containing human serum at 37 ° C. and 5% CO ₂ . The formed sheet-shaped cell culture was peeled off by temperature treatment to room temperature, washed with HBSS +, and transferred to the first container. The cells were cultured in the same medium as above at 37 ° C. and 5% CO ₂ for 1 hour, and the sheet-like cell culture was adhered to the container and further cultured overnight. The laminated sheet-shaped cell culture was prepared by placing the formed sheet-shaped cell culture on the sheet-shaped cell culture adhered to the container and culturing overnight. After culturing, the medium in the insert was changed to an HBSS (+) solution containing 100 μg / ml FITC-labeled dextran (SIGMA, average molecular weight 3000 to 5000), and the medium in the plate well was changed to 100 μg / ml unlabeled dextran (SIGMA The label was added by replacing each with an HBSS (+) solution containing ˜6000 molecular weight, and incubated at room temperature for 2 hours. Next, the insert was taken out, and the absorbance of FITC contained in the well on the plate side was read with a plate reader (485 nm / 538 nm). The results are shown in Table 4.

In the table, RFU represents relative fluorescence units with a measured value in a control experiment using an insert not containing a sheet-like cell culture as 100. From the above results, it can be seen that the quality of the already formed sheet-shaped cell culture can be evaluated by the method of the present invention. In addition, since the RFU is smaller in the case of the laminated sheet-shaped cell culture in which two sheet-shaped cell cultures are stacked, the RFU is smaller than the number of the sheet-shaped cell cultures. It turns out that it becomes small depending on this, and the number of laminated | stacked sheet-like cell cultures can be determined by the method of this invention.

Example 8 Detection of Cell Purity In order to examine whether the purity of the cells constituting the sheet cell culture can be detected by the method of the present invention, the following experiment was conducted.
Translucent PET 24-well culture insert (BD) having a pore diameter of 3 μm and a pore density of 2.0 ± 0.2 × 10 ⁵ / cm ² as the first container, and colorless and transparent polystyrene as the second container A 24-well plate (BD) was used.
Skeletal myoblasts prepared from human muscle tissue by a conventional method and fibroblasts (TIG-2M-30, Human Science Foundation) are mixed, and the ratio of CD56 positive cells, which are markers of skeletal myoblasts, is 69. %, 32% or 0% specimens were prepared. The ratio of CD56 positive cells was measured by a flow cytometry method using a PE-labeled anti-CD56 antibody (BD, 555516) (BD, FACS Calibur). Each specimen was seeded in a first container at a density of 7.93 × 10 ⁴ cells / well, and 6 in a DMEM-F12 medium (Life Technologies) containing 20% human serum at 37 ° C. and 5% CO ₂ . Time sheet culture was performed. After the culture, the absorbance of FITC contained in the well on the plate side was measured in the same manner as in Example 7. The results are shown in FIG. In the figure, RFU represents relative fluorescence units with the measured value in a control experiment using an insert not containing a sheet-like cell culture as 100. From the above results, it can be seen that as the ratio of skeletal myoblasts increases, RFU increases, that is, as the ratio of skeletal myoblasts increases, the amount of label transfer increases, and as the ratio of fibroblasts increases, the labeling increases. It can be seen that the amount of migration of the cells decreases, and that the label permeability of the sheet-shaped cell culture varies depending on the type and composition ratio of the constituent cells.

  The various features of the invention described herein can be combined in various ways, and all aspects obtained by such combinations, including combinations not specifically described herein, can Within range. Further, those skilled in the art understand that many various modifications are possible without departing from the spirit of the present invention, and equivalents including such modifications are also included in the scope of the present invention. Accordingly, it should be understood that the embodiments described herein are exemplary only, and are not intended to limit the scope of the present invention.

Claims (9)

(1) seeding cells in a first container having a semi-permeable portion through which a label passes but cells do not pass, and forming a sheet so as to coat the semi-permeable portion;
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) A method for detecting the number of laminated sheet-shaped cell cultures or the cell purity of the sheet-shaped cell culture, which may be performed simultaneously with step (1) or after step (1). (1) seeding cells in a first container having a semi-permeable portion through which a label passes but cells do not pass, and forming a sheet so as to coat the semi-permeable portion;
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) including a step of detecting the label transferred to the container to which the label is not added, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes step (1) At the same time as before step (1) or after step (1),
When the amount of label detected or increased by step (4) becomes almost constant in multiple consecutive measurements, or when the absolute value of the rate of change over time in the amount of label falls below the reference value To determine that the sheeting is complete ,
A method for detecting completion of sheeting, wherein the cell is a skeletal myoblast . (1) a step of seeding cells in a first container having a semipermeable portion through which a label passes but cells do not pass, and culturing the sheet so as to coat the semipermeable portion;
(2) adding a label to the first container or a liquid-impermeable second container that can be in fluid communication with the first container via the semi-permeable portion;
(3) In a state where the first container and the second container are in fluid communication via the semipermeable part, the label is a container to which the label is not added among the first container and the second container. Transitioning through a semi-permeable part,
(4) detecting the label transferred to the container to which the label is not added;
(5) Sheet culture when the amount or increase of the label becomes almost constant in a plurality of consecutive measurements, or when the absolute value of the rate of change over time in the amount of label is below the reference value And the step of recovering the sheet-shaped cell culture from the first container, wherein the label is inhibited from passing between cells by cell-cell adhesion, and step (2) includes steps (1) and simultaneous, rather it may also be carried out after the step (1) than before or step (1),
A method for producing a sheet-like cell culture for transplantation, wherein the cells are skeletal myoblasts .   4. A method according to any one of claims 1 to 3, wherein the label has a molecular weight of 100 to 1500,000.   The method according to any one of claims 1 to 4, wherein the label is selected from the group consisting of a fluorescent label, an enzyme label, a luminescent label, a radioactive label, a magnetic label and a colorimetric label.   6. The method according to any one of claims 1 to 5, wherein the semipermeable portion has pores with a pore size of about 0.01 [mu] m to about 50.0 [mu] m. The method of claim 1, wherein the cells are selected from the group consisting of myoblasts and mesenchymal stem cells. (1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semi-permeable portion through which the label passes but cells do not pass, and (3) a liquid-impermeable second container capable of containing the semi-permeable portion. The kit for using for the method as described in any one of Claims 1-7. (1) a label that inhibits passage between cells by cell-cell adhesion;
(2) a first container having a semipermeable portion through which the label passes but cells do not pass;
The method according to any one of claims 1 to 7, comprising (3) a liquid-impermeable second container capable of accommodating the semi-permeable portion, and (4) a detector for detecting a label. System for use in.