Cell culture equipment for cell sheet production, support for recovery, and cell sheet production kit

Abstract

The present invention provides a cultureware and a cell sheet production kit that prevent cells from detaching due to mechanical stimuli such as changing the culture medium. [Solution] A cell detachment-inhibiting region is provided around the entire inner circumference of the culture surface of the cell culture substrate to inhibit cell detachment, and a stimulus-responsive region is located inside this, whose adhesiveness to cells changes in response to a stimulus. Furthermore, a recovery support with specific characteristics is used for recovering cell sheets, allowing for easy recovery of cell sheets.

Description

Technical Field

[0001] The present invention relates to a recovery support and a cell culture device used for peeling and recovering sheet-like cells used in the fields of regenerative medicine, drug discovery research, etc. from a device, and a cell sheet production kit including these. In particular, it relates to cell culture devices used for culturing in a culture device with a relatively large area where cells are likely to peel off during culture, and for producing a cell sheet from cells having a property of being easily peeled off. Specific cell types include kits and cell culture devices used for producing cell sheets composed of epithelial cells, mesenchymal stem cells, myoblasts, cardiomyocytes, smooth muscle cells, chondrocytes, osteoblasts, vascular endothelial cells, fibroblasts, renal cells, and hepatocytes.

Background Art

[0002] In recent years, regenerative medicine, which promotes the self-regenerative ability of damaged organs and tissues and restores tissue repair and function by culturing cells collected from tissues with normal functions and transplanting them to patients, has been attracting increasing attention. Regenerative medicine is a medical treatment that regenerates damaged tissues and organs and restores lost functions by transplanting cells cultured outside the patient's body or tissues constructed from the cultured cells into the patient's body. A cell sheet is a sheet of cells cultured for use in regenerative medicine and is used for transplantation to patients or for constructing tissues. In addition, cell sheets play an important role not only in clinical applications but also in basic research such as the safety evaluation of pharmaceuticals and drug development.

[0003] As a treatment method using products for regenerative medicine and the like, there is a method of transplanting a cell sheet obtained by culturing cells into a sheet as described above. When adherent cells are cultured on a culture device, they adhere to the culture device and also adhere to each other, and when they become confluent, they form a sheet-like cell aggregate (cell sheet). Note that "confluent" refers to a state in which cells cover the culture surface. For example, epithelial cell sheets are used for extensive burns, osteoarthritis, suppression of esophageal stricture at the resection site after endoscopic submucosal dissection (ESD) for esophageal cancer, corneal regeneration, mesenchymal stem cells for brain diseases such as dementia, breast reconstruction, osteoarthritis and traumatic cartilage defects, ischemic heart disease, and myoblasts are transplanted to sites where cell regeneration is required such as ischemic heart disease to perform treatment. Depending on the treatment purpose, it is necessary to prepare various cells and various sizes of cell sheets for culture.

[0004] When transplanting cells in regenerative medicine, it is necessary to detach the cells from the cell culture device while keeping them in a sheet form. As a method for detaching the cell sheet from the cell culture device, there are a method of using a proteolytic enzyme such as dispase to weaken the bond between the culture surface of the culture device and the cells, and a method of using a cell culture device equipped with a culture surface whose cell adhesion changes.

[0005] When recovering using a proteolytic enzyme, not only the bond between the cells and the culture surface of the cell culture device but also the bond between the cells is weakened, so there is a problem that the cell sheet is damaged and the engraftment after transplantation is significantly inferior. To avoid this problem, a method of using a cell culture device equipped with a culture surface whose adhesion to cells changes, which recovers the cells without destroying the proteins present on the cell membrane, is recommended.

[0006] As a method of recovering a cell sheet using a culture surface with a changing cell adhesion force, there is a method using a stimulus-responsive polymer. Examples of the type of stimulus include temperature, pH, ions, light, potential, magnetism, etc. Among these, temperature stimulation using a temperature-responsive polymer can be preferably used because it is easy to apply the stimulus and has no effect on cells. Patent Document 1 discloses a culture device coated with a polymer having an upper critical solution temperature and a lower critical solution temperature. By using a temperature-responsive polymer, the environmental temperature can be changed to control the balance between hydrophobicity and hydrophilicity on the surface of the culture device.

[0007] In a culture device coated with a temperature-responsive polymer, at the temperature during cell culture, water desorbs from the polymer on the surface of the device, causing the polymer surface to contract and making the surface more adhesive to cells. On the other hand, below a predetermined temperature, the surface of the device becomes hydrophilic, and the cells lose the scaffold they were adhering to, causing the cells to detach from the device. According to the method using a temperature-responsive polymer, although the adhesiveness between cells and the device is lost, the adhesiveness between cells is not lost, so that the cells can be recovered in the form of a sheet.

[0008] Treatment by regenerative medicine, etc. is treatment using epithelial cells, endothelial cells, and stem cells, and a cell sheet required depending on the treatment area is prepared. Depending on the treatment, the type and size of the cells to be prepared also differ, and accordingly, the ease of handling the cells during culture also differs. For example, when used for suppressing esophageal stricture after endoscopic submucosal dissection (ESD) of esophageal cancer or for regeneration of the cornea, periodontal tissue, etc., a cell sheet of not such a large area is required. On the other hand, for treatment of extensive burns, etc., a cell sheet of a large area is required. A cell sheet of a large area is more likely to be peeled off from the culture device during the manufacturing process compared to a cell sheet of a small area. Also, there are cell types such as myoblasts that have very easily peelable properties. When manufacturing a cell sheet of a large area, or depending on the type of cells, as the cells grow and become confluent, mechanical stimulation such as pipetting during the replacement of the culture medium can make the cells more likely to be peeled off from the surface of the culture device.

[0009] When collecting as a cell sheet, cells are cultured until they reach a confluent state. However, if the cells to be collected as a cell sheet peel off when aspirating the culture medium, it will cause problems in transplantation. Even if the operation is carried out with sufficient care, if the cells peel off from the edge during medium replacement or the like during the culture period, they cannot be collected as a cell sheet and transplantation cannot be performed. Also, when using cells for the treatment of extensive fever or the like, since the transplantation area is large, it is necessary to use a culture device with a large culture area as much as possible and collect all the cultured cell sheets. As described above, since cells are likely to peel off when the culture area is large, the peeling of cells during medium replacement or the like has been a major problem.

[0010] In a method using a culture device having a stimulus-responsive surface, a cell culture container is disclosed in which a stimulus-responsive surface and a non-stimulus-responsive surface are arranged on the surface of the device to adjust cell adhesiveness (Patent Documents 2 and 3). Patent Document 2 discloses a cell culture device having a cell adhesion region having cell adhesiveness so as to surround a stimulus-responsive region as a culture container for stably holding a cell sheet on the culture surface and easily collecting the cell sheet even after stimulation. Patent Document 3 discloses a cell culture device including a stimulus-responsive region, a cell adhesion region, and a cell non-adhesion region, and having a boundary with the cell non-adhesion region at at least a part of the outer periphery of the stimulus-responsive region, in order to provide a cell culture device in which the back surface, which is the side adhered to the scaffold (surface of the culture device) of the cell, can be easily observed.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0012] However, the container described in Patent Document 2 holds a cell sheet on the culture surface by securing an area where cells can adhere after being detached from the stimulus-responsive area by the application of a stimulus, and easily recovers the cells by detaching the adhesion site with pipetting or forceps. That is, the cell culture container described in Patent Document 2 is configured such that, after the application of a stimulus, while holding a part of the cell sheet on the surface of the culture equipment, the cell sheet can be easily peeled off by pipetting or the like. Therefore, during the culture period, that is, it is not assumed that cells will be detached due to mechanical stimuli such as medium replacement before the application of a stimulus, and it is not intended to prevent cells from being detached during the cell culture period and becoming unable to be recovered as a cell sheet.

[0013] The culture equipment described in Patent Document 3 is for detaching cells in a part of an area in a sheet form by applying a stimulus and observing the back surface. Therefore, it is assumed that the cell adhesion area, which is the side for observing the cell surface, remains firmly adhered to the culture equipment. Therefore, it does not consider the detachment of cells due to mechanical stimuli during the culture.

[0014] As shown in the embodiment, it is an object to provide a culture equipment having a cell detachment suppression area where cells are difficult to be detached against mechanical stimuli in operations such as medium replacement, and capable of recovering as a cell sheet. Furthermore, when developing the culture equipment of the present embodiment, it has become clear that, while the provision of a cell detachment suppression area can prevent cells from being detached from the culture equipment during the culture period, on the other hand, there is also a problem that the cell sheet cannot be easily recovered. In order to solve this, the material of the recovery support used when detaching the cells while keeping them in a sheet form was devised. An object of the present invention is to provide a kit for manufacturing a cell sheet that can prevent the detachment of the cell sheet and also easily recover the cell sheet.

[0015] In addition, when used in basic research on regenerative medicine or drug discovery research, it has been required to analyze cells cultured under the same conditions by various methods. For example, cell sheets are transplanted into animal models, and the gene expression of cell sheets cultured under the same conditions is analyzed by PCR or Western blotting, or the morphology of the cells is observed with a microscope. Since the cells used in regenerative medicine are not usually cell lines but cells derived from tissues such as skin and mucosa obtained from patients, the properties of the cells may not necessarily be the same for each culture device. Therefore, there have been cases where it is questioned whether the resulting changes are due to cell variations or changes caused by treatment with drugs or the like. In the present embodiment, an object is to provide a cell sheet production kit and a culture device capable of recovering a plurality of culture sheets from one culture device. By analyzing cells cultured in the same culture device, it becomes possible to make the culture conditions exactly the same, and changes can be analyzed more precisely.

Means for Solving the Problem

[0016] The present invention includes the following cell culture devices, cell sheet recovery supports, and cell sheet production kits. (1) A cell culture device characterized in that a cell detachment suppression region for suppressing cell detachment is provided around the cell culture surface, and a stimulus-responsive region whose adhesiveness to cells changes by an external stimulus is arranged inside the cell detachment suppression region. (2) The cell culture surface of the cell culture device is 3.5 cm 2 or more and 300 cm 2 or less. The cell culture device according to (1). (3) The cell detachment suppression region is a cell adhesion surface in a temperature range of 1°C to 50°C. The cell culture device according to (1) or (2). (4) The cell adhesion surface is a culture surface that does not respond to external stimuli. The cell culture device according to (3). (5) The external stimulus is light, temperature, pH, or magnetic field. The cell culture device according to any one of (1) to (4). (6) The stimulus-responsive region is a temperature-responsive surface that responds to temperature stimuli, and is characterized in that cells detach from the surface at a temperature of 0°C or higher and 32°C or lower. The cell culture device according to any one of (1) to (5). (7) The cell detachment inhibition region and the stimulus-responsive region are characterized in that they are divided on the same plane. The cell culture device according to any one of (1) to (6). (8) The cell detachment inhibition region is characterized in that it has a width of 2 mm or more and 10 mm or less. The cell culture device according to any one of (1) to (7). (9) The cell detachment inhibition region is characterized in that it is provided so as to divide the cell culture surface in addition to the periphery of the cell culture surface. The cell culture device according to any one of (1) to (8). (10) The cell detachment inhibition region and the stimulus-responsive region are characterized in that they are processed so as to be visually distinguishable. The cell culture device according to any one of (1) to (9). (11) The cell detachment inhibition region and the stimulus-responsive region are characterized in that they have light permeability. The cell culture device according to any one of (1) to (10). (12) The cell adhesion property of the cell detachment inhibition region and the stimulus-responsive region under non-stimulating conditions is characterized in that the cell detachment inhibition region has stronger cell adhesion than the stimulus-responsive region. The cell culture device according to any one of (1) to (11). (13) It is for producing a cell sheet of epithelial cells, mesenchymal stem cells, or myoblasts. The cell culture device according to any one of (1) to (12). (14) The stimulus-responsive region and the cell detachment inhibition region are produced by coating a temperature-responsive monomer on the cell culture surface and irradiating only the stimulus-responsive region with an electron beam, γ-ray, or ultraviolet ray to form a stimulus-responsive layer. The cell culture device according to any one of (1) to (13). (15) A cell sheet production kit comprising the cell culture device according to any one of (1) to (14) and a recovery support used for cell sheet recovery. (16) The recovery support has a thickness of 30 μm or more and 300 μm or less, and a basis weight of 10 g / m 2 or more and 150 g / m 2The kit for producing a cell sheet according to (15), which is a paper, a cellulose film, or a plastic film having a wet tensile strength of 0.03 kN / m or more and 1.0 kN / m or less. (17) The kit for producing a cell sheet according to (15) or (16), wherein the recovery support has a diameter or a length in the vertical and horizontal directions smaller than that of the stimulus-responsive region. (18) It has a thickness of 30 μm or more and 300 μm or less, and a basis weight of 10 g / m 2 or more and 150 g / m 2 or less, and is a recovery support used for recovering a cell sheet, which is a paper, a cellulose film, or a plastic film having a wet tensile strength of 0.03 kN / m or more and 1.0 kN / m or less. (19) A method for producing a cell sheet, comprising a step of culturing cells in a cell culture device according to any one of (1) to (14) to form a cell sheet, and a step of recovering the cell sheet with the recovery support of (18). (20) A method for producing a cell sheet, comprising a step of culturing cells using the kit for producing a cell sheet according to any one of (15) to (17) to form a cell sheet, and a step of recovering the cell sheet. (21) A treatment method, characterized in that treatment is performed with a cell sheet produced using the cell culture device according to any one of (1) to (14). (22) A treatment method, characterized in that treatment is performed with a cell sheet produced using the kit for producing a cell sheet according to any one of (15) to (17). (23) The treatment method according to (21) or (22), wherein the treatment target is extensive burns, leukoderma, ulcers, decubitus ulcers, pneumothorax, osteoarthritis, esophageal stricture, colorectal stricture, endometriosis, osteoporosis, renal fibrosis, liver fibrosis, and ischemic heart disease.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0018] The cell culture device shown below can efficiently produce cell sheets for use in treatments such as regenerative medicine because cells do not detach until the cell sheet is recovered and the cell sheet can be easily recovered. In addition, it can be suitably used as a cell culture device suitable for research and development of regenerative medicine or drug discovery research.

[0019] The cell culture device included in the cell sheet production kit may have any shape and can be appropriately selected in consideration of the size and shape of the desired cell sheet. The cell culture device may have any shape as long as it is generally used for cell culture. For example, it is preferably in the shape of a container for culturing adherent cells, such as a petri dish, a flask, or a multi-well plate. When using a flask, if it is a peel-off type flask or a flask with a detachable upper part, the upper part of the flask can be removed, so that the cell sheet can be easily recovered using the support for recovery.

[0020] First, the cell culture device, which is a component of the cell sheet production kit, will be described with reference to the drawings below, but it is not limited to the following embodiments. [Embodiment 1] One embodiment of the cell culture device of the present invention is a cell culture device provided with a cell detachment inhibition region 1 on the inner circumference of the culture device and a stimulus-responsive region 2 inside thereof. FIG. 1 is a diagram schematically showing the culture surface of the cell culture device. Since the stimulus-responsive region 2 and the cell detachment inhibition region 1 are adjacent to each other, there is an adhesion / detachment boundary 3 which is a boundary portion. The culture surfaces of the cell culture devices shown in FIG. 1 are exemplified as being rectangular and circular, but may be of any shape such as square, and the size can also be appropriately selected as an optimal size depending on the location where the cell sheet is transplanted.

[0021] In the cell culture device, the stimulus-responsive region 2 is arranged in the center and the cell detachment inhibition region 1 is arranged on the outer edge thereof, and the cell detachment inhibition region 1 is provided over the entire circumference of the stimulus-responsive region 2. For medium replacement, suction is performed from a portion along the edge of the culture device and the medium is added. Therefore, if the cell adhesiveness at the outer edge of the culture surface is high, cell detachment is less likely to occur due to mechanical stimulation during medium replacement. Also, it is important that a cell detachment inhibition region is provided over the entire inner circumference of the culture device. Since the cell detachment inhibition region is provided over the entire circumference, cell detachment can be prevented regardless of the position where the medium is suctioned and added. Due to the cell detachment inhibition region 1 existing over the entire circumference, the cells adhere firmly to the surface of the culture device, and detachment due to mechanical stimulation during medium replacement can be avoided.

[0022] The cell detachment inhibition region 1 is a region composed of a surface that inhibits cell detachment regardless of the presence or absence of a stimulus. The cell detachment inhibition region desirably has high cell adhesiveness, but as the degree of cell adhesiveness, it is sufficient that the cell adhesiveness is maintained against mechanical stimulation such as suction and addition of the medium. Also, it is preferable that the adhesiveness to cells is higher than the cell adhesiveness of the stimulus-responsive region.

[0023] For example, the culture device used in the following embodiment has the mouse neuroblastoma cell line Neuro2a at 5×10 3 cells / cm 2 ~1×10 5 cells / cm 2When seeded and cultured using DMEM medium containing 10% FBS or FCS, the cell adhesion and spreading rate after 1 hour of culture is 5 - 20% of the culture surface in the stimulation response region and 60 - 75% in the cell detachment inhibition region, and it is processed to have a stronger cell adhesion and spreading rate in the cell detachment inhibition region. That is, even in the cell detachment inhibition region without applying a stimulus to the stimulation response region, it has strong cell adhesion compared to the stimulation response region in the range of 3 to 15 times. However, since it is known that cell adhesion varies depending on culture conditions such as cell type, seeding number, and medium used, without being restricted to this numerical value, it can be judged by the commonly used cell adhesion index and made to have stronger cell adhesion in the cell detachment inhibition region.

[0024] The cell detachment inhibition region may be a surface with improved cell adhesion and proliferation. Therefore, it may be composed of glass commonly used as a culture device or plastic with surface treatment. Examples of plastics include polystyrene, polypropylene, polyvinyl chloride, polycarbonate, etc. Also, as surface treatment, methods usually used for plastic modification such as corona discharge treatment, plasma treatment, UV ozone treatment, electron beam irradiation treatment, laser treatment, etc. can be used.

[0025] Also, in order to more actively prevent cell detachment, substances that physically and chemically enhance cell adhesion, such as hydrophilic polystyrene, basic polymers such as polylysine, basic compounds such as aminopropyltriethoxysilane and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and condensates containing these may be coated on the surface. Also, substances that enhance cell adhesion due to biochemical properties, such as fibrin, fibronectin, laminin, tenascin, hydro nectin, collagen, atelocollagen, gelatin, peptides containing the RGD (arginine-glycine-aspartic acid) sequence, peptides containing the YIGSR (tyrosine-isoleucine-glycine-serine-arginine) sequence, and mixtures thereof may be coated.

[0026] The stimulus-responsive region 2 may be any region where the cell adhesiveness changes upon stimulation, and a temperature-responsive polymer with an upper or lower critical solution temperature of 0 to 80°C can preferably be used. The temperature-responsive polymer has a high affinity for the surrounding water at a temperature above the upper critical solution temperature or below the lower critical solution temperature, and shows cell non-adhesiveness because the polymer takes in water and swells. A polymer that shows cell adhesiveness because water desorbs from the polymer at a temperature below the upper critical solution temperature or above the lower critical solution temperature is preferred. In particular, the upper critical solution temperature is preferably 37°C or higher, which is the temperature for culturing cells, and the lower critical solution temperature (T) is preferably 37°C or lower, which is the temperature for culturing cells. Specifically, it is preferably a polymer in which the surface of the culture equipment shows cell non-adhesiveness in the temperature range of 0°C or higher and 32°C or lower.

[0027] Examples of such temperature-responsive polymers include acrylic polymers or methacrylic polymers. More specifically, poly-N-isopropylacrylamide (T = 32°C), poly-N-n-propylacrylamide (T = 21°C), poly-N-n-propylmethacrylamide (T = 32°C), poly-N-ethoxyethylacrylamide (T = about 35°C), poly-N-tetrahydrofurfurylacrylamide (T = about 28°C), poly-N-tetrahydrofurfurylmethacrylamide (T = about 35°C), poly-N,N-diethylacrylamide (T = 32°C), etc. can be mentioned.

[0028] Monomers for forming these polymers include monomers that can be polymerized by radiation such as gamma rays, electron beams, ultraviolet irradiation, etc., for example, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives, N,N-dialkyl-substituted (meth)acrylamide derivatives, (meth)acrylamide derivatives having a cyclic group, vinyl ether derivatives, etc. These can be used alone or as a mixture of two or more.

[0029] The above-mentioned polymerizable monomer is coated on a cell culture vessel and irradiated with electron beams or radiation, so that the monomer polymerizes to form a polymer layer and a stimulus-responsive region is formed. The formation of the cell detachment-inhibiting region can be achieved by two methods: one is to form a stimulus-responsive region over the entire culture area and then remove the stimulus-responsive layer; the other is to shield the region where the cell detachment-inhibiting region is to be formed from electron beams or the like so that polymerization does not occur, thereby partially preventing the formation of the stimulus-responsive layer. Here, the portion where the cell detachment-inhibiting region is desired to be formed is shielded to prevent the formation of the polymer. When removing the stimulus-responsive region to form the cell detachment-inhibiting region, it is necessary not only to know the thickness of the layer where the polymer has been formed and completely remove it, but also to manage the removal of the removed polymer from the culture surface. If the method of shielding the cell detachment-inhibiting region from electron beams or the like is used, there is no need to remove the stimulus-responsive layer, and it is possible to form the cell detachment-inhibiting region and the stimulus-responsive region by a very simple process.

[0030] The width of the cell detachment-inhibiting region 1 is preferably 2 mm or more and 10 mm or less. The mechanical stimulation caused by the suction and addition of the culture medium does not depend on the size of the culture equipment, etc., and extends to a region with a certain width from the wall of the culture equipment. If the width of the cell detachment-inhibiting region is less than 2 mm, sufficient cell adhesion cannot be obtained, and the cell sheet may peel off. Therefore, if the cell detachment-inhibiting region is narrow, cell detachment cannot be prevented. On the other hand, if the width is wide, a stable adhesive force can be obtained, but the cell area that can be recovered will decrease. Also, if the width is wide, the cells will not detach from the culture equipment, making it difficult to recover them from the culture equipment. Therefore, as a result of considering ensuring a sufficiently stable adhesive force and the recovery efficiency of the cell sheet, the width of the cell detachment-inhibiting region is preferably 2 mm or more and 10 mm or less, more preferably 4 mm or more and 8 mm or less. As described above, the cell adhesion force in the cell detachment-inhibiting region only needs to be stronger than that in the stimulus-responsive region at the cell culture temperature, but it is necessary to have the same adhesive force regardless of the presence or absence of the stimulus for detaching the cells.

[0031] As described above, when using a cell culture device with a large culture area, it is likely to peel off during medium replacement. We actually examined whether the ease of peeling differed depending on the culture area, using primary cultured cells of ovine keratinocytes. The examination was carried out using a cell culture device with only a stimulus-responsive region, or a cell culture device having a cell detachment inhibition region with a width of 5 mm around the stimulus-responsive region. In any case of using the culture vessels, the seeding number of cells was adjusted according to the area, and the examination was carried out according to the schedule of medium replacement on the 4th and 7th days, and cell sheet recovery on the 10th day. After the start of cell culture, the cells had grown to a semi-confluent state in all culture vessels on the 7th day and to a confluent state on the 10th day. The material of any plate is polystyrene, which is coated with a temperature-responsive polymer and subjected to the same temperature-responsive surface treatment to form a stimulus-responsive region.

[0032]

Table 1

[0033] As shown in Table 1, even when the culture area was 3.5 cm 2 , cell detachment was confirmed at a high frequency of 3 out of 12 wells in the culture vessel without a cell detachment inhibition region. Furthermore, when the culture area was larger than 8.8 cm 2 , the cells detached during the medium replacement on the 7th day and could not be recovered as a cell sheet. In the case of a cell culture device equipped with a cell detachment inhibition region and a stimulus-responsive region, even in the culture vessel of 8.8 cm 2 , the cells could be recovered without problems. Therefore, although the confirmation at a culture area of 3.5 cm 2 has not been carried out, it is possible to recover the cells as a cell sheet without problems. Also, although a comparison study was carried out here using a plate with a culture area of 84 cm 2 , it has been confirmed that a cell sheet can be recovered without problems even when the culture area is 225 cm 2 .

[0034] In addition, the cell detachment inhibition region and the stimulus-responsive region are arranged on the same plane. This is because if there are irregularities in the cell detachment inhibition region and the stimulus-responsive region, when the cell sheet is collected and applied to the affected area as a cell sheet clinically, there will be parts that do not adhere, and it is considered that the engraftment property is poor.

[0035] Cell sheets obtained by culturing various cells can be transplanted to the affected area for treatment. For example, epidermal cell sheets can be used for the treatment of extensive burns, leukoderma, ulcers, pressure sores; cartilage sheets can be used for the treatment of osteoarthritis; mesenchymal stem cell sheets can be used for the treatment of brain diseases such as dementia, breast reconstruction, osteoarthritis and traumatic cartilage defects, ischemic heart diseases; fibroblast sheets can be used for the treatment of pulmonary air leakage; oral mucosal epithelial cell sheets can be used for the treatment of esophageal stricture, colorectal stricture, and endometriosis; osteoblast sheets can be used for the treatment of osteoporosis; renal cell sheets can be used for the treatment of renal fibrosis; hepatocyte sheets can be used for the treatment of liver fibrosis; myoblast, cardiomyocyte, and smooth muscle cell sheets can be used for the treatment of ischemic heart diseases. Note that the cells that can be used as cell sheets are not limited to the above, and also, any disease that can be improved by cell transplantation can be used for any disease as long as the disease can be improved by cell transplantation.

[0036] There has never been a culture device provided with a cell detachment inhibition region having a predetermined cell adhesiveness and width around the stimulus-responsive region to prevent cells from detaching during cell culture. For transplantation, it is necessary to culture the cells until they become confluent, but by providing the cell detachment inhibition region, it has become possible to culture the cells without detaching during the cell culture period.

[0037] [Embodiment 2] Next, a culture device with a subdivided culture surface will be described. FIG. 2 schematically shows the culture surface of a culture device with a subdivided culture surface. Here, examples of dividing the culture surface of the culture device into four parts (left in FIG. 2) or eight parts (right in FIG. 2) by the cell detachment inhibition region 11 are shown. Here, an example of subdividing a rectangular culture device is shown, but any shape of culture device such as a square or a circle can be similarly subdivided by the cell detachment inhibition region 11. Also, depending on the application, it is not necessary to divide equally, and the divided areas may have different sizes. In this form, the cell detachment inhibition region 11 will also exist inside the culture surface along the outer periphery of the stimulus-responsive region 12. The adhesion / detachment boundary 13 will exist as the outer periphery of each stimulus-responsive region 12 subdivided by the cell detachment inhibition region 11.

[0038] In the case of a culture device with a subdivided culture surface, the width of the cell detachment inhibition region 11 is preferably set to be 2 mm or more and 10 mm or less, more preferably 4 mm or more and 8 mm or less. When using a culture device provided with subdivided compartments, not only can different analyses be performed for each compartment as described above, but it is also possible to collect the cell sheets for each compartment and apply them to different affected parts. Furthermore, since the cell detachment inhibition region exists not only on the outer periphery but also inside the stimulus-responsive region, it is possible to more strongly inhibit the detachment of cells.

[0039] Since the culture device of this embodiment has a surface that is subdivided by the cell detachment inhibition region, it is possible to analyze cells cultured under the same culture conditions by different analysis methods. By using cells cultured in the same culture device, more accurate analysis results can be obtained. Also, according to this embodiment, it is possible to apply the cell sheet cultured in one culture device to a plurality of affected parts.

[0040] [Embodiment 3] Next, a cell sheet production kit 23 including a culture device 21 and a recovery support 22 for recovering the cell sheet will be described (Fig. 3(A)). In order to recover the cultured cells in a sheet form, it is preferable to use the recovery support 22. By attaching the cells to the recovery support 22, it can be easily grasped with tweezers or the like.

[0041] The cell sheet is recovered as follows. After the cells reach confluence, the culture medium is aspirated and removed. After aspirating the culture medium, a small amount of culture medium is added to prevent the cells from drying during the operation. The amount of the culture medium may be in the range of 5 μl / cm 2 to 30 μl / cm 2 or so. Next, the recovery support 22 is placed on the cells. The recovery support 22 is made to be slightly smaller in size than the stimulus-responsive region. For example, in the case of a rectangular or square culture device, the vertical and horizontal lengths of the stimulus-responsive region may be shortened by about 1 mm to 5 mm each. In the case of a circular shape, a diameter that is also about 1 mm to 5 mm shorter may be prepared.

[0042] For example, when the stimulus-responsive region is temperature-responsive and the lower critical solution temperature is 32°C, it is left standing at a temperature of 32°C or lower, preferably 0°C to 30°C for about 5 to 60 minutes. After standing, when the recovery support is grasped and lifted with tweezers or the like, the cells remain attached to the recovery support in a sheet form and are peeled off from the cell culture device. In the case of cells with high adhesiveness to the cell detachment inhibition region, the periphery of the recovery support may be peeled off with tweezers or the like. The cell sheet attached to the recovery support may be placed directly at the site where transplantation is desired. The surface opposite to the surface attached to the recovery support, that is, the exposed surface, is the surface that was adhered to the surface of the culture device. That is, it is the surface that was in contact with the scaffold, and this surface can be brought into close contact with the surface that requires transplantation.

[0043] The support for recovery 22 can be made of known materials such as paper, cellulose film, plastic film, etc. Since it has good adhesiveness to cells, it is preferable to use paper, cellulose film, or plastic film. However, since it should not break even when immersed in the culture solution and the work becomes delicate, it is preferably a flexible and easy-to-handle material. The specific material of the support for recovery has a thickness of 30 μm or more and 300 μm or less, and a basis weight of 10 g / m 2 or more and 150 g / m 2 or less, and is preferably paper, cellulose film, or plastic film having a wet tensile strength of 0.03 kN / m or more and 1.0 kN / m or less. With a thickness and basis weight within this range, it is easy to grip with tweezers and easy to perform delicate operations. Also, with a material having a wet tensile strength within this range, there is no fear of damage even when wet with the culture solution.

[0044] Also, although it depends on the size of the support for recovery 22, it is desirable to place it within 1 to 2 mm inside the adhesion / detachment boundary 24 formed by the stimulus-responsive region and the cell detachment suppression region (Fig. 3(C)). This is because the cell detachment suppression region prevents cells from detaching due to stimulation, and when cells are firmly adhered to the surface of the device, there is a risk of the cell sheet tearing. Supports for recovery are prepared in a size slightly smaller than the stimulus-responsive region, but it is very difficult to place the support for recovery inside the adhesion / detachment boundary. Especially when the culture surface is large, placing it inside both vertically and horizontally requires delicate techniques.

[0045] As shown in Figs. 3(B) and 3(C), by providing a boundary display mark 25 that can be visually confirmed in the region corresponding to the cell detachment suppression region, the adhesion / detachment boundary 24 can be identified. Since the surfaces of the device in the stimulus-responsive region and the cell detachment suppression region do not optically change, it is not known where the adhesion / detachment boundary is. However, since the boundary display mark 25 allows the location where the support for recovery 22 should be placed to be visually recognized, the support for recovery can be surely placed inside the adhesion / detachment boundary 24.

[0046] The boundary display mark 25 may be subjected to frosting or light coloring on the surface or bottom surface of the culture device at the cell detachment inhibition region or the location corresponding to the adhesion / detachment boundary. If it is a light-transmitting process, there will be no problem even when observing the growth and morphology of cells under a microscope. In addition, the growth of cells can be confirmed in the stimulus-responsive region, and even if the cell detachment inhibition region is processed or colored, there is no problem at all in performing the culture. The surface to be processed may be the surface where cells adhere as long as the process does not affect cell adhesion, or it may be the outside of the container.

[0047] The kit for manufacturing a cell sheet according to this embodiment suppresses the detachment of cells during the cell culture period and can be easily recovered when recovering the cell sheet. The kit for manufacturing a cell sheet equipped with a culture device and a recovery support has a configuration that has never existed before. By using the kit for manufacturing a cell sheet, the manufactured cell sheet can be easily grasped in a sheet form. Therefore, although some training was required to handle cell sheets in the past, even beginners can handle them.

Explanation of symbols

[0048] 1, 11... cell detachment inhibition region, 2, 12... stimulus-responsive region, 3, 13, 24... adhesion / detachment boundary, 21... culture device, 22... recovery support, 23... kit for manufacturing a cell sheet, 25... boundary display mark

Claims

1. A cell detachment inhibition region having a stronger cell adhesion force than a stimulus-responsive region is provided around the cell culture surface, regardless of the presence or absence of a stimulus that suppresses cell detachment over the entire circumference, a stimulus-responsive region whose adhesiveness to cells changes by an external stimulus is disposed inside the cell detachment inhibition region, the cell detachment inhibition region has a cell adhesiveness stronger than that of the stimulus-responsive region in a range of 3 times or more and 15 times or less even in a state where no stimulus is applied to the stimulus-responsive region, and a cell culture device characterized by this.

2. The cell culture surface of the cell culture device is, 3.5 cm ２ 300 cm or more ２ The cell culture device according to claim 1, which is 300 cm or less.

3. The cell detachment inhibition region is, The cell culture device according to claim 1 or 2, which is a cell adhesion surface in a temperature range of 1°C to 50°C.

4. The cell culture device according to claim 3, wherein the cell adhesion surface is a culture surface that does not respond to an external stimulus.

5. The cell culture device according to any one of claims 1 to 4, wherein the external stimulus is light, temperature, pH, or magnetic field.

6. The stimulus-responsive region is, a temperature-responsive surface that responds to a stimulus by temperature, The cell culture device according to any one of claims 1 to 5, wherein the surface is a surface where cells detach at a temperature of 0°C or higher and 32°C or lower.

7. The cell detachment inhibition region and the stimulus-responsive region are, The cell culture device according to any one of claims 1 to 6, characterized in that they are divided on the same plane.

8. The cell detachment inhibition region is, The cell culture device according to any one of claims 1 to 7, characterized in that the width is 2 mm or more and 10 mm or less.

9. The cell detachment inhibition region is provided so as to divide the cell culture surface in addition to the periphery of the cell culture surface, and the cell culture device according to any one of claims 1 to 8 is characterized by this.

10. The cell detachment inhibition region and the stimulus-responsive region are, The cell culture device according to any one of claims 1 to 9, characterized in that they are processed so as to be distinguishable by visual inspection.

11. The cell detachment inhibition region and the stimulus-responsive region are, The cell culture device according to any one of claims 1 to 10, characterized in that they have light transmissibility.

12. The cell adhesiveness under the condition of not stimulating the cell detachment inhibition region and the stimulus-responsive region is, The cell culture device according to any one of claims 1 to 11, characterized in that the cell detachment inhibition region has a stronger cell adhesiveness than the stimulus-responsive region.

13. The cell culture device according to any one of claims 1 to 12, which is for producing a cell sheet of epithelial cells, mesenchymal stem cells, or myoblasts.

14. The stimulus-responsive region and the cell detachment inhibition region coat the cell culture surface with a temperature-responsive monomer, The cell culture device according to any one of claims 1 to 13, which is produced by forming a stimulus-responsive layer by irradiating only the stimulus-responsive region with electron beams, γ-rays, or ultraviolet rays.

Images