JP6756502B2 - Cell culture and its production method - Google Patents

Description

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

In recent years, attempts have been made to transplant various cells for repairing damaged tissues and the like. For example, use of fetal myocardial cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for repair of myocardial tissue damaged by ischemic heart disease such as angina and myocardial infarction. Has been attempted (Non-Patent Documents 1 to 4).

As part of such an attempt, a cell structure formed by using a scaffold and a sheet-shaped cell culture in which cells are formed in a sheet shape have been developed (Patent Document 1).
Regarding the therapeutic application of sheet-shaped cell culture, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet-shaped cell culture for corneal damage, oral mucosal sheet-like for endoscopic resection of esophageal cancer Studies on the use of cell cultures are underway, and some of them are in the stage of clinical application.

In the clinical application of such cell cultures, the quantity and quality of the cell cultures determine the correctness of treatment, so it is necessary to properly control the manufacturing process and quality thereof. For that purpose, it is important to detect changes and abnormalities in the cell culture during the manufacturing process as soon as possible and take necessary measures. In addition, if it is possible to monitor whether the cell culture is functioning properly after being applied to the subject, it is possible to respond promptly when signs of dysfunction are observed and prevent the occurrence of adverse events. It will be possible. However, a technique capable of solving the above-mentioned problems related to clinical application of cell culture is not yet known.

Special Table 2007-528755

Haraguchi et al., Stem Cells Transl Med. 2012; 1 (2): 136-41 Sawa et al., Surg Today. 2012; 42 (2): 181-4 Matsuura et al., Biomaterials. 2011; 32 (30): 7355-62 Kawamura et al., Circulation. 2012; 126 (11 Suppl 1): S29-37

An object of the present invention is to provide a cell culture capable of proper control of a manufacturing process and quality, and / or monitoring after being applied to a subject, a manufacturing method thereof, and the like.

While studying sheet-shaped cell cultures, the present inventor has noticed that the sheet-shaped cell cultures may spontaneously exfoliate from the culture substrate during the sheet-forming culture period. In such a case, if the sheet-shaped cell culture is left as it is without noticing the peeling from the culture substrate, the sheet-shaped cell culture is deformed and becomes unsuitable for clinical use. The occurrence of such early exfoliation is considered to be due in part to the strong cell-cell binding in the sheet-shaped cell culture, but the strong cell-cell binding is rather a preferable property in the treatment with the sheet-shaped cell culture. It is not desirable to discard such sheet cell cultures. Therefore, if the above-mentioned early exfoliation can be detected early, the present inventor can take measures such as collecting the exfoliated sheet-shaped cell culture and placing it on a support until it is used to prevent deformation. We thought that it would lead to effective utilization of such sheet-shaped cell culture, and as a result of further research, we found that by mixing a sensor chip with the cell culture during sheet-forming culture, the sheet-shaped cell culture was peeled off from the culture substrate. Was found to be able to be detected at an early stage. It was also found that the state of the sheet-shaped cell culture applied to the subject can be monitored by mixing the sensor chip with the cell culture during sheet-forming culture and embedding the sensor chip in the sheet-shaped cell culture as described above.

That is, one aspect of the present invention relates to the following.
<1> (1) A step of mixing a sensor chip and a cell suspension to prepare a cell suspension containing the sensor chip.
(2) A step of forming a sheet-like cell culture containing a sensor chip by forming a sheet of cells contained in a cell suspension together with a sensor chip on a culture substrate.
(3) A step of detecting exfoliation of a sheet-shaped cell culture from a culture medium based on information from a sensor chip.
A method for detecting exfoliation of a sheet-like cell culture from a culture medium, which comprises.
<2> (1) A step of mixing a sensor chip and a cell suspension to prepare a cell suspension containing the sensor chip.
(2) A step of forming a sheet-like cell culture containing a sensor chip by forming a sheet of cells contained in a cell suspension together with a sensor chip on a culture substrate.
(3) A step of detecting exfoliation of a sheet-shaped cell culture from a culture medium based on information from a sensor chip.
(4) A method for producing a sheet-shaped cell culture, which comprises a step of collecting the sheet-shaped cell culture in which peeling from the culture substrate is detected.

<3> The method according to <1> or <2> above, wherein the sensor chip has a communication function.
<4> The item according to any one of <1> to <3> above, wherein the sensor chip can measure an item selected from the group consisting of tension, pressure, displacement, ion concentration and bioactive substance concentration. Method.
<5> The method according to any one of <1> to <4> above, wherein the sensor chip stores individual identification information.
<6> A system for detecting peeling of a sheet-shaped cell culture from a substrate, which comprises a sensor chip embedded in the sheet-shaped cell culture and a reading device for reading information from the sensor chip.

<7> A sheet-like cell culture containing a sensor chip.
<8> The sheet-shaped cell culture according to <7> above, wherein the sensor chip has a communication function.
<9> The sheet-like cell culture according to <7> or <8> above, wherein the sensor chip can measure an item selected from the group consisting of tension, pressure, displacement, ion concentration and bioactive substance concentration. ..
<10> The sheet-shaped cell culture according to any one of <7> to <9> above, wherein the sensor chip stores individual identification information.
<11> The cell culture according to any one of <7> to <10> above for treating a disease related to tissue abnormality.
<12> A kit for producing a sheet-shaped cell culture containing a sensor chip, which comprises a cell suspension containing the sensor chip and an element used for producing the sheet-shaped cell culture.
<13> The kit according to <12> above, wherein the sensor chip stores individual identification information.

<14> The sheet-shaped cell culture comprising a step of reading information transmitted from a sensor chip contained in the sheet-shaped cell culture according to any one of the above <8> to <11> applied to the subject. How to inspect the condition of an object.
<15> Examining the effect of treating a disease by the sheet-shaped cell culture, which comprises a step of reading information transmitted from a sensor chip contained in the sheet-shaped cell culture according to <11> applied to the subject. how to.
<16> The step of reading the identification information stored in the sheet-shaped cell culture according to any one of the above <7> to <11> or the sensor chip included in the kit according to claim 12 or 13 is included. , A method of identifying a target to which a sheet cell culture is applied.
<17> A method for treating a disease related to a tissue abnormality in a subject, which requires an effective amount of the sheet-shaped cell culture according to any one of <7> to <11>. The method comprising administering to the subject.

<18> Cardiomyocyte culture containing a sensor chip.
<19> The cell culture according to <18> above, wherein the sensor chip has a communication function.
<20> The cell culture according to <18> or <19>, which is in the form of a sheet.
<21> The cell culture according to any one of <18> to <20> above, wherein the cardiomyocytes are derived from iPS cells.
<22> Any one of <18> to <21> above, wherein the sensor chip can measure an item selected from the group consisting of the beat, action potential, ion concentration and physiologically active substance concentration of the cell culture. The cell culture according to the section.
<23> The cell culture according to any one of <18> to <22> above, wherein the sensor chip stores individual identification information.
<24> The cell culture according to any one of <18> to <23> above for treating heart disease.

<25> The method for producing a cell culture according to any one of <18> to <24> above, which comprises a step of mixing a cell suspension containing cardiomyocytes and a sensor chip.
<26> A kit for producing the cell culture according to any one of <18> to <24> above, which comprises a cardiomyocyte suspension containing a sensor chip.
<27> The kit according to <26> above, wherein the cell culture is in the form of a sheet and further contains elements used for producing the sheet-like cell culture.
<28> The kit according to <26> or <27> above, wherein the sensor chip stores individual identification information.

<29> The state of the cell culture including the step of reading the information transmitted from the sensor chip contained in the cell culture according to any one of the above <19> to <24> applied to the subject. How to inspect.
<30> A method for examining the effect of treatment of heart disease by the cell culture, which comprises a step of reading information transmitted from a sensor chip contained in the cell culture according to the above <24> applied to a subject.
<31> A cardiomyocyte culture comprising the step of reading the identification information stored in the cell culture according to the above <23> or <24> or the sensor chip included in the kit according to the above <28> is applied. How to identify the target.
<32> A method for treating a heart disease in a subject, wherein an effective amount of the cell culture according to any one of <18> to <24> is administered to the subject in need thereof. Included, said method.

INDUSTRIAL APPLICABILITY According to the present invention, peeling of a sheet-shaped cell culture from a culture substrate can be detected at an early stage, unwanted deformation of the sheet-shaped cell culture can be prevented, and the efficiency of production of the sheet-shaped cell culture can be improved. Is possible. Further, according to the present invention, it is possible to monitor the cell culture after application to the subject, and it is possible to obtain useful information for evaluating the therapeutic effect and determining the necessity of adjunctive treatment. Furthermore, according to the present invention, it is possible to track the correspondence between the cell culture and the subject from the initial stage of the production process of the cell culture to the series of work steps from the application to the subject, and the cell culture is applied. It is possible to effectively prevent the mistake of the target to be performed.

FIG. 1 is a view showing a cross section of a sheet-shaped cell culture containing a sensor chip. FIG. 2 is a view showing the bottom surface of a sheet-shaped cell culture containing a sensor chip. FIG. 3 is a diagram showing an example of a method of embedding a sensor chip in a sheet-shaped cell culture. FIG. 4 is a diagram showing an example of a method for inspecting a sheet-shaped cell culture applied to a target heart from outside the body. FIG. 5 is a diagram showing an example of a system for detecting exfoliation of a sheet-shaped cell culture from a culture medium. FIG. 6 is a diagram showing changes in the position of the sensor chip before and after the sheet-shaped cell culture is exfoliated from the culture substrate. FIG. 7 is a diagram showing a change in the position of the sensor chip after a part of the sheet-shaped cell culture is exfoliated from the culture substrate. FIG. 8 is a diagram showing a change in the position of the sensor chip after the entire sheet-shaped cell culture is exfoliated from the culture substrate.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those 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.
Hereinafter, embodiments of the present invention will be described with reference to the drawings. It should be noted that the embodiments described below are intended to illustrate the present invention, and are not intended to limit the present invention.

<Cell culture containing sensor chip>
FIG. 1 is a cross-sectional view of a sheet-shaped cell culture containing the sensor chip according to the present invention. The sensor chip 1 exists in a form of being embedded in the extracellular matrix layer 21 produced by the sheet-shaped cell culture 2 under the sheet-shaped cell culture 2. FIG. 2 is a conceptual diagram showing the bottom surface of the sheet-shaped cell culture 2.

The sensor chip is a chip having a sensor function. The chip typically comprises an integrated circuit (IC) formed on a semiconductor substrate (eg, a silicon wafer) or a non-semiconductor substrate (eg, a biodegradable substrate made of cellulose nanofibers or the like). The sensor chip is preferably smaller in size than the cell culture. IC chips are becoming smaller, and many micrometer-sized IC chips have been developed (Burke and Rutherglen, Biomed Microdevices. 2010; 12 (4): 589-96). Sensors are also becoming smaller, and nano-sized sensors using carbon nanotubes have been developed (Tilmaciu and Morris, Front Chem. 2015; 3:59, Lerner et al., ACS Nano. 2012; 6). (6): 5143-9).

The sensor chip 1 shown in FIGS. 1 and 2 has a thin and flat shape according to the shape of the sheet-shaped cell culture 2, but has various shapes such as a columnar shape, a spherical shape, and a dice shape depending on the shape of the cell culture. You can. The sensor chip may be completely encapsulated in the cell culture or part of it may be exposed outside the cell culture. Here, "complete inclusion" means that the sensor chip is covered with components (cells, extracellular matrix, etc.) contained in the cell culture and is not exposed to the outside. When the sensor chip is completely encapsulated in the cell culture, the risk of the sensor chip falling out of the cell culture can be minimized. On the other hand, when obtaining information outside the cell culture, it may be preferable that a part of the sensor, for example, an electrode for measurement or the like is exposed outside the cell culture. Therefore, it is preferable that the sensor chip is encapsulated in the cell culture in order to prevent the sensor chip from falling off within a range that does not interfere with the function of the sensor. As a result, when the sensor chip is integrated with the cell culture and applied to the subject, the location of the cell culture can be detected more reliably, and the function of the cell culture can be directly monitored. Further, when the sheet-shaped cell culture is peeled from the culture substrate, the sensor chip is released from the culture substrate together with the sheet-shaped cell culture, and the sheet-shaped cell culture is shrunk according to the contraction of the sheet-shaped cell culture. Since it changes its position, it is also useful for early detection of exfoliation of sheet-like cell cultures. The sensor chip may have a hydrophilic treatment on the surface or may be coated with an extracellular matrix, a cell adhesion factor, fibrin, or the like in order to increase the adhesion to cells and reduce the risk of falling off.

The sensor chip preferably has a communication function. RFID tags are well known as chips with communication functions, and wireless sensors applying this technology have also been developed (Dai et al., Sensors (Basel). 2009; 9 (11): 8748-60, Deng et al., Sensors (Basel). 2015; 15 (3): 6872-84). The sensor chip may be an active type having a built-in battery or a passive type having no battery, but the passive type is preferable because the size can be reduced. The passive type typically includes an antenna for receiving power, converts radio waves transmitted from an external power supply device into electric power, and uses this for various processes.

The sensor can be configured to measure various items depending on the purpose of the sensor chip. Such items include, but are not limited to, pulsation, action potential, tension, pressure, displacement, ion concentration, physiologically active substance concentration, and the like of the cell culture. Measurement of the beat and / or action potential of the cell culture is useful, for example, to monitor its function when the cell culture is a cardiomyocyte culture. Measurements of tension, pressure and / or displacement are useful, for example, to detect signs or development of detachment when the cell culture is a sheet-like cell culture. The measurement of the ion concentration is useful, for example, when the cell culture is a myocardial cell culture, it is useful for detecting the generation of an active potential and monitoring the function of the myocardial cell culture. In the case of a sheet-shaped cell culture, the entry of a medium such as a medium between the culture substrate and the sheet-shaped cell culture that occurs when the sheet-shaped cell culture is peeled off is detected, and the sheet-shaped cell culture is cultured. It is useful for quickly detecting peeling. The measurement of the concentration of physiologically active substances has been suggested to produce or induce the production of cytokines such as VEGF, bFGF, and HGF as one of the therapeutic mechanisms for heart diseases such as myocardial cell cultures and myoblast cultures. (Non-Patent Documents 2 and 4), it is useful for evaluating or predicting the therapeutic effect of a myocardial cell culture or a myoblast culture.

The sensor chip may preferably store individual identification information. Such identification information is useful, for example, for identifying the target to which the cell culture is applied, tracking the cell culture, and identifying each sensor chip when the cell culture contains a plurality of sensor chips. The identification information may be rewritable or non-rewritable.

The number of sensor chips 1 shown in FIGS. 1 and 2 is 2 to 3 per sheet-shaped cell culture, but the number is not limited to this, and one cell culture may have one or more sensor chips. It may be included. To prepare for unexpected peeling, breakage or failure of the sensor chips, to detect the degree of peeling of the sheet-shaped cell culture from the culture substrate, and between the sensor chips contained in one cell culture. In order to use the information related to the relative positional relationship of, for example, to detect the contraction of the sheet-shaped cell culture due to exfoliation, the cell culture preferably contains two or more sensor chips.

Sheet-shaped cell culture refers to cells that are connected to each other to form a sheet. 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 capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediators are preferably derived from cells, particularly those derived from the cells that make up the cell culture. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminated (multilayer), for example, two layers, three layers, four layers. Layers, 5 layers, 6 layers, etc.) may be used. The sheet-shaped cell culture may be attached to the culture substrate or released (peeled), but is typically released from the culture substrate while maintaining the sheet shape (isolation). It may also be referred to as a sheet-like cell culture or a free sheet-like cell culture).

Although the sheet-shaped cell culture 2 is shown in FIGS. 1 and 2, the present invention can be applied to cell cultures other than the sheet-shaped cell culture. The cell culture means a culture obtained by culturing cells, and can take various forms such as a cell suspension and a cell structure having a predetermined shape and structure. The cell structure can have various shapes such as a columnar shape, a lump shape, and a plug shape in addition to the sheet shape. Of these, sheet-shaped cell cultures are preferable from the viewpoint of high therapeutic effect.

The cell culture may or may not contain a scaffold (support), but it is preferable that the cell culture does not contain a scaffold (support) from the viewpoint of biocompatibility and high therapeutic effect. Scaffolds may be used in the art to attach cells to and / or inside the scaffold to maintain the physical integrity of the cell culture, eg, polyvinylidene fluoride (PVDF). Although manufactured membranes and the like are known, preferred cell cultures are capable of maintaining their physical integrity without such scaffolds. In addition, the cell culture preferably comprises only the cell-derived substances constituting the cell culture, and does not contain any other substances.

The cells contained in the cell culture include any type of cells, but cells used for medical purposes are preferable. Such cells are not limited, and include, for example, hematopoietic stem cells used for treatment of blood diseases, lymphocytes used for immunotherapy, immune cells such as dendritic cells, and cells capable of forming sheet-like cell cultures. Can be mentioned. The cells capable of forming a sheet-like cell culture include, but are not limited to, for example, adherent cells (adherent cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocellular cells, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, heart stem cells, adipose stem cells, hepatic stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, It includes mesenchymal stem cells, etc.). Somatic cells may be differentiated from stem cells, especially iPS cells. Non-limiting examples of cells capable of forming 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, endometrial membrane, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, heart stem cells, adipose stem cells, hepatic stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (For example, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.) ), Renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin cells and the like.

Examples of preferred cells include cardiomyocytes, particularly cardiomyocytes derived from pluripotent stem cells (ie, cells having the characteristics of cardiomyocytes derived from pluripotent stem cells). Pluripotent stem cells are a well-known term in the art and mean cells that have the ability to differentiate into various tissues of the body. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Of these, iPS cells with less ethical problems are preferable. The characteristics of cardiomyocytes include, but are not limited to, expression of cardiomyocyte markers, presence of autonomous pulsation, and the like. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like. ..

Cardiomyocytes derived from pluripotent stem cells can be obtained by inducing cardiomyocytes from pluripotent stem cells, that is, by subjecting the pluripotent stem cells to cardiomyocyte induction treatment. Various methods for inducing cardiomyocytes from pluripotent stem cells are known (for example, Non-Patent Documents 3-4, Burridge et al., Cell Stem Cell. 2012 Jan 6; 10 (1): 16-28). .. Non-limiting examples of such an induction method include, for example, a method by embryoid body formation, a method by monolayer differentiation culture, a method by forced aggregation, and the like. Cardiomyocytes derived from pluripotent stem cells can be purified after induction to increase their purity. Purification methods include various separation methods using cardiomyocyte-specific markers (eg, cell surface markers), such as magnetic cell separation method (MACS), flow cytometry method, affinity separation method, and specific methods. Examples include a method of expressing a selectable marker (for example, an antibiotic resistance gene) by a promoter, and a combination of these methods (see, for example, Burridge et al. Above). Examples of cell surface markers specific to cardiomyocytes include CD172a, KDR, PDGFRA, EMILIN2, VCAM and the like. In addition, examples of promoters specific to cardiomyocytes include NKX2-5, MYH6, MLC2V, ISL1 and the like.

The cells can be derived from any multicellular organism (eg, animal, etc.) that can be treated with cell culture. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Includes mammals. Further, although only one type of cell may be used for forming the cell culture, two or more types of cells may be used. In a preferred embodiment, when there are two or more types of cells forming a cell culture, the ratio (purity) of the most abundant cells is 60% or more, preferably 70% or more, more preferably 75 at the end of cell culture production. % Or more.

The cell may be a heterologous cell or an allogeneic cell. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), i.e., recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferable from the viewpoint of clinical application because they do not cause rejection even when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In addition, in this specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells. In one embodiment, the cell is an autologous cell or an allogeneic cell. In one embodiment, the cell is an autologous cell. In another embodiment, the cell is an allogeneic cell.

Cell cultures are useful in the treatment of various diseases, especially those associated with tissue abnormalities. Therefore, in one embodiment, the cell culture is for use in the treatment of diseases associated with tissue abnormalities. Tissues to be treated include, but are not limited to, for example, myocardium, cornea, retina, esophagus, skin, articular cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, etc. Be done. In addition, the diseases to be treated are not limited, for example, heart diseases (for example, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.), corneal diseases (for example, corneal epithelial stem cell exhaustion, corneal membrane). Injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens Johnson syndrome, ocular herbitis, etc.), retinal disease (eg, retinal pigment degeneration, age-related yellow spot degeneration, etc.) , Esophageal disease (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin disease (eg, skin injury (trauma, burn), etc.), joint disease (eg, osteoarthritis) , Cartilage disease (eg, cartilage damage), Liver disease (eg, chronic liver disease), Pancreatic disease (eg, diabetes), Dental disease (eg, periodontal disease), Kidney disease (eg, renal failure) , Renal anemia, renal osteodystrophy, etc.), thyroid disease (eg, hypothyroidism), muscle disease (eg, muscle injury, myitis, etc.), middle ear disease (eg, middle ear inflammation, etc.), bone marrow disease (For example, leukemia, poor regeneration anemia, immunodeficiency disease, etc.).

The usefulness of cell cultures for the above diseases is described, for example, in Patent Document 1, Non-Patent Documents 1 to 4, Arauchi et al., Tissue Eng Part A. 2009; 15 (12): 3943-9, Ito et al. ., Tissue Eng. 2005; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007; 127 (10) 1038-44, Watanabe et al., Transplantation. 2011; 91 (7): 700-6, Shimizu et al., Biomaterials. 2009; 30 (30): 5943-9, Ebihara et al., Biomaterials. 2012; 33 (15): 3846-51, Takagi et al., World J Gastroenterol. 2012; 18 (37): 5145-50, etc.

Cell cultures can be applied to the tissue to be treated and used to repair and regenerate it, but as a source of bioactive substances such as hormones, sites other than the tissue to be treated ( It can also be transplanted into subcutaneous tissue (eg, Arauchi et al., Shimizu et al., Etc.).

Cell cultures include a variety of additional ingredients, such as pharmaceutically acceptable carriers, ingredients that enhance the viability, engraftment and / or function of cell cultures, and other effects useful in the treatment of the disease of interest. It may further contain an ingredient or the like. Any known additional component can be used as such additional component, and those skilled in the art are familiar with these additional components. In addition, the cell culture can be used in combination with an ingredient that enhances the viability, engraftment and / or function of the cell culture, and other active ingredients that are useful for treating a disease of interest.

The cell culture containing the sensor chip can be produced by a method including a step of mixing the sensor chip and the cell suspension for forming the cell culture. When the cell culture is formed by seeding the cell suspension on the culture substrate, a mixed solution of the sensor chip and the cell suspension may be seeded on the culture substrate, or the cell suspension may be seeded. May be seeded on the culture substrate on which the sensor chip is placed, or the sensor chip may be added to the cell suspension on the culture substrate after seeding the cell suspension on the culture substrate. By mixing and culturing the sensor chip with the cell suspension before, at the same time as seeding, or early after seeding, the sensor chip can be easily wrapped in the extracellular matrix produced by the cells. , Efficient embedding is possible.

When the cell culture is produced by layering a plurality of cell layers, as shown in FIG. 3, the sensor chip 1 is placed on the lower cell layer 22, and the upper cell layer 23 is placed on the sensor chip 1. By layering, the sensor chip can also be embedded in the cell culture. In addition, when the cell culture contains a support, the sensor chip can be attached to the cell culture by attaching the sensor chip to the support or by placing the sensor chip on the support and seeding the cells on the sensor chip. Can be embedded. Further, it is also possible to embed the sensor chip in the cell culture by placing the sensor chip on the cell culture and coating it with an adhesive component such as fibrin glue.

The cells can be cultured under the conditions normally used in the art. For example, typical culture conditions include culturing at 37 ° C. and 5% CO ₂ . The cell culture medium used for culturing (sometimes referred to simply as "culture medium" or "medium") is not particularly limited as long as it can maintain the survival of cells, but is typically amino acids, vitamins, and electrolytes. Can be used with the main component of. In one embodiment, the culture medium is based on a basal medium for cell culture. Such basal medium is not limited to, for example, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80. -7 etc. are included. Many of these basal media are commercially available, and their compositions are also known.
The basal medium may be used as it has a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and culture conditions. Therefore, the basal medium is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased.

The culture substrate is not particularly limited as long as the cells can form a cell culture on the cells, and includes, for example, containers of various materials, solid or semi-solid surfaces in the containers, and the like. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices. The culture substrate may be prepared using the above-mentioned materials, or a commercially available one may be used. Preferred culture substrates include, but are not limited to, for example, substrates with an adhesive surface suitable for the formation of sheet-like cell cultures. Specifically, a base material having a hydrophilic surface, for example, a base material coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin. , Vitronectin, proteoglycan, glycosaminoglycan and other extracellular matrices, and base materials coated with cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface. In addition, such substrates are commercially available (eg, Corning ^(R) TC-Treated Culture Dish, Corning, etc.).

The surface of the culture substrate may be coated with a material whose physical properties change in response to irritation, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl (Amid, etc.), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), having cyclic groups (eg, Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidin, 1- (1-oxo-2-propenyl) -piperidin, 4- (1-oxo-2-propenyl) -morpholin, 1, -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methyl vinyl ether), or a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-211865 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhered to the materials can be promoted. Culture dishes coated with temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell ^(R) ) and can be used.

The sheet-shaped cell culture can be formed by sheet-culturing a cell suspension containing a sensor chip on a culture substrate. "Sheet culture" means culturing the cells seeded on the culture medium so as to form a sheet-like cell culture (that is, to form a sheet). In sheet culture, cells that can form a sheet-like cell culture are typically seeded on a culture medium and for a predetermined period (for example, 8-48 hours, 10-36 hours, 11-30 hours, 12). (~ 26 hours, etc.), the cells are cultured under conditions that form cell-cell adhesion, the cells interact with each other, and the cells are connected to each other. The conditions for forming cell-cell adhesion include, but are not limited to, any conditions that can form cell-cell adhesion, including, for example, general cell culture conditions. Such conditions include, for example, culturing at 37 ° C. and 5% CO ₂ . Further, those skilled in the art can select the optimum conditions according to the type of cells to be seeded. Non-limiting examples of sheeted culture are described in, for example, Patent Document 1, JP 2010-081829, JP 2010-226991, JP 2011-110368, JP 2011-172925, WO 2014/185517 and the like.

The seeding density of the cells is not particularly limited, but in the sheet culture, for example, the density may be such that the cells can form a sheet-like cell culture without substantially proliferating. "The density at which cells can form a sheet-like cell culture without substantially proliferating" means that a sheet-like cell culture is obtained when cultured in a non-proliferative culture medium containing substantially no growth factor. It means the cell density that can be formed. This seeding density is higher than that in the method using a culture medium containing a growth factor, and may be higher than the density at which cells reach confluence. Non-limiting examples of such densities are, for example, 1.0 × 10 ⁵ pieces / cm ² or more. The upper limit of the seeding density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not undergo differentiation, but may be, for example, less than 3.4 × 10 ⁶ cells / cm ² .

The “density at which cells can form a sheet-like cell culture without substantially proliferating” is 1.0 × 10 ^{5 to} 3.4 × 10 ⁶ cells / cm ² in one embodiment, and 3. In another embodiment. 0 × 10 ^{5 to} 3.4 × 10 ⁶ pieces / cm ² , 3.5 × 10 ^{5 to} 3.4 × 10 ⁶ pieces / cm ² in yet another aspect, 1.0 × 10 ⁶ in yet another aspect. ~ 3.4 × 10 ⁶ pieces / cm ² , in yet another aspect 3.0 × 10 ^{5 to} 1.7 × 10 ⁶ pieces / cm ² , in another aspect 3.5 × 10 ^{5 to} 1.7 × 10 ⁶ pieces / cm ² , and in yet another aspect, 1.0 × 10 ^{6 to} 1.7 × 10 ⁶ pieces / cm ² . The above range may include both the upper limit and / or the lower limit as long as the upper limit is less than 3.4 × 10 ⁶ pieces / cm ² . Therefore, the above densities are, for example, 3.0 × 10 ⁵ pieces / cm ² or more and 3.4 × 10 ⁶ pieces / cm less than ² (including the lower limit and not including the upper limit), 3.5 × 10 ⁵ pieces / cm. ² or more 3.4 x 10 ⁶ pieces / cm less than ² (including lower limit, not including upper limit), 1.0 x 10 ⁶ pieces / cm ² or more and less than 3.4 x 10 ⁶ pieces / cm ² (including lower limit) , Upper limit not included), 1.0 × 10 ⁶ pieces / cm ^{2 or} more 3.4 × 10 ⁶ pieces / cm less than ² (not including lower limit or upper limit), 1.0 × 10 ⁶ pieces / cm ^{2 or} more 1 .7 × 10 ⁶ pieces / cm ² or less (the lower limit is not included, but the upper limit is included).

The sheeting medium used for sheeting is not particularly limited as long as it can induce cell sheeting, and includes, for example, physiological saline, various physiological buffers (for example, PBS, HBSS, etc.), and various cells. Those based on the basal medium for culture can be used. Such basal medium is not limited to, for example, DMEM, MEM, F12, DME, RPMI1640, 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 as it has a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and culture conditions. Therefore, the basal medium is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased. The sheeting medium may contain additives such as serum (eg, fetal bovine serum such as bovine serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).

The cell culture can be used in the production of pharmaceutical compositions containing the cell culture. Such pharmaceutical compositions may include various additional ingredients, such as pharmaceutically acceptable carriers, ingredients that enhance the viability, engraftment and / or function of cell cultures, and other ingredients useful in the treatment of a disease of interest. It may contain an active ingredient or the like. Any known additional component can be used as such additional component, and those skilled in the art are familiar with these additional components. In addition, the pharmaceutical composition can be used in combination with an ingredient that enhances the viability, engraftment and / or function of the cell culture, and other active ingredients that are useful for treating a target disease. In one aspect, the pharmaceutical composition is for use in the treatment of diseases associated with tissue abnormalities. The tissues and diseases to be treated are as described above for cell cultures.

The cell culture and the pharmaceutical composition may be provided as a kit. Such kits include some or all of the elements used in the production of cell cultures or pharmaceutical compositions. The kit is not limited to, for example, cells used for producing cell cultures, sensor chips, culture solutions, culture dishes, instruments (for example, pipettes, droppers, tweezers, etc.), methods for producing and using cell cultures. (For example, a usage manual, a medium on which information on a manufacturing method and a usage method is recorded, for example, a flexible disc, a CD, a DVD, a Blu-ray disc, a memory card, a USB memory, etc.) may be included.

In the kit, the cells can be provided as a cell suspension contained in a container suitable for storing the cells, such as a tube, vial, flask. The cell suspension may be in a liquid state or may be frozen. When the cells are provided frozen, the cell suspension preferably contains a cryoprotectant. The sensor chip may be provided in the form contained in the cell suspension or as a component separate from the cell suspension. For example, by providing a sensor chip in which information on the origin of cells is stored in a cell suspension, the sensor chip moves together with the cells, and the cell culture is performed from the process of producing a cell culture or a pharmaceutical composition. It is possible to identify the origin of cells at each stage leading to the use of a substance or a pharmaceutical composition. Therefore, mistakes such as cell mistakes can be effectively prevented.

A specific sensor chip (or a chip that does not have a sensor function that stores information about the origin of cells) that stores information about the origin of cells can be attached to other elements (part or all) included in the kit. Elements included in a kit that are expected to be used in a subject can also reduce the risk of being accidentally used in another subject. This makes it possible to avoid contamination by other target cells and other components. The "information on the origin of cells" may be an identification number or the like that can identify individual sensor chips. In this case, the origin of the cell can be identified from the sensor chip by associating the identification number with the information on the origin of the cell in a database or the like.

The cell culture or pharmaceutical composition can be used in a method of treating a disease in a subject. Such treatment methods include administering to a subject in need thereof an effective amount of cell culture or pharmaceutical composition. The tissues and diseases to be treated are as described above for cell cultures. In addition, in such a treatment method, a component that enhances the viability, engraftment and / or function of the cell culture, other active ingredients useful for treating the target disease, and the like may be used in combination with the cell culture. it can.

The treatment method may further include the step of producing a cell culture. Such a treatment method may further include the step of collecting the cells for producing the cell culture from the subject or the tissue to be supplied with the cells before the step of producing the cell culture. In one embodiment, the subject from which the cell or tissue from which the cell is source is harvested is the same individual to whom the cell culture is administered. In another embodiment, the subject from which the cell or tissue from which the cell is sourced is harvested is a separate entity of the same species as the subject to whom the cell culture is administered. In another embodiment, the subject from which the cell or tissue from which the cell is sourced is harvested is an individual heterologous to the subject to whom the cell culture is administered.

As used herein, the term "subject" means any individual organism, preferably an animal, more preferably a mammal, even more preferably a human. The subject may be healthy or suffer from any disease, but is typically suffering from or at risk of suffering from the disease if treatment of the disease is intended. Means the object.

Also, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, transient remission or prevention of disease. For example, the term "treatment" includes medically acceptable interventions for a variety of purposes, including delaying or stopping the progression of a disease, regressing or eliminating lesions, preventing the onset or recurrence of the disease, and the like. ..

The effective amount in the treatment method is, for example, an amount capable of suppressing the onset or recurrence of the disease, alleviating the symptoms, or delaying or stopping the progression (for example, the number of cells contained in the cell culture, the cell culture). Size, weight, etc.), preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

Cell cultures and pharmaceutical compositions can be administered by various routes such as intravenous, intramuscular, subcutaneous, local, intraarterial, intraportal, intraventricular, intraperitoneal and the like. In the case of sheet-shaped cell culture, the administration method typically includes direct application to tissues, but when using a fragment of sheet-shaped cell culture, various administration methods that can be administered by injection are possible. It may be administered by a route such as intravenous, intramuscular, subcutaneous, local, intraarterial, intraportal, intraventricular, or intraperitoneal.
The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained.

<Test method for cell culture applied to the subject>
FIG. 4 shows an aspect of a method for inspecting a cell culture applied to a subject. FIG. 4 shows a step of reading the information from the sensor chip 1 embedded in the sheet-shaped cardiomyocyte culture 24 applied to the heart 31 of the subject 3 with the reading device 4. The cardiomyocytes constituting the sheet-shaped cardiomyocyte culture 24 are derived from iPS cells. The sensor chip 1 is a passive type, and converts radio waves transmitted from the reading device 4 into electric power, thereby performing measurement of a predetermined item and transmission of measured values. Passive chips and readers of such chips are well known in the field of RFID technology. Items to be measured include items related to the state of the cell culture and the function of the cell culture related to the treatment of the disease. More specifically, without limitation, for example, pulsation, action potential, ion concentration, physiologically active substance concentration, etc. of the cell culture can be mentioned. Examples of the type of ion include those related to action potential, such as sodium ion and potassium ion. The physiologically active substances to be measured include not only those produced by the myocardial cells themselves, but also those produced by the myocardial cells by acting on other cells in the target body to induce the production, and examples thereof include, for example, VEGF. , BFGF and other angiogenic cytokines, HGF and other cell death inhibitory cytokines, SDF-1 and other stem cell homing cytokines, and the like. The information from the sensor chip also includes information about the position of the sensor chip. The change in the position of the sensor chip is an index such as deviation or dropout from the application position of the cell culture, and dropout of the sensor chip.

The sensor chip 1 can also be configured to transmit identification information unique to each sensor chip together with the measured value. This makes it possible to confirm the origin of the cell culture 24 applied to the subject 3. Further, by forming the sensor chip with a biodegradable material, the influence on the target 3 can be minimized, and the recovery of the sensor chip 1 becomes unnecessary.

FIG. 4 shows an inspection method when the sheet-shaped cardiomyocyte culture 24 is used for the heart 31 of the subject 3, but the inspection method can be applied to other cell cultures and to other tissues. Needless to say, it can be applied even when used. For example, in the above-mentioned test method, when a sheet-like skeletal myoblast culture is used for the heart, when a pancreatic islet cell culture is used for the pancreas, when a sheet-like epithelial cell is used for the skin, a sheet-like oral mucosal epithelium is used. When stem cells are used in the esophagus, sheet-like corneal epithelial stem cell cultures are used in the eyes, sheet-like root membrane cell cultures are used in teeth, chondrocyte cultures are used in osteoarthritis, etc. Can also be applied. In addition, the sensor chip can be configured to measure items suitable for each case. For example, in the case of islet cell cultures, it can be configured to measure the concentration of hormones such as insulin and glucagon.

By the above test, the state of the cell culture applied to the subject can be grasped in real time, and based on such information, the correctness or evaluation of the treatment with the cell culture can be predicted or evaluated, and the necessity of adjuvant therapy can be determined. It becomes possible to do. For example, if the above test confirms dysfunction of the sheet-shaped cardiomyocyte culture (for example, loss of pulsation, abnormal action potential, low cytokine concentration), the function of the sheet-shaped cardiomyocyte culture is improved. It is possible to perform adjuvant therapy (for example, administration of cytokines and the like), application of additional sheet-shaped cardiomyocyte cultures, and the like.

<System for detecting exfoliation of sheet-shaped cell culture>
FIG. 5 shows an aspect of a system for detecting exfoliation of a sheet-shaped cell culture from a culture substrate (hereinafter, may be abbreviated as exfoliation detection system). The peeling detection system 5 in this embodiment includes a sensor chip 1 embedded in a sheet-shaped cell culture 2 and a reading device 40 that reads information from the sensor chip 1. The sensor chip 1 is a passive type, converts radio waves emitted by a communication device 41 provided in the reading device 40 into electric power, and performs various processes such as measurement of predetermined items and transmission of measured values. The communication device 41 receives the information transmitted by the sensor chip 1 and sends it to the processor 421 of the reading device main body 42. The processor 421 analyzes the data sent from the communication device 41 and outputs the analysis result via the display 422 and the speaker 423. The reading device 40 includes a user interface 424 for controlling the reading device 40 and a power supply 425 for supplying power to the reading device 40.

The communication device 41 does not necessarily have to be installed under the culture base 6 as shown in FIG. 5, and is installed at an arbitrary position such as the upper part of the culture base 6 where communication with the sensor chip 1 can be performed. can do. Further, in FIG. 5, the reading device main body 42 and the communication device 41 are separated and connected by a cable 43, but the reading device 42 and the communication device 41 do not necessarily have to be separated and are integrated with each other. It may be.

The sensor chip 1 is configured to be able to detect one or more items that change as the sheet-shaped cell culture 2 in the medium 7 is exfoliated from the culture substrate 6. For example, the sheet-shaped cell culture 2 contracts when peeled from the culture substrate 6, but the tension associated with the contraction, the increase in the pressure generated in the cell culture 2, and the components of the cell culture around the sensor chip 1 By detecting displacement or the like, such contraction can be detected. Further, as the sheet-shaped cell culture 2 contracts, the position of the sensor chip 1 can change from the position 8 before peeling as shown in FIG. 6, so that the sheet-shaped cell culture 1 is based on the change in the position. It is also possible to detect peeling from the culture substrate 6. Since the radio wave transmitted from the sensor chip 1 is attenuated according to the distance, the change in the position of the sensor chip 1 is, for example, the sensor chip 1 received by each communication device 42 by arranging a plurality of communication devices 41 at different positions. It can be detected by analyzing the intensity of radio waves from.

When the sheet-shaped cell culture 2 begins to peel off from the culture base 6, as shown in FIG. 7, the medium flows between the bottom surface of the sheet-shaped cell culture 2 and the culture base 6 which have been adhered until then. 11 is generated, and the physicochemical environment on the bottom surface of the sheet-shaped cell culture 2 is changed. Therefore, by detecting the concentration of a substance having a difference in concentration between the cell culture 2 and the medium 7, for example, an ion, the inflow 11 of the medium can be detected quickly. Further, when the distance between the sensor chip 1 and the communication device 41 changes with the peeling of the sheet-shaped cell culture 2 from the culture substrate 6, the radio wave intensity from the sensor chip 1 received by the communication device 41 also changes. .. Therefore, the peeling of the sheet-shaped cell culture 2 from the culture substrate 6 can be detected based on the radio wave intensity itself. When the radio wave intensity itself from the sensor chip 1 is used, the sensor chip 1 does not necessarily have to have a sensor function by itself, and the sensor chip 1 and the reading device 40 may cooperate to perform the sensor function.

When the cell culture 2 contains a plurality of sensor chips 1, a more precise analysis of the exfoliated state becomes possible by comparing the information from each sensor chip 1. For example, when the processor 421 of the reader 40 receives information indicating exfoliation from a part of the plurality of sensor chips 1 (for example, FIG. 7), a part of the sheet-shaped cell culture 2 is exfoliated and a part is exfoliated. It can be determined that the cell culture has not been exfoliated, while when information indicating exfoliation is received from all the sensor chips 1 (for example, FIG. 8), it is determined that the sheet-shaped cell culture 2 has completely exfoliated. Can be done. Then, these analysis results can be notified to the user via the display 422 and the speaker 423. In addition, the degree of exfoliation can be notified, for example, as a percentage of exfoliation, based on the ratio of the sensor chips that transmit the information indicating exfoliation to all the sensor chips contained in the cell culture.

The exfoliation detection system can be incorporated into an apparatus for producing a sheet-shaped cell culture such as a cell culture apparatus, and an exfoliation detection function can be imparted thereto. By adding the detachment detection function, it becomes possible to quickly collect the sheet-shaped cell culture exfoliated from the culture substrate, and there is a risk that the sheet-shaped cell culture exfoliated from the culture substrate is left unattended at an early stage. , It is possible to reduce the trouble of diligently checking the state of the sheet-shaped cell culture.

Similarly, a step of detecting peeling of a sheet-shaped cell culture from a culture substrate using a sensor chip embedded in the sheet-shaped cell culture can be incorporated into a step of producing the sheet-shaped cell culture. By incorporating the exfoliation detection step, it becomes possible to quickly recover the sheet-like cell culture exfoliated from the culture substrate, and there is a risk that the sheet-like cell culture exfoliated from the culture substrate is left unattended at an early stage. It is possible to reduce the trouble of diligently checking the state of the sheet-shaped cell culture.

The various features described herein can be combined in various ways, and the embodiments obtained by such combinations are all within the scope of the present invention, including combinations not specifically described herein. is there. Those skilled in the art also understand that a number of various modifications that do not deviate from the spirit of the present invention are possible, and equivalents containing such modifications are also included in the scope of the present invention. Therefore, it should be understood that the embodiments described herein are merely exemplary and are not intended to limit the scope of the invention.

1 Sensor chip 2 Sheet cell culture 21 Extracellular matrix layer 22 Cell layer (lower layer)
23 cell layer (upper layer)
24 Sheet-shaped myocardial cell culture 3 Target 31 Heart 4, 40 Reader 41 Communication device 42 Reader body 421 Processor 422 Display 423 Speaker 424 User interface 425 Power supply 43 Cable 5 Peeling detection system 6 Culture base 7 Medium 8 Before peeling Position of sensor chip 9 State before peeling 10 State after peeling 11 Inflow of medium

Claims (13)

(1) A step of mixing a plurality of sensor chips that store individual identification information and a cell suspension to prepare a cell suspension containing the plurality of sensor chips.
(2) A step of forming a sheet-like cell culture containing a plurality of sensor chips by forming a sheet of cells contained in the cell suspension together with a plurality of sensor chips on a culture substrate.
(3) A step of detecting exfoliation of a sheet-shaped cell culture from a culture medium based on information from a plurality of sensor chips.
A method for detecting exfoliation of a sheet-like cell culture from a culture medium, which comprises. (1) A step of mixing a plurality of sensor chips that store individual identification information and a cell suspension to prepare a cell suspension containing the plurality of sensor chips.
(2) A step of forming a sheet-like cell culture containing a plurality of sensor chips by forming a sheet of cells contained in the cell suspension together with a plurality of sensor chips on a culture substrate.
(3) A step of detecting exfoliation of a sheet-shaped cell culture from a culture medium based on information from a plurality of sensor chips.
(4) A method for producing a sheet-shaped cell culture, which comprises a step of collecting the sheet-shaped cell culture in which peeling from the culture substrate is detected. The method according to claim 1 or 2, wherein the plurality of sensor chips have a communication function. The method according to any one of claims 1 to 3, wherein the plurality of sensor chips can measure an item selected from the group consisting of tension, pressure, displacement, ion concentration and bioactive substance concentration. A sheet-shaped cell culture provided with a plurality of sensor chips that store individual identification information for peeling detection embedded in the sheet-shaped cell culture and a reading device that reads information from the plurality of sensor chips. A system for detecting peeling of an object from the substrate. A sheet-like cell culture containing multiple sensor chips that store individual identification information for exfoliation detection . The sheet-shaped cell culture according to claim 6 , wherein the plurality of sensor chips have a communication function. The sheet cell culture according to claim 6 or 7 , wherein the plurality of sensor chips can measure an item selected from the group consisting of tension, pressure, displacement, ion concentration and bioactive substance concentration. The sheet-like cell culture according to any one of claims 6 to 8 , for treating a disease associated with a tissue abnormality. Comprising a cell suspension containing a plurality of sensor chips which stores individual identification information for peeling detection, and elements used in the production of sheet-like cell culture sheet cell culture comprising a plurality of sensor chips A kit for manufacturing things. The state of the sheet-shaped cell culture, which comprises the step of reading information transmitted from a plurality of sensor chips contained in the sheet-shaped cell culture according to any one of claims 7 to 9 applied to the subject. How to inspect. A method for examining the effect of treating a disease by the sheet-shaped cell culture, which comprises a step of reading information transmitted from a plurality of sensor chips contained in the sheet-shaped cell culture according to claim 9 applied to the subject. .. A sheet-shaped cell culture according to any one of claims 6 to 9 , or a sheet-shaped cell culture comprising a step of reading identification information stored in a plurality of sensor chips included in the kit according to claim 10 . How to identify what applies to.