Novel nasal mucosal cell sheet

Abstract

The present invention addresses the problem of providing a cultured cell sheet which can solve problems associated with the defects of mucosal membrane tissues including the occurrence of an inflammation in a mucosal membrane tissue that covers a bone tissue and the formation of granulation tissues.　In order to solve the above-mentioned problem, the present inventors have made studies and developments based on exhaustive considerations from various angles. As a result, it is found that a cultured cell sheet, which is produced by culturing cells collected from a nasal mucosal membrane tissue that serves as a raw material under specified conditions and then reconstituting the cultured product in a sheet-like form, is engrafted in a defected part in a mucosal membrane tissue, including parts in which an inflammation occurs in the surface of a bone tissue in a middle ear or a granulation tissue is formed, to accelerate the regeneration of a mucosal membrane tissue such as a mucosal membrane tissue that covers a bone tissue, and effectively prevent the occurrence of an inflammation in a middle ear bone tissue, and prevent the occurrence of fibrosis, the formation of a granulation tissue and the formation of an unhealthy epidermis which are characteristic of an inflammation of a bone tissue, and it is demonstrated that the cultured cell sheet can solve the above-mentioned problem. It is also found that the cultured cell sheet according to the present invention, which is produced using cells collected from a nasal mucosal membrane tissue as a raw material, is also useful for the prevention of an inflammation in a bone tissue in a part other than the middle ear.

Description

[Technical Field]

[0001] This invention relates to a cultured cell sheet, a method for manufacturing the sheet, and a method for using the same, using cells collected from nasal mucosal tissue that is useful in the field of medicine as raw material. [Background technology]

[0002] Humans perceive sound as air vibrations with frequencies of approximately 20 to 20,000 Hz that reach the auricle. Sound enters the ear canal as air vibrations, is transmitted from the eardrum to the three ossicles in the middle ear cavity, where the vibrations are amplified and transmitted to the cochlea in the inner ear. These vibrations cause the lymphatic fluid filling the inner ear to move, and this stimulus is transmitted from the hair cells in the cochlea to the cochlear nerve. The signal transmitted to the cochlear nerve travels through the brainstem and diencephalon to the auditory cortex of the cerebral cortex, where it is perceived as sound. Damage to any of these conduction pathways can lead to hearing loss. The middle ear cavity is lined with middle ear mucosa, which also serves as the periosteum. The mucosa has gas ventilation capabilities and maintains good air content, thus efficiently transmitting sound to the inner ear. The function of the mucosa plays a crucial role in the middle ear, but there is no established method to regenerate damaged middle ear mucosa.

[0003] Adhesive otitis media and cholesteatoma are well-known as representative intractable middle ear diseases that cause hearing loss. Adhesive otitis media is a disease in which the eardrum cannot maintain its normal position and adheres to the bone wall in the middle ear cavity, resulting in a loss of air in the middle ear cavity. Because the adhered eardrum's vibrations are hindered, sound is not transmitted properly, resulting in hearing loss. It also becomes susceptible to infection, leading to repeated ear discharge, and if it progresses, it can develop into cholesteatoma. Cholesteatoma is a disease in which keratinized stratified squamous epithelium, continuous with the skin of the external auditory canal, invades the middle ear, containing keratinized material and desquamation, and destroys the surrounding bone tissue, leading to various complications. Destruction of the ossicles causes hearing loss, and if the inflammation and bone destruction spread to the inner ear, it can lead to irreversible inner ear hearing loss. Furthermore, due to the anatomical characteristics of the middle ear, it can cause various other serious complications, including dizziness, facial nerve paralysis, meningitis, and intracranial complications such as brain abscesses.

[0004] The definitive treatment for adhesive otitis media and cholesteatoma is middle ear surgery called tympanoplasty, one of which aims to improve hearing. For this to happen, it is necessary for air to be present in the middle ear cavity, allowing vibrations of the eardrum to be transmitted to the inner ear via the ossicles without loss. To create an ideal middle ear cavity after surgery, it is important to regenerate the middle ear mucosa, thereby restoring physiological gas ventilation capacity and preventing adhesion of the eardrum. However, in adhesive otitis media and cholesteatoma, the function of the middle ear mucosa is inherently impaired, so postoperative regeneration of the middle ear mucosal epithelium is delayed, and it is often difficult to create an effective air-filled cavity. In particular, in cases of adhesive otitis media where the eardrum is recessed and adheres to the inner wall of the middle ear cavity, the bone surface of the middle ear is exposed when the lesion is peeled away during surgery, making it difficult to preserve the middle ear mucosa. For this reason, postoperative hearing improvement in adhesive otitis media is very poor compared to other middle ear diseases.

[0005] On the other hand, surgical procedures for cholesteatoma include posterior auditory canal preservation tympanoplasty, which preserves the posterior wall of the external auditory canal, and posterior auditory canal removal tympanoplasty, which removes the posterior wall. Posterior auditory canal preservation tympanoplasty is superior in that it preserves the physiological morphology of the external auditory canal, but the drawback of this procedure is that postoperative recurrence is common. Residual recurrence due to incomplete removal of the lesion during surgery can now be prevented by using endoscopy, but it is difficult to reliably prevent replastic recurrence caused by postoperative re-adhesion or re-indentation. On the other hand, posterior auditory canal removal tympanoplasty can prevent replastic recurrence, but it impairs the physiological morphology of the external auditory canal and may lead to postoperative mastoid cavity problems, also known as postoperative cavity problems. The ideal surgical procedure would be tympanoplasty that preserves the posterior wall of the external auditory canal and creates a middle ear cavity with good postoperative aeration. However, a definitive surgical method does not yet exist, and conventional tympanoplasty alone has its limitations, making it impossible to expect better treatment results than before.

[0006] Given these circumstances, it is believed that if it becomes possible to regenerate the middle ear mucosa damaged after surgery, it will be possible to prevent re-adhesion of the tympanic membrane in adhesive otitis media, and to prevent replastic recurrence while preserving the posterior wall of the external auditory canal in cholesteatoma, thus significantly improving the prevention of recurrence, which has been difficult until now. How to regenerate the middle ear mucosa early has been a major challenge, and various methods of direct mucosal transplantation have been attempted by various researchers in the hope of preventing re-indentation and re-adhesion of the tympanic membrane after surgery and regenerating the middle ear mucosa (Non-patent Literature 1), but there is still no established treatment method.

[0007] Against this backdrop, the inventors have been working on the clinical application of a novel treatment using cell sheet engineering aimed at regenerating the middle ear mucosa for intractable middle ear diseases. The inventors have been researching the regeneration of the middle ear mucosa for some time, and have confirmed that good mucosal regeneration is observed after transplanting a three-dimensionally artificial middle ear mucosa (Non-Patent Literature 2) into a rabbit model of middle ear mucosal damage (Non-Patent Literature 3). Subsequently, with a view to the clinical realization of regenerative medicine for the middle ear mucosa in humans, they have been working on the regeneration of the middle ear mucosa using somatic stem cells with a cell culture substrate coated with a temperature-responsive polymer based on cell sheet engineering (Patent Literature 1, Non-Patent Literature 4) (Non-Patent Literature 5). Regenerative medicine using cell sheet engineering has already been successfully applied to humans in the cornea, esophagus, etc., and good therapeutic effects have been obtained. As a cell source for cell sheet fabrication, we focused on nasal mucosa, which can be harvested minimally in an outpatient setting, is anatomically continuous with the middle ear mucosa, and is histologically similar. We have developed a novel treatment method in which we create a sheet of the patient's own cultured epithelial cells using a culture substrate coated with a temperature-responsive polymer from nasal mucosa, with the aim of promoting the regeneration of the middle ear mucosa after surgery and preventing re-adhesion of the tympanic membrane, and then transplant it into the middle ear cavity after surgery. We have obtained results in preclinical animal experiments using rabbits with cell culture inserts coated with temperature-responsive polymer (Non-Patent Literature 6), and have also succeeded in fabricating human nasal mucosal epithelial cell sheets (Patent Literature 2).

[0008] It is known that when the mucosal tissue on the surface of the middle ear bone is damaged, problems such as inflammation of the bone tissue and formation of granulation tissue occur. Once a problem occurs, treatment by means such as removal of granulation tissue and suppression of inflammation on the surface of the bone tissue is effective, but there are problems such as recurrence. Even when using the conventional human nasal mucosa epithelial cell sheet (Patent Document 2) developed by the present inventors, this problem could not be solved.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention aims to provide a cultured cell sheet that can solve the problems associated with the loss of mucosal tissue, such as inflammation of the bone tissue surface and granulation tissue formation, as described above. [Means for solving the problem]

[0012] The inventors have conducted research and development from various angles to solve the above problems. As a result, they have found that a cultured cell sheet obtained by culturing cells collected from nasal mucosal tissue under specific conditions and reconstructing it into a sheet engrafts on areas of mucosal tissue defects such as inflammation and granulation tissue formation on the surface of bone tissue in the middle ear, promotes the regeneration of mucosal tissue covering bone tissue, efficiently suppresses inflammation occurring in the bone tissue of the middle ear, and inhibits characteristic fibrosis, granulation tissue formation, and poor epithelial formation during bone tissue inflammation, thus demonstrating that the aforementioned problems can be solved. Furthermore, they have found that the cultured cell sheet made from cells collected from nasal mucosal tissue of the present invention is also useful for suppressing inflammation in bone tissue in areas other than the middle ear.

[0013] More specifically, this application provides the following embodiments to solve the aforementioned problems: [1]: A cultured cell sheet made from cells collected from nasal mucosal tissue, containing 50-90% undifferentiated cells relative to the total number of cells; [2]: Undifferentiated cells positive for p63, as described in [1], cell culture sheet; [3]: A cultured cell sheet as described in [1] or [2], wherein the undifferentiated cells are nasal mucosal epithelial stem cells or nasal mucosal epithelial progenitor cells; [4]: A cell culture sheet as described in [1] or [2] in which undifferentiated cells do not have cilia; [5]: The cell sheet described in [1] or [2], wherein the cell sheet is a mixture of nasal mucosal epithelial cells and nasal mucosal epithelial stem cells or nasal mucosal epithelial progenitor cells, in addition to one or more other epithelial cells or other epithelial stem cells, mesenchymal stem cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, and adipocytes; [6]: A cultured cell sheet as described in [5], wherein the proportion of fibroblasts contained in the cell sheet is 0-12%; [7]: A cultured cell sheet according to [1] or [2], wherein the nasal mucosal tissue from which the raw material cells are derived is from the inferior turbinate mucosa, middle turbinate mucosa, superior turbinate mucosa, nasal cavity mucosa, or paranasal sinus tissue; [8]: Cell culture sheets as described in [1] or [2], wherein the nasal mucosal tissue is derived from human, rabbit, dog, cat, pig, monkey, chimpanzee, rat, or mouse; [9]: Cell culture sheets as described in [1] or [2], intended for the treatment of chronic otitis media, adhesive otitis media, cholesteatoma, chronic sinusitis, sinus tumors, external auditory canal tumors, external auditory canal atresia, chronic tonsillitis, pharyngeal cancer, laryngeal cancer, oral cancer, tongue cancer, tracheal granulation tissue, tracheal scarring, tracheal stenosis, tracheal injury, esophageal cancer, vocal cord lesions, inflammatory bowel disease, anal ulcers, anal fissures, hemorrhoids, or anal fistulas;

[10] : The following steps: (1) A step of preparing mucosal epithelial cells to be used as raw material from nasal mucosal tissue; (2) A step of culturing the prepared mucosal epithelial cells to increase the number of raw material mucosal epithelial cells; (3) A step of seeding the obtained cells onto a cell culture substrate and culturing them, thereby creating a cultured cell sheet in which the cells cover the surface of the culture substrate; (4) A step of peeling off and collecting the cultured cell sheet prepared on the culture substrate while it remains in sheet form; A method for producing a cultured cell sheet containing 50-90% undifferentiated cells relative to the total number of cells, using cells collected from nasal mucosal tissue as raw material;

[11] : In process (1), A process for preparing raw material mucosal epithelial cells by obtaining cells using an explorer culture method, in which nasal mucosal tissue collected from a subject is left standing on a cell culture substrate without enzymatic treatment, and cultured in a serum-containing culture medium or a serum-free culture medium, thereby causing the cells in the tissue to migrate into the culture medium; The method according to

[10] , which prepares mucosal epithelial cells to be used as raw material;

[12] : A method for producing a cultured cell sheet according to

[10] or

[11] , wherein the cell culture substrate used in steps (1) and (2) is a dish-type culture substrate, a flask-type culture substrate, or a multi-well type culture substrate;

[13] : Processes (3) and (4) (3) A step of preparing a cell culture sheet on a cell culture substrate coated with a temperature-responsive polymer whose surface changes in hydration capacity in the temperature range of 0 to 80°C, by culturing the prepared mucosal epithelial cells in a temperature range where the hydration capacity of the temperature-responsive polymer is weak; (4) A step of obtaining a cultured cell sheet in sheet form by changing the temperature of the culture medium to a temperature at which the hydration force of the temperature-responsive polymer is strong, thereby detaching the cultured cell sheet from the cell culture substrate; The method according to

[10] or

[11] for preparing raw material mucosal epithelial cells, carried out by

[10] or

[11] ;

[14] : A method for producing a cultured cell sheet according to

[10] or

[13] , wherein the cell culture substrate used in step (3) is coated on its surface with one or more of the following: type I collagen, laminin, fibronectin, or Matrigel;

[15] : A method for producing a cell culture sheet according to

[10] or

[13] , wherein the temperature-responsive polymer used in steps (3) and (4) is poly-(N-isopropylacrylamide);

[16] : A method for producing a cultured cell sheet according to

[10] or

[13] , wherein the peeling step in step (4) is a method of adhering a carrier to cultured cells and peeling them off together with the carrier;

[17] : A method for producing a cultured cell sheet according to

[10] or

[13] , further comprising the step of stacking the cell sheet detached in step (4) onto another cell sheet detached in step (4), or repeating this operation to stack cell sheets;

[18] : The number of mucosal epithelial cells to be seeded at the start of culture in step (3) is 3 × 10 4 ~2×10 5 cells / cm 2 A method for producing a cultured cell sheet as described in

[10] or

[13] ;

[19] : A method for producing a cultured cell sheet according to

[10] or

[13] , using feeder cells during the culture in step (2) and / or step (3);

[20] : A method for producing a cultured cell sheet according to

[10] or

[13] , wherein serum-free medium is used for culturing in step (3). [Effects of the Invention]

[0014] The cultured cell sheet made from cells collected from nasal mucosal tissue as described in this invention efficiently engrafts onto the surface of the middle ear bone, promoting the regeneration of mucosal tissue covering the bone tissue and suppressing fibrosis, granulation tissue formation, and poor epithelial formation in the middle ear cavity. Furthermore, this cultured cell sheet made from cells collected from nasal mucosal tissue can also be used as a useful mucosa in the case of mucosal loss in the nasal cavity and oral cavity. In addition, the cultured cell sheet made from cells collected from nasal mucosal tissue of this invention is also useful for suppressing inflammation of bone tissue in areas other than the middle ear. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows that fibroblast-like cells and undifferentiated cells migrated from nasal mucosal tissue using the serum explorer culture method. [Figure 2] Figure 2 shows a cell sheet prepared using feeder cells on a cell culture substrate coated with a temperature-responsive polymer. [Figure 3] Figure 3 shows a specific example of observations regarding p63 expression when counting the number of cells in tissue sections using immunohistochemical staining. [Figure 4] Figure 4 shows a cell sheet (left) recovered from a cell culture substrate coated with a temperature-responsive polymer without using feeder cells, and a cell sheet (right) obtained from a cell insert coated with a temperature-responsive polymer, after being detached from the cell culture substrate. [Figure 5] Figure 5 shows that fibroblast-like cells and undifferentiated cells migrated from nasal mucosal tissue using the serum-free explorer culture method. [Figure 6] Figure 6 shows a cell sheet prepared on a cell culture substrate coated with a temperature-responsive polymer, without the use of feeder cells. [Figure 7] Figure 7 shows a cell sheet, recovered from a cell culture substrate coated with a temperature-responsive polymer without the use of feeder cells, after being detached from the cell culture substrate. [Figure 8] Figure 8 shows examples of observations of p63 expression and PDGF Rα expression in tissue sections by immunohistochemical staining. [Figure 9] Figure 9 shows the work process for confirming the effect of the bone tissue regeneration cell sheet of the present invention by transplanting the nasal mucosa-like cell sheet of the present invention into rabbits from which the middle ear mucosa has been removed in advance. [Figure 10] Figure 10 shows an overview of the process of transplanting a cultured cell sheet, prepared using cells collected from nasal mucosal tissue by the method of Example 1 and without using feeder cells, into a human patient. [Figure 11] Figure 11 shows the postoperative course in a patient after transplantation of the nasal mucosal epithelial cell sheet of the present invention. [Modes for carrying out the invention]

[0016] This invention provides a cultured cell sheet made from cells collected from nasal mucosal tissue. Furthermore, this invention provides a method for recovering cells from nasal mucosal tissue under specific culture conditions and ultimately producing a sheet-like cultured cell sheet on the surface of a cell culture substrate. Moreover, this invention provides a method for utilizing the obtained cultured cell sheet.

[0017] <Cell Sheet> According to the present invention, it is possible to provide a cultured cell sheet made from cells collected from nasal mucosal tissue, in which undifferentiated cells make up 50-90% of the total number of cells. As a result of the inventors' research, it was found that undifferentiated cells proliferate easily during culture, have good adhesion to the transplant site, and perform the function of regenerating epithelial tissue in the transplant site after transplantation. Initially, the inventors thought that fully differentiated nasal mucosal epithelial cells, that is, not undifferentiated cells, which have abundant cilia, were more useful, and focused on cells with cilia, stating that "if the number of cells with cilia is less than 5%, the transplanted cell sheet will not be able to fully exert the effects of the present invention and is undesirable" (Patent Document 2). However, subsequent research revealed that undifferentiated cells (cells without cilia) have a better effect as cell sheets, and that by including 50-90% undifferentiated cells in the total number of cells in the cell sheet, the engraftment rate of the cell sheet at the transplant site increases, and the epithelial tissue of the superficial layer of the middle ear bone can be regenerated, leading to the present invention.

[0018] In this invention, the undifferentiated cells constituting the cultured cell sheet, which is made from cells collected from nasal mucosal tissue, constitute 50% or more, preferably 55% or more, more preferably 60% or more, and even more preferably 65% ​​or more, of the total number of cells in the cell sheet.

[0019] These undifferentiated cells can proliferate not only during culture but also at the transplant site after transplantation, and play a role in regenerating the epithelial tissue at the transplant site. Therefore, if the proportion of undifferentiated cells is less than 15% of the total number of cells in the cell sheet, the cell sheet transplanted at the transplant site will not be able to fully exert the effects of the present invention. In the present invention, undifferentiated cells refer to cells that do not possess pluripotency like iPS cells or ES cells, but maintain the ability to differentiate into epithelial cells, ciliated cells, goblet cells, etc.

[0020] Undifferentiated cells in this invention are characterized by being positive for p63 as a marker. p63 is known to be a transcription factor essential for epithelial morphogenesis and stem cell properties, and in this invention, it is a marker indicating that cells collected from nasal mucosal tissue are highly stem cell cells (i.e., undifferentiated cells). Whether cells contained in a cultured cell sheet are positive for p63 can be determined by detecting the cells using an antibody against p63. Specifically, detection can be performed by preparing tissue sections from a cultured cell sheet and detecting p63-positive cells by immunostaining; by enzymatically separating cells from a cultured cell sheet and then detecting cells that bind to an antibody against p63 as p63-positive cells using flow cytometry; or by enzymatically separating cells from a cultured cell sheet and then detecting the p63 protein or p63 gene using methods such as Western blotting or real-time PCR. When confirming how many p63-positive cells (undifferentiated cells) are present in multiple cells, it is preferable to use either the immunostaining method or the flow cytometry method from the above-mentioned methods.

[0021] In the present invention, the undifferentiated cells are preferably epithelial stem cells, and examples include, but are not limited to, nasal mucosal epithelial stem cells, nasal mucosal epithelial progenitor cells, and mesenchymal stem cells. The cells contained in the cultured cell sheet can be defined as epithelial stem cells by detecting the expression of p63, cytokeratin 5, and cytokeratin 14 as described above. Furthermore, the undifferentiated cells contained in the cultured cell sheet of the present invention have characteristics such as not having cilia and not containing goblet cells as they can be observed under a microscope.

[0022] As described above, the cultured cell sheet in the present invention preferably contains a large amount of undifferentiated cells, but the cell sheet in the present invention may also contain cells other than epithelial stem cells, and there are no restrictions on the type of cells. For example, one or more of the following cells can be used: epithelial cells other than nasal mucosal epithelial cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, bone marrow-derived cells, adipocyte-derived cells, and mesenchymal stem cells. Furthermore, there are no particular restrictions on the proportion of each of these cells contained.

[0023] Fibroblasts are one example of cells that may be included in the cultured cell sheet of the present invention, but their proportion should be very low, in the range of 0% to 12%, and preferably 0% to 5%. Generally, fibroblasts have the effect of strengthening the structure of the cell sheet in a cultured cell sheet, and it is thought that including them in the range of 0.1% to 1% increases the strength of the cell sheet.

[0024] In the present invention, the content of cilia-retaining nasal mucosal cells (i.e., one of the differentiated cells) is required to be less than 5% of the total number of cells in the cultured cell sheet made from cells collected from the nasal mucosal tissue ultimately obtained, preferably less than 4%, more preferably less than 3%, and most preferably less than 1%. Cultured cell sheets with a content of cilia-retaining nasal mucosal cells of 5% or more are undesirable because they have poor engraftment onto the surface of the middle ear bone when transplanted to the affected area, and the effects of the present invention cannot be fully exhibited.

[0025] The cells used as raw materials for manufacturing the cultured cell sheets of the present invention are collected from nasal mucosal tissue. The nasal mucosal tissue can be any mucosal tissue from within the nasal cavity, paranasal sinuses, or mastoid sinuses. For example, the nasal mucosa used in the present invention includes the inferior turbinate mucosa, middle turbinate mucosa, superior turbinate mucosa, nasal cavity mucosa, and paranasal sinus tissue. Among these, the inferior turbinate mucosa is particularly preferred because it is easy to collect. The nasal mucosal tissue used as raw material in the present invention is abundant in the body, its collection is easy enough to be done on an outpatient basis, and it is preferable because it places less burden on the patient and raises fewer ethical concerns for the patient.

[0026] The cells used as raw materials for manufacturing the cultured cell sheet in this invention may be cells themselves collected from nasal mucosal tissue, cells cultured and proliferated under culture conditions after collection, or cell lines, but there are no restrictions on the type of cells used.

[0027] Furthermore, there are no particular restrictions on the animals from which these raw materials cells originate, but examples include humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or immunodeficient animals thereof. However, when using the cultured cell sheet made from cells collected from nasal mucosa tissue of the present invention for human treatment, it is preferable to use cells derived from humans, pigs, monkeys, or chimpanzees. In order to reduce side effects associated with cell transplantation, such as immune reactions, it is particularly preferable to use human-derived cells, and most preferably to use autologous cells.

[0028] The size of the cultured cell sheet of the present invention can be freely set according to the site of transplantation. The size of the cultured cell sheet is determined by the size of the culture substrate used for culture. For example, when using commercially available culture substrates, the diameter of the cultured cell sheet can be up to approximately 35 mm when using a 6-well plate or a 35 mm dish, and up to approximately 60 mm when using a 60 mm dish. When creating a cultured cell sheet using these plates and using the cultured cell sheet for a transplantation site smaller than the size of the cell sheet, the cultured cell sheet can be cut as appropriate.

[0029] The cultured cell sheet of the present invention may consist of two or more cultured cell sheets stacked on top of each other. In another embodiment, a cell sheet containing one or more of the following cells—vascular endothelial cells, vascular endothelial progenitor cells, adipocytes, or mesenchymal stem cells—may be stacked on top of the cultured cell sheet. The number of stacked sheets is not particularly limited, but the number of stacks should be 10 or less, preferably 5 or less, and more preferably 2 or less. Stacking cultured cell sheets made from cells collected from nasal mucosal tissue can improve the cell density per sheet area. Furthermore, in the cultured cell sheet made from cells collected from nasal mucosal tissue of the present invention, as long as the function of the cell sheet of the present invention as described above is not impaired, it may contain a mixture of one or more of the following cells in addition to mucosal cells: vascular endothelial cells, vascular endothelial progenitor cells, adipocytes, or mesenchymal stem cells. These cell sheets may also be stacked on top of the cultured cell sheet made from cells collected from nasal mucosal tissue.

[0030] The cultured cell sheet of the present invention can be prepared using any culture substrate, but as an example, it can be prepared using a culture substrate coated with a temperature-responsive polymer. When using a culture substrate coated with a temperature-responsive polymer, mucosal epithelial cells collected from nasal mucosal tissue are cultured on the culture substrate coated with the temperature-responsive polymer to prepare a cultured cell sheet on the culture substrate, and then the temperature is changed to a state in which the hydration power of the temperature-responsive polymer is strong, thereby peeling the cultured cell sheet from the culture substrate coated with the temperature-responsive polymer, and thus providing a cultured cell sheet in sheet form. More specifically, a method for producing a nasal mucosal tissue-derived cultured cell sheet can be provided, characterized by culturing cells collected from nasal mucosal tissue on a cell culture substrate coated on its surface with a temperature-responsive polymer whose hydration power changes in the temperature range of 0 to 80°C at a temperature range in which the polymer's hydration power is weak, and then changing the temperature of the culture medium to a state in which the polymer's hydration power is strong, thereby peeling the cultured cell sheet while it remains in sheet form.

[0031] In the present invention, a cultured cell sheet made using a culture substrate coated with a temperature-responsive polymer and derived from cells collected from nasal mucosal tissue is characterized by not being damaged by proteolytic enzymes such as dispase and trypsin when the cultured cell sheet is detached. Therefore, the cultured cell sheet detached from the culture substrate retains the adhesive proteins that were present in the basal layer when the cell sheet was adhered to the culture substrate. For example, in the case of a cultured cell sheet made by culturing mucosal cells in a sheet form, the desmosome structure between cells is retained when detached. As a result, when transplanting the cultured cell sheet, the cultured cell sheet can adhere well to the tissue at the transplant site, enabling efficient transplantation. Generally, it is known that dispase, a proteolytic enzyme, can detach cells while retaining 10-40% of the desmosome structure between cells, but it destroys most of the basal membrane-like proteins between cells and the substrate, resulting in a cell sheet with low strength. In contrast, the cultured cell sheet produced by the method of the present invention, using cells collected from nasal mucosal tissue as raw material, retains both the desmosome structure and basement membrane-like proteins, and thus can obtain the various effects described above.

[0032] The cultured cell sheet in this invention, which is made from cells collected from nasal mucosal tissue, is produced using epithelial cells collected from nasal mucosal tissue as raw material. Therefore, the cell sheet of this invention has the effect of substituting for the function of mucosal epithelial tissue and has several characteristic functions, such as promoting the regeneration of mucosal tissue covering bone tissue. In other words, when the cell sheet of this invention is transplanted into the body surface in the middle ear cavity, nasal cavity, or oral cavity, it promotes the regeneration of mucosal tissue covering bone tissue. The purpose of this invention is to regenerate biological functions by producing a cell sheet with such functions and transplanting it into the body. Furthermore, the cultured cell sheet obtained in this invention, which is made from cells collected from nasal mucosal tissue, may secrete mucosal-specific substances such as mucin, hyaluronic acid, and polysaccharides, and the type and amount of these secretions are not particularly limited. Moreover, the cell sheet used in this invention may have genes introduced in order to enhance the function of the cell sheet, and the method of introduction can be in accordance with conventional methods.

[0033] The cultured cell sheet of the present invention has the characteristics described above and can be used to treat diseases occurring on the epithelial surface by transplanting it into, for example, the middle ear cavity or mastoid cavity in the body. Therapeutic applications of the cultured cell sheet of the present invention include, but are not limited to, the treatment of diseases such as chronic otitis media, adhesive otitis media, cholesteatoma, chronic sinusitis, paranasal sinus tumors, external auditory canal tumors, external auditory canal atresia, chronic tonsillitis, pharyngeal cancer, laryngeal cancer, oral cancer, tongue cancer, tracheal granulation tissue, tracheal scarring, tracheal stenosis, tracheal injury, esophageal cancer, vocal cord lesions, inflammatory bowel disease, anal ulcers, anal fissures, hemorrhoids, or anal fistulas, or the reconstruction of the middle ear cavity when reconstruction of the middle ear cavity is necessary after surgery or for other reasons.

[0034] <Method for manufacturing cultured cell sheets> In one aspect of the present invention, a method for producing the cultured cell sheet described above can be provided. Specifically, a method for producing a cultured cell sheet using cells collected from nasal mucosal tissue as raw material is provided, characterized by comprising: (1) a step of preparing mucosal epithelial cells to be used as raw material from nasal mucosal tissue; (2) a step of culturing the prepared mucosal epithelial cells to increase the number of cells of the mucosal epithelial cells to be used as raw material; (3) a step of culturing on a cell culture substrate to produce a cultured cell sheet in which the cells cover the surface of the cell culture substrate; and (4) a step of peeling off and collecting the cultured cell sheet produced on the cell culture substrate while it is still in sheet form.

[0035] Process (1): Obtaining raw material cells from tissue The cultured cell sheet in this invention is made from cells collected from nasal mucosal tissue. In other words, in this invention, it is necessary to separate cells from tissue collected from the mucosa of the nasal cavity, paranasal sinuses, or mastoid sacs through a specific process. One such processing method is the explorer culture method, in which nasal mucosal tissue collected from a subject is left standing on a cell culture substrate without enzymatic treatment, and cultured in a serum-containing culture medium or a serum-free culture medium to induce cell migration in the tissue.

[0036] The culture medium used during the explicit culture in this process may be either a serum-containing medium or a serum-free medium (so-called serum-free medium), and the method should be carried out appropriately according to the standard procedures depending on the cells being used; there are no particular limitations. For example, when using a serum-containing medium, the base medium may be DMEM medium, F-12 medium, KCM medium, or a mixture thereof. In this case, additives commonly used in cell culture, such as serum, may be added to the medium. Alternatively, when using a serum-free medium, various commercially available serum-free media for epithelial cells, such as UltraCULTURE serum-free medium (Lonza), PC-1 serum-free medium (Lonza), EpiCult Medium (VERITAS), or EpiLife Medium (ThermoFisher), may be used as the serum-free medium. In this case, additives commonly used in serum-free cell culture, such as Supplement S7 (Gibco) or human serum albumin, may be added to the medium.

[0037] In this process, when adding serum to the culture medium, fetal bovine serum or human serum can be used. In this case, the concentration of the serum used in the culture medium should be 0.5 to 35%, preferably 1 to 20%, more preferably 5 to 15%, and most preferably 8 to 12%.

[0038] In this process, a ROCK (Rho-binding kinase) inhibitor can be added to the culture medium to prevent apoptosis (programmed cell death) of nasal mucosal epithelial cells migrating from the nasal mucosal tissue. Examples of ROCK inhibitors that can be used in this invention include, but are not limited to, Y-27632, ripasudil hydrochloride hydrate (Kowa Co., Ltd.), thiazovivin, fasudil, GSK429286A, RKI-1447, H-1152, and Azaindole 1.

[0039] Furthermore, to increase cell adhesion, the surface of the cell culture substrate used may be pre-coated with collagen, laminin, fibronectin, Matrigel, serum, or mixtures thereof as needed, or a positively charged cell culture substrate surface may be used. The present invention is not particularly limited by these conditions.

[0040] In this process, the number of days for the explore culture can be appropriately determined while checking the shape and number of cells that have migrated from the nasal mucosa tissue into the culture medium over time. Typically, the explore culture is performed for 10 to 14 days, and the cells are harvested after it is confirmed that the cells have migrated and that they cover 70% of the culture surface of the culture substrate. However, the number of days for the explore culture can be extended or shortened, for example, while observing the culture status.

[0041] When obtaining nasal epithelial cells that have migrated using Explant culture, the nasal epithelial cells attached to the cell culture substrate are treated with enzymes to suspend them from the substrate and then recovered. Trypsin, protease, and collagenase can be used as enzymes for this process.

[0042] Examples of culture substrates that can be used in step (1) of the present invention include dish-type culture substrates, flask-type culture substrates, and multi-well-type culture substrates. As for the material of the substrate for cell culture to be coated, any material that can be shaped, such as glass, modified glass, polystyrene, polymethyl methacrylate, and other compounds commonly used in cell culture, can be used, as well as any other polymer compounds and ceramics.

[0043] Step (2): Cell proliferation The epithelial cells obtained as raw materials in step (1) may have a small number of cells for subsequent culture depending only on the number of cells obtained in step (1). Therefore, as necessary, in step (2), the mucosal epithelial cells prepared in step (1) can be cultured to increase the number of mucosal epithelial cells as raw materials. Note that the number of passages can be determined based on the relationship between the number of cells obtained in step (1) and the number of desired cells. However, since the properties of cells generally change as the number of passages increases, it is preferable to culture with as few passages as possible. Therefore, if a sufficient amount of cells is obtained in step (1) for the sheet culture of the cells in step (3) described below, step (2) may not be performed.

[0044] In step (2), similar to step (1-a) and step (1-b), serum can be added to the culture medium, and a ROCK (Rho-associated coiled-coil containing protein kinase) inhibitor can be added to the culture medium for the purpose of preventing cell death (apoptosis) of nasal mucosal epithelial cells during the culture period.

[0045] The culture substrate that can be used in step (2) of the present invention can be the same as that in step (1). Also, the culture medium that can be used in step (2) may be a serum-containing culture medium or a serum-free culture medium, similar to the case of step (1).

[0046] Step (3): Sheet culture of cells In the present invention, the cells thus obtained are seeded on a cell culture substrate and cultured to prepare a cell sheet in which the cells cover the surface of the cell culture substrate on the cell culture substrate. At this time, the number of cells to be seeded is generally 3.0×10 4 ~2.0×10 5 cells / cm 2 is preferably set, preferably 4.0×10 4 ~1.5×10 5 cells / cm 2 and more preferably 5.0×10 4 ~1.0×10 5 cells / cm 2It is preferable to do so. On the other hand, if the seeding concentration is 3.0 × 10 4 cells / cm 2 If the number of cells to be seeded is less than 2.0 × 10⁶, the proliferation of mucosal cells will be poor, and the degree of functional expression of the resulting nasal mucosal tissue-derived cultured cell sheet will be impaired, which is undesirable in terms of carrying out the present invention. Conversely, if the number of cells to be seeded is 2.0 × 10⁶, 5 cells / cm 2 Too much of it is undesirable because it impairs the proliferation of mucosal cells.

[0047] In this step of the present invention, a cell culture substrate coated on its surface with a temperature-responsive polymer can be used as the cell culture substrate. When a cell culture substrate coated on its surface with a temperature-responsive polymer is used as the cell culture substrate, step (3) can be carried out by culturing the prepared mucosal epithelial cells on a cell culture substrate coated on its surface with a temperature-responsive polymer whose hydration power changes in the temperature range of 0 to 80°C, in a temperature range where the hydration power of the temperature-responsive polymer is weak, and preparing a cell culture sheet on the cell culture substrate.

[0048] The culture medium used for cell culture in this invention may be either a serum-containing medium or a serum-free medium (so-called serum-free medium), and is not particularly limited, as it can be carried out appropriately according to the conventional method depending on the cells used. For example, in the case of nasal mucosal epithelial cells, KCM medium, DMEM medium, F-12 medium, or mixtures thereof can be used. In this case, additives that are normally used in cell culture, such as serum, may be added to the medium. Alternatively, commercially available serum-free culture media for epithelial cells, such as UltraCULTURE serum-free medium (Lonza), PC-1 serum-free medium (Lonza), EpiCult Medium (VERITAS), and EpiLife Medium (ThermoFisher), may be used. When using a serum-containing medium, fetal bovine serum (FBS), human allogeneic serum, or human autologous serum can be used, and it is preferable to use human autologous serum from the viewpoint of cell proliferation and in vivo immune response after transplantation of cultured cell sheets.

[0049] When creating the cultured cell sheet of the present invention, feeder cells may be used to promote cell proliferation and cell sheet formation of nasal mucosal epithelial cells. In this case, the cultured cell sheet may be prepared by mixing feeder cells with raw material mucosal epithelial cells and seeding them on a cell culture substrate, or by first culturing feeder cells on a cell culture substrate to form a monolayer, and then seeding the raw material mucosal epithelial cells on top of that to prepare the cultured cell sheet. When creating a cultured cell sheet using feeder cells, feeder cells can be mouse-derived fibroblasts treated with gamma irradiation or antibiotics, mouse fetal-derived fibroblasts, autologous cells, or more specifically, 3T3-J2 cells. Although feeder cells may induce an immune response in vivo after transplantation, the impact is low because they are eliminated to the surface during culture.

[0050] On the other hand, when preparing the cultured cell sheet of the present invention, it is also possible to prepare the cultured cell sheet by seeding raw cells onto a cell culture substrate without using feeder cells, culturing them in serum-free medium until completely confluenced, then changing to serum-containing medium or the like and culturing for 4 to 7 days to create a layered structure. In this case, from the viewpoint of the immune response that may occur in vivo after transplantation, the risk is lower and therefore preferable.

[0051] The inventors found that when culturing human nasal mucosal epithelial cells, regardless of the form of the culture substrate described later, adding human serum promotes the proliferation of the cultured human nasal mucosal epithelial cells, resulting in a human nasal mucosal epithelial cell sheet that possesses physical strength and flexibility. When using human serum, it is possible to use serum from another person, but it was found that using the patient's own serum from which the nasal mucosal tissue was collected further enhances cell proliferation and results in a human nasal mucosal epithelial cell sheet that is physically strong and flexible, which is relatively preferable for the present invention. If serum is used, the concentration of the serum in the culture medium is preferably 0.5 to 35%, more preferably 1 to 20%, more preferably 5 to 15%, and most preferably 8 to 12%. If the serum concentration is higher than 30%, the strength of the cultured cells may weaken, which is undesirable for the present invention.

[0052] In step (3) of the present invention, the culture substrate is preferably in the shape of a flat plate. When cells are cultured using a culture substrate of this shape, the resulting cultured cell sheet of the present invention can be easily peeled off the culture substrate by step (4) described later, and the structure including the base structure tends to remain after peeling, resulting in a physically strong cell sheet that is also highly flexible.

[0053] Furthermore, to increase cell adhesion to the culture substrate surface, the substrate can be coated with various extracellular matrices (ECMs), serum, or combinations thereof, as needed, regardless of whether or not it has been treated with a temperature-responsive polymer. ECMs such as type I collagen, type IV collagen, laminin, fibronectin, Matrigel, poly-D-lysine, and mixtures thereof may be used, and the present invention is not particularly limited to these conditions.

[0054] In step (3) of the present invention, the mucosal epithelial cells prepared in steps (1) and (2) are cultured on a cell culture substrate coated with a temperature-responsive polymer whose hydration capacity changes in the temperature range of 0 to 80°C, at a temperature range in which the hydration capacity of the temperature-responsive polymer is weak. The temperature in which the hydration capacity of the temperature-responsive polymer is weak is preferably 37°C, which is the temperature at which cells are cultured. The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such polymers include the polymer described in Japanese Patent Application Publication No. 2-211865. Specifically, for example, the temperature-responsive polymer used in the present invention can be obtained by the homopolymerization or copolymerization of the following monomers. Examples of monomers that can be used include (meth)acrylamide compounds, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, or vinyl ether derivatives, and in the case of copolymers, any two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or mixtures of polymers and copolymers may also be used. Furthermore, crosslinking is possible within a range that does not impair the inherent properties of the polymer. In this case, since the substances being cultured and detached are cells, the separation is carried out in the range of 5°C to 50°C, so the temperature-responsive polymers are poly-(n-propylacrylamide) (lower limit critical dissolution temperature of the homopolymer 21°C), poly-(Nn-propylmethacrylamide) (same 27°C), poly-(N-isopropylacrylamide) (same 32°C), poly-(N-isopropylmethacrylamide) (same 43°C), poly-(N-cyclopropylacrylamide) Examples include poly(N-ethoxyethylacrylamide) (at approximately 45°C), poly(N-ethoxyethylmethacrylamide) (at approximately 35°C), poly(N-ethoxyethylmethacrylamide) (at approximately 45°C), poly(N-tetrahydrofurfurylacrylamide) (at approximately 28°C), poly(N-tetrahydrofurfurylmethacrylamide) (at approximately 35°C), poly(N,N-ethylmethylacrylamide) (at approximately 56°C), and poly(N,N-diethylacrylamide) (at approximately 32°C).Examples of monomers used for copolymerization to produce the temperature-responsive polymer used in the present invention include, but are not particularly limited to, polyacrylamide, poly-(N,N-diethylacrylamide), poly-(N,N-dimethylacrylamide), polyethylene oxide, polyacrylic acid and its salts, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose, carboxymethylcellulose, and other hydrated polymers.

[0055] The method for coating the substrate surface with the above-mentioned temperature-responsive polymers used in step (3) of the present invention is not particularly limited, but for example, the substrate and the temperature-responsive polymers can be coated by electron beam irradiation (EB), gamma ray irradiation, ultraviolet irradiation, plasma treatment, powder treatment, organic polymerization reaction, or by physical adsorption such as coating or kneading. The amount of temperature-responsive polymer coating on the culture substrate surface is 1.1 to 2.3 μg / cm². 2 A range of 1.4 to 1.9 μg / cm³ is preferable. 2 And more preferably 1.5 to 1.8 μg / cm³ 2 It is 1.1 μg / cm³. 2 When the coating amount is smaller, the cells on the polymer are difficult to remove even when stimulated, resulting in significantly reduced work efficiency, which is undesirable. Conversely, 2.3 μg / cm³ is undesirable. 2 If the surface area exceeds this level, cells will have difficulty adhering to that area, making it difficult to ensure sufficient cell adhesion. In such cases, if a cell adhesion protein is further coated on top of the temperature-responsive polymer coating layer, the amount of temperature-responsive polymer coating on the substrate surface will be 2.3 μg / cm². 2 The above is also acceptable, and in that case the coating amount of the temperature-responsive polymer is 9.0 μg / cm². 2 The following is good, preferably 8.0 μg / cm³ 2 The following is good: 7.0 μg / cm³ 2 The following is advantageous: The coating amount of the temperature-responsive polymer is 9.0 μg / cm². 2If the temperature-responsive polymer coating layer is excessively high, it becomes difficult for cells to adhere, which is undesirable. The type of cell adhesion protein is not limited, but examples include collagen, laminin, laminin 5, fibronectin, and Matrigel, either individually or in mixtures of two or more. The coating method for these cell adhesion proteins can be conventional, and typically involves applying an aqueous solution of the cell adhesion protein to the substrate surface, then removing the aqueous solution and rinsing. Step (3) of the present invention is a step that uses a technology that utilizes the cell sheet itself, which is made from a cell culture substrate coated with a temperature-responsive polymer. Therefore, it is undesirable for the amount of cell adhesion protein coating on the temperature-responsive polymer layer to be excessively high. The amount of temperature-responsive polymer coating and the amount of cell adhesion protein coating can be measured by conventional methods, such as directly measuring the cell attachment area using FT-IR-ATR, or immobilizing a pre-labeled polymer in a similar manner and estimating the amount from the amount of labeled polymer immobilized on the cell attachment area; any of these methods may be used.

[0056] Step (4): Detachment of the cell sheet from the cell culture substrate. In this invention, a cultured cell sheet made on a cell culture substrate in step (3) can be peeled off and recovered while remaining in sheet form in step (4), thereby obtaining a cultured cell sheet made from cells collected from nasal mucosal tissue. Step (4), when using a cell culture substrate coated with a temperature-responsive polymer, involves changing the temperature of the culture medium to a state where the hydration power of the temperature-responsive polymer is strong, thereby peeling the cultured cell sheet from the cell culture substrate and obtaining the cultured cell sheet while remaining in sheet form; It is characterized by being carried out by [this method].

[0057] In the method of step (4) of the present invention, to detach and recover the cultured cell sheet from the culture substrate coated with a temperature-responsive polymer, the cell sheet can be detached by raising the temperature of the culture substrate to which the cultured cells are attached above the upper critical dissolution temperature or below the lower critical dissolution temperature of the coating polymer on the culture substrate. This can be done in the culture medium or in other isotonic solutions, and the method can be selected according to the purpose. In order to detach and recover the cells more quickly and efficiently, methods such as lightly tapping or shaking the substrate, stirring the culture medium with a pipette, detaching with tweezers, detaching with a cell scraper, detaching by adding 4°C culture medium, detaching after leaving it on ice, detaching after leaving it at 20°C, or detaching using a carrier for cultured cell sheets may be used individually or in combination.

[0058] The above will be explained using poly(N-isopropylacrylamide) as an example of a temperature-responsive polymer. Poly(N-isopropylacrylamide) is known as a polymer with a lower critical dissolution temperature of 31°C. In its free state, it undergoes dehydration in water at temperatures above 31°C, causing the polymer chains to aggregate and the solution to become cloudy. Conversely, at temperatures below 31°C, the polymer chains hydrate and become dissolved in water. In this invention, this polymer is coated and fixed to the surface of a substrate such as a petri dish. Therefore, at temperatures above 31°C, the polymer on the substrate surface also undergoes dehydration, but because the polymer chains are coated and fixed to the substrate surface, the substrate surface becomes hydrophobic. Conversely, at temperatures below 31°C, the polymer on the substrate surface hydrates, but because the polymer chains are coated and fixed to the substrate surface, the substrate surface becomes hydrophilic. The hydrophobic surface at this time is a suitable surface for cells to attach and proliferate, while the hydrophilic surface is a surface that cells cannot attach to, and cultured cells or cell sheets can be detached simply by cooling.

[0059] Furthermore, in step (4) of the present invention, when peeling and recovering a cultured cell sheet made from cells collected from nasal mucosal tissue from the culture substrate, a carrier may be attached to the cultured cells at the end of the culture period as needed, and the cultured cell sheet made from cells collected from nasal mucosal tissue may be recovered together with the carrier. Typically, in step (4), when peeling and recovering a cultured cell sheet from a culture substrate coated with a temperature-responsive polymer, after attaching a carrier to the cultured cells at the end of the culture period, the cultured cell sheet made from cells collected from nasal mucosal tissue can be peeled off together with the carrier by raising the temperature of the coating polymer of the culture substrate above the upper critical dissolution temperature or below the lower critical dissolution temperature.

[0060] The carrier material used as needed may be, for example, hydrophilized polyvinylidene difluoride (PVDF), polypropylene, polyethylene, polylactic acid, cellulose and its derivatives, chitin, chitosan, collagen, paper such as Japanese paper, urethane, spandex, or other net-like or stockinette-like polymer materials.

[0061] In the present invention, cultured cell sheets made from cells collected from two or more nasal mucosal tissues recovered in step (4) can be used by laminating them. The method for laminating cultured cell sheets in the present invention is not particularly limited, but for example, it can be obtained by peeling off the cultured cell sheets while they are still in sheet form and, if necessary, using a jig for moving cultured cells to laminate the peeled cultured cell sheets together. When laminating cultured cell sheets, the culture can be carried out under the normal culture conditions used when creating the cultured cell sheets so that the peeled cultured cell sheets adhere to each other. For example, the culture medium used may be a medium to which serum such as human serum or fetal bovine serum (FBS) has been added, or it may be a serum-free medium to which serum has not been added. Furthermore, if necessary, jigs may be used to move the cell sheets. Such jigs are not limited in material or shape, as long as they can capture the detached cell sheets. For example, materials such as polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, polylactic acid, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, and fibrin glue are commonly used, and the jigs can be in the form of a membrane, porous membrane, nonwoven fabric, or woven fabric, and can be used in contact with the cultured cell sheet.

[0062] The cultured cell sheet of the present invention, manufactured in this manner, can be transplanted to a desired site in the living body. Examples of transplantation sites include mucosal defects in the middle ear cavity, mastoid sac, nasal cavity, and oral cavity. Among these, transplantation to mucosal defects in the middle ear cavity is a particularly promising site for demonstrating the effects of the present invention.

[0063] The condition of the transplant site is not particularly limited, and in order to enhance the engraftment of the cultured cell sheet to the transplant site, vascular induction may be performed in advance, and intercellular adhesion components such as fibrin glue and collagen may be applied. Here, for example, when vascular induction is performed on the transplant site before transplantation, the method of performing vascular induction on the transplant site is also not particularly limited, and examples include a method in which FGF, an angiogenic growth factor, is embedded in microspheres and the composition, size, and injection range of these microspheres are changed while allowing them to act on the body for 8 to 10 days, or a method in which polyethylene terephthalate mesh is cut to an arbitrary size to make a bag-like structure, FGF dissolved in a high-concentration agarose solution is placed inside the bag, and after 8 to 10 days the bag is removed to create a space in which blood vessels have been induced.

[0064] The present invention will be described in more detail below based on examples, but these are not intended to limit the invention in any way. Furthermore, the following examples were basically carried out with the permission of the university's ethics committee. [Examples]

[0065] Example 1: Preparation of raw material cells by serum-containing explant culture method In this example, mucosal tissue was collected from the inferior turbinate mucosa in the nasal cavity of surgical patients using an endoscope, and raw material cells for creating cultured cell sheets were prepared using serum-containing explorer culture.

[0066] The collected tissue was sterilized by immersion in an iodine solution, and then sliced ​​so that the thickness of the stromal tissue layer was roughly equal to the thickness of the epithelial tissue layer (a thickness ratio of approximately 1.0). Subsequently, the tissue was divided into 1-2 mm square sections, and four of these sections were placed on one Primarya (Corning) culture substrate without enzymatic treatment. After culturing for 5 days in KCM medium or FAD medium (both containing DMEM medium:Ham's F12=3:1, with 10% FBS and ROCK inhibitor (10 μM Y-27632 (WAKO))), the culture medium was changed to KCM medium with 10% FBS and ROCK inhibitor. Then, on days 8 and 11, the culture medium was changed again using the aforementioned medium, and an exploratory culture was performed to induce cell migration from the tissue pieces. Culturing was continued until the migrated cells covered approximately 70% of the surface of the cell culture substrate.

[0067] As a result, fibroblast-like cells and undifferentiated cells migrated from the nasal mucosal tissue (Figure 1). The migrated cells were harvested by detaching them from the cell culture substrate after trypsin treatment. The number of cells was 34.3 × 10⁶. 4 It was cells / dish.

[0068] Example 2: Preparation of mucosal epithelial cell sheets by serum culture using feeder cells In this example, using the cells obtained in Example 1 as raw materials, a mucosal epithelial cell sheet was prepared using feeder cells under serum-added culture conditions.

[0069] Mouse-derived 3T3-J2 cells (donated by J-TEC) were used as feeder cells. These feeder cells were subcultured and grown using KCM medium containing the ROCK inhibitor (10 μM Y-27632 (WAKO)). The cultured and grown cells were harvested by trypsin treatment to prepare a cell suspension.

[0070] Next, 1-2 × 10⁶ cells obtained by the Explant culture method of Example 1 4 cells / cm 2The feeder cells were mixed with the nasal mucosal epithelial cells, and these mixtures were seeded onto a cell culture substrate (Corning Primary). The cells were cultured for 4 days in KCM medium supplemented with a ROCK inhibitor (10 μM Y-27632 (WAKO)). The cultured and proliferated nasal mucosal epithelial cells were harvested by trypsin treatment, and the cell density was 26.4 × 10⁶. 4 A cell suspension was prepared by adjusting the concentration to cells / mL.

[0071] To the nasal mucosal epithelial cells prepared as described above, feeder cells were mixed and seeded onto two cell culture substrates (35 mm, manufactured by CellSeed) coated with a temperature-responsive polymer (poly-N-isopropylacrylamide). For comparison, nasal mucosal epithelial cells prepared as described above were seeded onto cell inserts coated with the temperature-responsive polymer used in Patent Document 2. In both cases, the seeding density of nasal mucosal epithelial cells was 3 × 10⁶ for the cell culture substrate coated with the temperature-responsive polymer. 4 cells / cm 2 For cell inserts coated with temperature-responsive polymer, the dimensions are 3 × 10 4 cells / cm 2 In all cases, the culture medium used was the same KCM medium (containing 10% FBS and a ROCK inhibitor (10 μM Y-27632 (WAKO)) as in the primary culture in Example 1). Cells were cultured for 8 days in KCM medium without the ROCK inhibitor, with the culture medium being changed on days 3, 5, and 7, to prepare sheet-like cells on the culture substrate (Figure 2). Subsequently, the culture medium was heated to 20°C, allowing the nasal mucosa cell sheets to be detached from the culture substrate coated with their respective temperature-responsive polymers.

[0072] The strength of the obtained cell sheets was compared. Cell sheets recovered from cell culture substrates coated with temperature-responsive polymer were stronger than those obtained from cell inserts coated with temperature-responsive polymer. Cell sheets recovered from cell culture substrates coated with temperature-responsive polymer could be peeled off while being grasped with tweezers, but those obtained from cell inserts coated with temperature-responsive polymer were so structurally damaged that they could not be grasped with tweezers.

[0073] Furthermore, regarding the removal of cell sheets, using a cell culture substrate coated with a temperature-responsive polymer resulted in significantly easier removal and shorter removal times compared to using cell inserts coated with a temperature-responsive polymer.

[0074] Furthermore, no significant differences were observed in the number of viable cells, cell viability, or pancytic keratin positivity between cell culture substrates coated with temperature-responsive polymers and cell inserts coated with temperature-responsive polymers.

[0075] Next, the cell composition of the obtained cell sheets was examined by immunohistochemical staining. The cells detected were mouse-derived feeder cells, undifferentiated cells expressing p63 (a stem cell / progenitor cell marker for epithelial cells), and fibroblasts. The cell sheets were prepared using nasal mucosal tissue collected from four human subjects (subject IDs: #1, #2, #3, and #4) as raw material, and a cell culture substrate coated with a temperature-responsive polymer. After detaching the cell sheets, they were fixed with paraformaldehyde and then embedded in paraffin to prepare tissue sections by vertically oriented thin sections that allowed the cross-section of the cultured cell sheet to be visible. From each cell sheet, a total of 32 tissue sections were randomly selected, and mouse-derived feeder cells, undifferentiated cells expressing p63 (a stem cell / progenitor cell marker for epithelial cells), and fibroblasts were detected in each cell sheet.

[0076] Mouse-derived feeder cells were detected by immunohistochemical staining using an anti-rabbit PDGFRα antibody (Cell Signaling).

[0077] Undifferentiated cells expressing p63 were detected by reacting tissue sections with an anti-p63 antibody (Abcam) as the primary antibody, followed by color development using ENVISION (DAKO) and DAB (DAKO), and then observation.

[0078] Fibroblasts were detected by reacting an antibody against platelet-derived growth factor receptor α (PDGFRα), a fibroblast marker (Cell Signaling), as the primary antibody, developing the reaction using ENVISION (DAKO) and DAB (DAKO), and then observing the results.

[0079] Mouse-derived feeder cells were not detected in 96 fields of view on tissue sections of the cell sheet.

[0080] Next, p63 expression was confirmed by immunohistochemical staining at three locations (96 locations in total) on each of the 32 tissue sections. Specific observation examples are shown in Figure 3. The numbers in the lower right corner of each 20x field of view image (33 / 48, 27 / 37, 48 / 61) indicate that out of a total of 48, 37, and 61 cells in each field of view, 33, 27, and 48 cells were p63-positive. Measurements taken for all fields of view (96 locations for each subject) showed that the undifferentiated cell content in each field of view ranged from 65% to 82% of the total number of cells in the cell sheet prepared using a cell culture substrate coated with a temperature-responsive polymer (see Table 1).

[0081] [Table 1]

[0082] On the other hand, when the obtained cell sheets were examined by immunohistochemical staining to confirm the proportion of fibroblasts, only 0 to 1 PDGFRα-positive cell (i.e., fibroblast) was confirmed per field of view in cell sheets prepared using a cell culture substrate coated with a temperature-responsive polymer, relative to the total number of cells in each field of view (average number of cells in all fields of view for all subjects = 34.51 cells). The fibroblast proportion ranged from 0 to 2.9%.

[0083] Based on the above findings, we discovered that using a cell culture substrate in the form of a petri dish coated with a temperature-responsive polymer results in nasal mucosal cell sheets produced on this substrate having higher strength and superior structure compared to those obtained using conventional techniques.

[0084] Example 3: Preparation of mucosal epithelial cell sheets by serum culture without the use of feeder cells In this example, using the cells obtained in Example 1 as raw materials, a mucosal epithelial cell sheet was prepared under serum-added culture conditions without using feeder cells.

[0085] Cells obtained by the Explant culture method of Example 1 were seeded onto a cell culture substrate (Corning Primary) and cultured for 4 days using KCM medium supplemented with a ROCK inhibitor (10 μM Y-27632 (WAKO)).

[0086] The cultured and proliferated cells were harvested by trypsin treatment, and the cell density was set to 19 × 10⁶. 4 A cell suspension was prepared at a concentration of cells / ml and seeded onto two cell culture substrates (35 mm, manufactured by CellSeed) coated with a temperature-responsive polymer (poly-N-isopropylacrylamide). For comparison, cells were seeded similarly using cell inserts coated with the temperature-responsive polymer used in Patent Document 2. In both cases, the seeding density was 10 × 10 for the cell culture substrate coated with the temperature-responsive polymer. 4 cells / cm 2 For cell inserts coated with temperature-responsive polymer, the dimensions are 20 × 10 4 cells / cm 2 The culture medium used was the same KCM medium (containing 5% FBS) as in the primary culture in Example 1, with the addition of a ROCK inhibitor (10 μM Y-27632 (WAKO)). The cells were cultured for 8 days in KCM medium without the ROCK inhibitor, with the culture medium being changed on days 3, 5, and 7, to prepare sheet-like cells on the culture substrate. Subsequently, the medium was heated to 20°C, allowing the nasal mucosa cell sheets to be detached from the culture substrate coated with their respective temperature-responsive polymers.

[0087] The strength of the obtained cell sheets was compared. Cell sheets recovered from cell culture substrates coated with temperature-responsive polymer were stronger than those obtained from cell inserts coated with temperature-responsive polymer. Cell sheets recovered from cell culture substrates coated with temperature-responsive polymer could be peeled off while being grasped with tweezers, but those obtained from cell inserts coated with temperature-responsive polymer were so structurally damaged that they could not be grasped with tweezers (Figure 4).

[0088] Furthermore, regarding the removal of cell sheets, using a cell culture substrate coated with a temperature-responsive polymer resulted in significantly easier removal and shorter removal times compared to using cell inserts coated with a temperature-responsive polymer.

[0089] Furthermore, no significant differences were observed in the number of viable cells, cell viability, or pancytic keratin positivity between cell culture substrates coated with temperature-responsive polymers and cell inserts coated with temperature-responsive polymers.

[0090] Next, the cell composition of the obtained cell sheets was examined by immunohistochemical staining. The target cells for detection were undifferentiated cells and fibroblasts expressing p63, a stem cell / progenitor cell marker for epithelial cells. The cell sheets were prepared using nasal mucosal tissue collected from three human subjects (subject IDs: #5, #6, and #7) as raw material, and a cell culture substrate coated with a temperature-responsive polymer. Undifferentiated cells and fibroblasts expressing p63 were detected by the method described in Example 2.

[0091] Immunohistochemical staining revealed that p63 expression was observed in 3 locations (96 locations in total) on each of 32 tissue sections. In cell sheets prepared using a cell culture substrate coated with a temperature-responsive polymer, the undifferentiated cell content in each field of view ranged from approximately 64% to 81% of the total number of cells (see Table 2).

[0092] [Table 2]

[0093] On the other hand, when the obtained cell sheets were examined by immunohistochemical staining to confirm the proportion of fibroblasts, only 0 to 3 PDGFRα-positive cells (i.e., fibroblasts) were identified per field of view in cell sheets prepared using a cell culture substrate coated with a temperature-responsive polymer, relative to the total number of cells in each field of view (average number of cells in all fields of view for all subjects = 70.05 cells). The fibroblast proportion ranged from 0 to 4.3%.

[0094] Based on the above findings, we discovered that using a cell culture substrate in the form of a petri dish coated with a temperature-responsive polymer results in nasal mucosal cell sheets produced on this substrate having higher strength and superior structure compared to those obtained using conventional techniques.

[0095] Example 4: Preparation of raw material cells by Explant culture method using serum-free medium In this example, mucosal tissue was collected from the inferior turbinate mucosa in the nasal cavity of surgical patients using an endoscope, and raw material cells for producing cultured cell sheets were prepared using the Explant culture method with EpiLife Medium (Thermo Fisher) supplemented with Supplement S7 (Thermo Fisher).

[0096] The collected tissue was sterilized by immersion in an iodine solution, and then sliced ​​so that the thickness of the stromal tissue layer was roughly equal to the thickness of the epithelial tissue layer (a thickness ratio of approximately 1.0). Subsequently, the tissue was divided into 1-2 mm square sections, and 2-3 of these sections were placed in one well of a 6-well TrueLine Cell Culture plate without enzymatic treatment. Cell migration was observed in KCM medium or FAD medium (both DMEM medium without ROCK inhibitors: culture medium containing Ham's F12 = 3:1, with 10% FBS added) (Figure 5, day 7 of culture).

[0097] Subsequently, the culture medium was changed to serum-free medium (EpiLife Medium (Thermo Fisher) with Supplement S7 (Thermo Fisher) added), and then explore culture was performed, changing the culture medium with serum-free medium every 1-2 days, and the culture was continued for 11 days until migrating cells covered approximately 70% of the surface of the cell culture substrate.

[0098] As a result, fibroblast-like cells and undifferentiated cells migrated from the nasal mucosal tissue (Figure 5, 11 days into culture). The migrated cells were harvested by detaching them from the cell culture substrate using recombinant cell dissociation enzymes. The number of cells was 2.7 × 10⁶. 5 It was cells / dish.

[0099] Example 5: Without using feeder cells Using EpiLife Medium Preparation of mucosal epithelial cell sheets by culture In this example, a mucosal epithelial cell sheet was prepared using the cells obtained in Example 4 as raw materials, without using feeder cells.

[0100] Cells obtained by the Explant culture method of Example 4: 2000-5000 cells / cm 2 The cells were seeded onto a polystyrene cell culture dish (Greiner Bio-One) and cultured for 6 days until confluence using serum-free medium (EpiLife Medium (Thermo Fisher) supplemented with Supplement S7 (Thermo Fisher)) (Figure 6 left). The cultured and proliferated nasal mucosal epithelial cells were harvested by recombinant cell dissociation enzyme treatment and a cell suspension was prepared.

[0101] The nasal mucosal epithelial cells prepared as described above were seeded onto two cell culture substrates (35 mm, manufactured by CellSeed) coated with a temperature-responsive polymer (poly-N-isopropylacrylamide). The seeding density of the nasal mucosal epithelial cells was 3 × 10⁻⁶. 4 cells / cm 2The culture medium used was serum-free medium (EpiLife Medium (Thermo Fisher) with Supplement S7 (Thermo Fisher) added). The cells were cultured for 7 days until full confluence was reached, with the medium being changed every 1-2 days. After that, to induce differentiation, the medium was changed to KCM medium or FAD medium (both DMEM medium without ROCK inhibitors: culture medium containing Ham's F12 = 3:1, with 10% FBS added), the medium was changed again after 3 days, and on the 4th day the temperature was raised to 20°C to detach the nasal mucosa cell sheets (Figure 6 right) from the culture substrate coated with a temperature-responsive polymer.

[0102] The resulting cell sheet maintained its shape (Figure 7) and could be peeled off while being grasped with tweezers. This indicates that it possesses similar strength to the mucosal epithelial cell sheet obtained in Example 2 using serum culture with feeder cells.

[0103] Next, the cell composition of the obtained cell sheets was examined by immunohistochemical staining. The target cells for detection were undifferentiated cells and fibroblasts expressing p63, a stem cell / progenitor cell marker for epithelial cells. The cell sheets were prepared using nasal mucosal tissue collected from three human subjects (subject IDs: #8, #9, and #10) as raw material, and a cell culture substrate coated with a temperature-responsive polymer. After detaching the cell sheets, they were fixed with paraformaldehyde and then embedded in paraffin to prepare tissue sections by vertically oriented thin sections that allowed the cross-section of the cultured cell sheet to be visible. Undifferentiated cells and fibroblasts expressing p63, a stem cell / progenitor cell marker for epithelial cells, were detected.

[0104] Undifferentiated cells expressing p63 were detected by reacting tissue sections with an anti-p63 antibody (Abcam) as the primary antibody, followed by color development using ENVISION (DAKO) and DAB (DAKO), and then observation.

[0105] Immunohistochemical staining revealed that, regarding p63 expression, the undifferentiated cell content in cell sheets prepared on cell culture substrates coated with a responsive polymer (Figure 8) was in the range of 88-89%.

[0106] [Table 3]

[0107] On the other hand, when the obtained cell sheets were examined for PDGFRα by immunohistochemical staining (Figure 8), the fibroblast ratio was found to be in the range of 0-2.9% in cell sheets prepared using a cell culture substrate coated with a temperature-responsive polymer.

[0108] Based on the above, nasal mucosal cell sheets prepared using serum-free culture medium and without feeder cells have the same characteristics as cell sheets prepared using serum culture and with feeder cells.

[0109] Example 6: Transplantation of cultured cell sheets into rabbits In this embodiment, the objective was to confirm the effect of the bone tissue regeneration cell sheet of the present invention by transplanting the nasal mucosa-like cell sheet prepared according to the present invention into rabbits from which the middle ear mucosa had been removed in advance.

[0110] The work procedure is shown in Figure 9. The cell culture sheet prepared in Example 3 was used as the cell culture sheet for transplantation. After peeling this cell sheet from the cell culture substrate coated with a temperature-responsive polymer, the cell sheet was attached to a blunt-tipped metal needle using forceps and transplanted onto the exposed bone surface in the middle ear cavity of rabbits from which the middle ear mucosa had been removed in advance.

[0111] As a result, in the group where the middle ear mucosa was removed and nothing was transplanted, narrowing of the middle ear cavity progressed and clear bone proliferation was observed. However, in the group where the bone tissue regeneration cell sheet of the present invention was transplanted, narrowing of the middle ear cavity did not progress, and no bone proliferation was observed. This result was also observed by coronal CT or by taking tissue from the middle ear cavity, decalcifying the sections, and staining them. Furthermore, analysis of the decalcified sections of the transplantation site confirmed that the engrafted nasal mucosa-like cell sheet remained.

[0112] To evaluate the function of the middle ear cavity after transplantation of the bone tissue regeneration cell sheet of the present invention, the middle ear cavity of rabbits was closed by plugging the Eustachian tube. As a result, the group transplanted with the mucosal epithelial cell sheet of the present invention showed significantly better results than the group in which the middle ear mucosa was removed, and the values ​​were close to normal. It is thought that the air pressure inside and outside the eardrum was maintained at a state approximately equal to that of normal tissue, and that optimal sound transmission function to the inner ear could be maintained. From the above, it was confirmed that the bone tissue regeneration cell sheet of the present invention can replace the middle ear mucosa.

[0113] Example 7: Transplantation of cultured cell sheets into humans In this embodiment, the objective was to confirm the effect of the bone tissue regeneration cell sheet of the present invention by transplanting the nasal mucosa-like cell sheet produced according to the present invention into human patients after surgery for cholesteatoma or adhesive otitis media.

[0114] After obtaining sufficient informed consent from the target human patients both verbally and in writing, blood samples were first collected. This blood was used to confirm the absence of infectious diseases, and at the same time, autologous serum was prepared from the patient's blood to be added to the culture medium.

[0115] Next, human nasal mucosal tissue measuring approximately 10 × 10 mm was collected endoscopically in an outpatient setting. Following the methods of Example 1 and Example 3, human nasal mucosal epithelial cell sheets were prepared at the cell processing facility (CPF) of Tokyo Jikei University School of Medicine using autologous serum-containing KCM (Keratinocyte Culture Medium) medium, in accordance with the previously prepared manufacturing instructions and manufacturing records.

[0116] Specifically, following the method described in Example 1, the collected nasal mucosal epithelium was cultured for two weeks using the explant culture method. Then, following the method described in Example 3, the proliferated epithelial cells were seeded onto a cell culture substrate coated with a temperature-responsive polymer, and subcultured for 12 days to produce cell sheets. During the culture process, it was confirmed that there was no infection, and tests were performed on cell purity and cell viability. All of the prepared autologous cultured epithelial cell sheets met the established quality standards. The results of various quality tests were checked by the day before transplantation, and cell sheets that met the standard values ​​were transplanted onto the exposed bone surface where the mucosa was missing during tympanoplasty after removal of cholesteatoma (Figure 10).

[0117] To date, tympanoplasty combined with autologous nasal mucosal epithelial cell sheet transplantation has been performed on four patients with cholesteatoma and one patient with adhesive otitis media. During standard tympanoplasty, autologous cultured epithelial cell sheets were transplanted onto the exposed bone surface where the middle ear mucosa was missing, with the aim of promoting postoperative middle ear mucosal regeneration and preventing re-adhesion of the eardrum. It was possible to transplant the cell sheets onto the bone surface within the narrow middle ear cavity using a transplantation device.

[0118] In all cases, the post-transplant course was very good, hearing improved, and postoperative CT showed aeration of the middle ear cavity corresponding to the cell sheet transplantation site. No recurrence of cholesteatoma or re-adhesion of the tympanic membrane was observed (Figure 11). The longest observation period was more than 3 years, and no adverse events from cell sheet transplantation have been observed. [Industrial applicability]

[0119] The cultured cell sheet made from cells collected from nasal mucosal tissue as described in this invention efficiently engrafts onto the surface of the middle ear bone, promoting the regeneration of mucosal tissue covering the bone tissue and suppressing fibrosis, granulation tissue formation, and poor epithelial formation in the middle ear cavity. Furthermore, this cultured cell sheet made from cells collected from nasal mucosal tissue can also be used as a useful mucosa in the case of mucosal loss in the nasal cavity and oral cavity. In addition, the cultured cell sheet made from cells collected from nasal mucosal tissue of this invention is also useful for suppressing inflammation of bone tissue in areas other than the middle ear.

Claims

1. The sample contains cells collected from nasal mucosal tissue, cultured cells thereof, or cell lines thereof, with 60-90% of the total number of p63-positive undifferentiated cells, and exhibiting desmosome structure and basement membrane-like structures. A cultured cell sheet with residual protein and capable of engraftment.

2. The cultured cell sheet according to claim 1, wherein the cultured cell sheet is detached from the cell culture substrate.

3. The cultured cell sheet according to claim 1, wherein the undifferentiated cells are nasal mucosal epithelial stem cells or nasal mucosal epithelial progenitor cells.

4. The cultured cell sheet according to claim 1, wherein the undifferentiated cells do not have cilia.

5. The cell sheet according to claim 1, wherein the cell sheet is a mixture of nasal mucosal epithelial cells and nasal mucosal epithelial stem cells or nasal mucosal epithelial progenitor cells, in addition to one or more other epithelial cells or other epithelial stem cells, mesenchymal stem cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, and adipocytes.

6. The culture according to claim 5, wherein the proportion of fibroblasts contained in the cell sheet is 0 to 12%. Cell sheet.

7. The cultured cell sheet according to claim 1, wherein the nasal mucosal tissue is derived from the inferior turbinate mucosa, the middle turbinate mucosa, the superior turbinate mucosa, the nasal cavity mucosa, or paranasal sinus tissue.

8. The cultured cell sheet according to claim 1, wherein the nasal mucosal tissue is derived from any of the following: human, rabbit, dog, cat, pig, monkey, chimpanzee, rat, or mouse.

9. Chronic otitis media, adhesive otitis media, cholesteatoma, chronic sinusitis, sinus tumors, external auditory canal tumors, external auditory canal atresia, chronic tonsillitis, pharyngeal cancer, laryngeal cancer, oral cancer, tongue cancer, tracheal granulation tissue, tracheal scarring, tracheal stenosis, tracheal injury, esophageal cancer, vocal cord lesions, inflammatory bowel disease, anal ulcers, anal fissures, hemorrhoids A cultured cell sheet according to any one of claims 1 to 8, which is intended for the treatment of anal fistulas.

10. The following steps: (1) A step of preparing mucosal epithelial cells isolated from nasal mucosal tissue using the Explant culture method; (2) A step of culturing the prepared mucosal epithelial cells to increase the number of mucosal epithelial cells; (3) Seed the obtained cells onto a cell culture substrate and culture them, and the cells on the culture substrate surface A step of preparing a cell culture sheet that covers a surface, wherein the cell culture substrate is coated with a temperature-responsive polymer on its surface; (4) The process of peeling off and collecting the cultured cell sheet prepared on the culture substrate while it remains in sheet form. The process involves the removal of the cultured cell sheet by a temperature change; Includes, Use feeder cells during the culture in step (2) and / or step (3), or use serum-free medium during the culture in step (3). A method for producing a cultured cell sheet containing cells derived from nasal mucosal tissue, cultured cells thereof, or cell lines thereof, wherein 60-90% of the total number of cells are p63-positive undifferentiated cells.

11. The cell culture substrate used in step (3) is a dish-type culture substrate, a flat A method for producing a cultured cell sheet according to claim 10, wherein the culture substrate is of the SCO type or the multiwell type.

12. In step (3), Prepared mucosal epithelial cells are treated with a temperature-responsive polymer whose hydration capacity changes in the temperature range of 0 to 80°C. Cells are cultured on a cell culture substrate coated with - at a temperature range where the hydration power of the temperature-responsive polymer is weak, and a cultured cell sheet is prepared on the cell culture substrate. In the above step (4), A method for producing a cultured cell sheet according to claim 10, wherein the culture medium is changed to a temperature at which the hydration force of the temperature-responsive polymer is strong, thereby peeling the cultured cell sheet from the cell culture substrate and obtaining a cultured cell sheet while it remains in sheet form.

13. The method for producing a cultured cell sheet according to claim 10, wherein the cell culture substrate used in step (3) is coated on its surface with a mixture of one or more of type I collagen, laminin, fibronectin, and Matrigel.

14. The temperature-responsive polymer used in steps (3) and (4) is poly-(N-I A method for producing a cultured cell sheet according to claim 10, wherein the material is sopropylacrylamide.

15. The peeling step in step (4) is to adhere the carrier to the cultured cells and the carrier A method for producing a cultured cell sheet according to claim 10, wherein both are peeled off.

16. The method for producing a cultured cell sheet according to claim 10, further comprising the step of stacking the cell sheet detached in step (4) onto another cell sheet detached in step (4), or stacking cell sheets by repeating the operation.

17. The number of mucosal epithelial cells seeded at the start of culture in step (3) is 3 × 10 4 ~2 x 10 5 cells / cm 2 The method for producing a cultured cell sheet according to claim 10.

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