Novel nasal mucosal cell sheet

Abstract

We provide cultured cell sheets. [Solution] A cultured cell sheet is provided, which is made from cells collected from nasal mucosal tissue, and contains 50-90% undifferentiated cells relative to the total number of cells, wherein, in one embodiment, the undifferentiated cells are positive for p63, and the cultured cell sheet is a mixture of nasal mucosal epithelial cells and nasal mucosal epithelial stem cells or nasal mucosal epithelial progenitor cells, as well as one or more of the following cells: other epithelial cells or other epithelial stem cells, mesenchymal stem cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, and adipocytes. [Effects] The cultured cell sheet of the present invention engrafts on areas of mucosal tissue defects such as inflammation and granulation tissue formation on the surface of bone tissue in the middle ear, promoting the regeneration of mucosal tissue, including mucosal tissue covering bone tissue, efficiently suppressing inflammation occurring in the bone tissue of the middle ear, and inhibiting characteristic fibrosis, granulation tissue formation, and poor epithelial formation that occur during bone tissue inflammation.

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 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 results in hearing loss. The middle ear cavity is lined with middle ear mucosa, which also serves as the periosteum, and maintains good air content due to the gas ventilation capacity of the mucosa, 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. While residual recurrence due to incomplete removal of the lesion during surgery can now be prevented with the use of endoscopy, it is difficult to reliably prevent replastic recurrence caused by postoperative re-adhesion or re-indentation. On the other hand, while replastic recurrence can be prevented with posterior auditory canal removal tympanoplasty, the physiological morphology of the external auditory canal is impaired, and there is a possibility of postoperative mastoid cavity problems, also known as postoperative cavity problems. The ideal surgical procedure is to preserve the posterior auditory canal wall using posterior auditory canal preservation tympanoplasty and to create a middle ear cavity with good postoperative aeration, but there is still no definitive surgical method, and there are limitations to simply performing conventional tympanoplasty. Currently, the above treatment outcomes cannot be expected.

[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 conducting research on the regeneration of the middle ear mucosa for some time, and have fabricated an artificial middle ear mucosa in three dimensions (non-patented). Reference 2) Transplantation into a rabbit model of middle ear mucosal damage resulted in good postoperative mucosal regeneration. It has been confirmed that this is possible (Non-Patent Document 3). Subsequently, clinical trials of regenerative medicine for the middle ear mucosa in humans have been conducted. With a view to realization, we have been conducting research on the regeneration of middle ear mucosa using somatic stem cells with cell culture substrates coated with temperature-responsive polymers based on cell sheet engineering (Patent Document 1, Non-Patent Document 4) (Non-Patent Document 5). Regenerative medicine using cell sheet engineering has already been used for the cornea, esophagus, etc. This method has been successfully applied clinically, yielding favorable therapeutic effects. We focused on nasal mucosa as a cell source for cell sheet fabrication, as it can be harvested minimally invasively 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 autologous cultured epithelial cell sheets using nasal mucosa coated with a temperature-responsive polymer culture substrate, with the aim of promoting postoperative middle ear mucosal regeneration and preventing re-adhesion of the tympanic membrane, and then transplant these sheets into the postoperative middle ear cavity. We have achieved results in preclinical animal experiments using rabbits with cell culture inserts coated with temperature-responsive polymers (Non-Patent Literature 6), and have also successfully fabricated human nasal mucosal epithelial cell sheets. It has been successful (Patent Document 2).

[0008] It is known that when the mucosal tissue on the surface of the middle ear bone is damaged, problems such as bone tissue inflammation and granulation tissue formation occur. Once a problem occurs, treatment by means such as removal of granulation tissue and suppression of inflammation on the surface of bone tissue is effective, but there are problems such as recurrence. Even when using the conventional human nasal mucosal 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, the present application provides the following embodiments to solve the aforementioned problems:[1]: a device prepared using cells taken from nasal mucosal tissue as raw material, with respect to the total number of cells undivided Cell culture sheets containing 50-90% cultured 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 contains nasal mucosal epithelial cells and nasal mucosal epithelial stem cells or pre-nasal mucosal epithelial cells. A cultured cell sheet as described in [1] or [2], comprising a mixture of progenitor cells and one or more other cells from among epithelial cells or other epithelial stem cells, mesenchymal stem cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, and adipocytes; [6]: The culture medium described in [5] contains 0-12% fibroblasts in the cell sheet. Cell nourishment sheets; [7]: The nasal mucosal tissue from which the raw material cells are derived includes the inferior turbinate mucosa, the middle turbinate mucosa, the superior turbinate mucosa, and the nose. A cultured cell sheet according to [1] or [2], derived from the mucous membrane or paranasal sinus tissue; [8]: Nasal mucosal tissue from humans, rabbits, dogs, cats, pigs, monkeys, chimpanzees, rats, A cultured cell sheet, as described in [1] or [2], derived from either mouse; [9]: Chronic otitis media, adhesive otitis media, cholesteatoma, chronic sinusitis, sinus tumor, outer ear Cell culture sheets as described in [1] or [2], intended for the treatment of tract 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) The prepared mucosal epithelial cells are cultured to increase the number of raw material mucosal epithelial cells. The process of filling; (3) The obtained cells are seeded onto a cell culture substrate and cultured, and the cells on the culture substrate surface A process for preparing a cell culture sheet that covers a surface; (4) The cultured cell sheet prepared on the culture substrate is peeled off and collected while still in sheet form. Length; 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

[0010] 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) By changing the temperature of the culture medium to a temperature at which the hydration capacity of the temperature-responsive polymer is strong, A process of detaching the cultured cell sheet from the cell culture substrate and obtaining the cultured cell sheet while it remains in sheet form; 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] : The temperature-responsive polymer used in steps (3) and (4) is poly(N- A method for producing a cultured cell sheet according to

[10] or

[13] , wherein the cell sheet is isopropylacrylamide;

[16] : The detachment step in step (4) causes the carrier to adhere to the cultured cells, A method for producing a cultured cell sheet according to

[10] or

[13] , which involves peeling off together with;

[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] : Use serum-free medium for the culture in step (3), as described in

[10] or

[13] . A method for manufacturing cell sheets. [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 account for 50-90% of the total number of cells. As a result of studies by the inventors, it has become clear 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 believed that fully differentiated nasal mucosal epithelial cells, i.e., not undifferentiated, with abundant cilia, were more useful, and therefore, "the number of cells with cilia is less than 5%." Furthermore, the transplanted cell sheet is undesirable because it cannot fully exhibit the effects of the present invention, and the focus was on cells with cilia (Patent Document 2), but subsequent studies revealed that undifferentiated cells (with cilia) are also undesirable. We discovered that cells without certain characteristics (i.e., cells without these characteristics) exhibit better results as cell sheets, and that when undifferentiated cells make up 50-90% of the total number of cells in the cell sheet, the engraftment rate of the cell sheet at the transplant site increases, enabling the regeneration of epithelial tissue on the surface of the middle ear bone. This led 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 include epithelial cells, ciliated cells, and goblet cells. This means that the cell maintains the pluripotency to differentiate into cells and other structures.

[0020] In this invention, undifferentiated cells are positive for p63 as a marker characteristic. It is characterized by the fact that p63 is a transcription factor essential for epithelial morphogenesis and stem cell properties. In this invention, p63 is a marker that indicates cells with high stem cell potential (i.e., undifferentiated cells) among cells collected from nasal mucosal tissue. 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, tissue sections are prepared from the cultured cell sheet and immunosuppression is performed. A method for detecting p63-positive cells by epidemic staining, and a method for enzymatically separating cells from a cultured cell sheet into fine particles. After separating cells, cells that bind to antibodies against p63 are detected as p63-positive cells using flow cytometry. Alternatively, cells from a cultured cell sheet are enzymatically separated into individual cells, and then p63 protein or p63 is detected using methods such as Western blotting or real-time PCR. It can be detected by methods such as gene detection. How many p63s are present in multiple cells? To confirm the presence of positive cells (undifferentiated cells), it is preferable to use either the immunostaining method or the flow cytometry method from the methods described above.

[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 fact that the cells contained in the cultured cell sheet are epithelial stem cells is stated in p63 above. To detect the expression of, to detect the expression of cytokeratin 5, to detect the expression of cytokeratin 14 It can be defined as detecting the present. 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 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 examples 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, in cultured cell sheets, fibroblasts reinforce the structure of the cell sheet. It has the effect of increasing the strength of the cell sheet when included in a concentration of 0.1% to 1%.

[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 final nasal mucosal tissue, preferably less than 4%, more preferably less than 3%, and most preferably less than 1%. Cultured cell sheets with a percentage of % or more are undesirable because they have poor engraftment onto the surface of the middle ear bone when transplanted to the affected area, and therefore cannot fully exhibit the effects of the present invention.

[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 cultured cell sheets in this invention may include 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, for example. Examples of suitable animals 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 mucosal 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 responses, 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. A cell culture sheet can be prepared using the plate, and if the cell culture sheet is to be used for a transplant site smaller than the size of the cell sheet, the cell culture sheet can be cut as needed.

[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 a mixture of one or more cells from among vascular endothelial cells, vascular endothelial progenitor cells, adipocytes, and mesenchymal stem cells may be laminated onto a cultured cell sheet. The number of laminated sheets is not particularly limited, but the number of laminates should be 10 or less, preferably 5 or less, and more preferably 2 or less. Laminating cultured cell sheets made from cells collected from nasal mucosal tissue can exhibit the characteristic of improving 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, a mixture of one or more cells from among vascular endothelial cells, vascular endothelial progenitor cells, adipocytes, and mesenchymal stem cells may be used in addition to mucosal cells, and these cell sheets may be laminated onto a 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, 0 This invention provides a method for producing a nasal mucosal tissue-derived cultured cell sheet, characterized by culturing cells collected from nasal mucosal tissue on a cell culture substrate coated with a temperature-responsive polymer whose hydration power changes in a temperature range of ~80°C, at a temperature range where the polymer's hydration power is weak, and then changing the temperature of the culture medium to a state where the polymer's hydration power is strong, thereby allowing the cultured cell sheet to be peeled off while remaining in sheet form.

[0031] In the present invention, a cultured cell sheet made using a culture substrate coated with a temperature-responsive polymer, and made from cells collected from nasal mucosal tissue, is characterized in that it is not damaged by proteolytic enzymes such as dispase and trypsin when the cultured cell sheet is peeled off. Therefore, the cultured cell sheet peeled off 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 peeled off. As a result, the cultured cells When transplanting the sheet, the cultured cell sheet and the tissue at the transplant site must adhere well. This will enable more efficient transplantation. Generally, with regard to dispase, a proteolytic enzyme, 10-40% of the desmosome structure between cells is retained. It is known that it can be detached by basement membrane-like proteins between cells and substrates, etc. Because the desmosome structure is almost completely destroyed, the resulting cell sheet is of low strength. In contrast, the cultured cell sheet produced by the method of the present invention using cells collected from nasal mucosal tissue 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, (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, and (3) a step of culturing on a cell culture substrate so that the cells cover the surface of the cell culture substrate. (4) The process of preparing a cultured cell sheet, A method for producing a cultured cell sheet using cells collected from nasal mucosal tissue as raw material is provided, characterized by including a step of peeling and recovering the sheet 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 explant 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 procedure depending on the cells being used; there are no particular limitations. For example, blood When using culture media containing phosphate, the base media can be DMEM medium, F-12 medium, or KCM medium. Examples include soil, or mixtures thereof. In this case, additives commonly used in cell culture, such as serum, may be added to the culture medium. Alternatively, if a serum-free medium is used, various commercially available serum-free media such as UltraCULTURE serum-free medium (Lonza), PC-1 serum-free medium (Lonza), EpiCult Medium (VERITAS), and EpiLife Medium (ThermoFisher) are available. Serum-free culture medium for cells may be used. 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 0.5-35%. Preferably used at a concentration of 1-20%, more preferably 5-15%, and most preferably 8-12%. .

[0038] In this process, ROCK (Rho-binding kinase) inhibitor is added to the culture medium with the aim of preventing apoptosis (programmed cell death) of nasal mucosal epithelial cells migrating from the nasal mucosal tissue. It can be added. Examples of ROCK inhibitors that can be used in the present invention include, but are not limited to, Y-27632, lipasudil 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] The culture substrate that can be used in step (1) of the present invention is a dish Examples include cell 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, in addition to the compounds such as glass, modified glass, polystyrene, and polymethyl methacrylate commonly used in cell culture, any substance that can be shaped, such as polymer compounds other than those mentioned above and ceramics, can be used.

[0043] Step (2): Cell proliferation The number of epithelial cells obtained in step (1) may be insufficient for subsequent culture. Therefore, if necessary, in step (2), the mucosal epithelial cells prepared in step (1) can be cultured to increase the number of mucosal epithelial cells to be used as raw material. The number of passages can be determined based on the relationship with the number of cells, but by increasing the number of passages... Generally, since the properties of cells change, it is preferable to culture them with as few passages as possible. Therefore, in step (1), if a sufficient amount of cells for the sheet culture of cells in step (3) described below is obtained, 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. In addition, for the purpose of preventing cell death (apoptosis) of nasal mucosal epithelial cells during the culture period, a ROCK (Rho-associated coiled-coil forming protein kinase) inhibitor can be added to the culture medium. This can be done.

[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. At this time, the number of cells to be seeded is generally 3.0×10 4 ~2.0×10 5 cells / cm 2 and preferably is 4.0×10 4 ~1.5×10 5 cells / cm 2 , more preferably 5.0×10 4 ~1.0×10 5 cells / cm 2 and preferably. On the other hand, when the seeding concentration is less than 3.0×10 4 cells / cm 2 , the growth of mucosal cells is poor, the degree of expression of the function of the cultured cell sheet derived from nasal mucosal tissue obtained is deteriorated, which is not preferable in implementing the present invention. Conversely, when the number of cells to be seeded is 2.0×105 cells / cm 2 Yo 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 with a temperature-responsive polymer can be used as the cell culture substrate. When a cell culture substrate coated with a temperature-responsive polymer is used as the cell culture substrate, step (3) is performed on the prepared mucosa. Epithelial cells are coated on the surface with a temperature-responsive polymer whose hydration capacity changes in the temperature range of 0 to 80°C. This can be done by culturing cells on a cell culture substrate at a temperature range where the hydration power of a temperature-responsive polymer is weak, and then preparing a cultured cell sheet on the cell culture substrate.

[0048] The culture medium used for cell culture in the present 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, examples include KCM medium, DMEM medium, F-12 medium, or mixtures thereof. In this case, additives commonly used in cell culture, such as serum, may be added to the culture medium. Alternatively, commercially available serum-free media such as UltraCULTURE serum-free medium (Lonza) or PC-1 may be used. You may use various commercially available serum-free culture media for epithelial cells, such as serum-free medium (Lonza), EpiCult Medium (VERITAS), and EpiLife Medium (ThermoFisher). If using a medium containing serum, you may use fetal bovine serum (FBS), human allogeneic serum, or human autologous serum. From the standpoint of cell proliferation and in vivo immune response after transplantation of cultured cell sheets, it is preferable to use human autologous serum.

[0049] When preparing 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 preparing a cultured cell sheet using feeder cells, feeder cells can include mouse-derived fibroblasts treated with gamma irradiation or antibiotics, mouse fetal-derived fibroblasts, autologous cells, and more specifically, 3T3-J2 cells. Feeder cells may potentially trigger an immune response in the body after transplantation, but the impact is minimal 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 material cells onto a cell culture substrate without using feeder cells, culturing them in serum-free medium until fully confluent, 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 the shape described above, the resulting cultured cell sheet of the present invention can be easily peeled off from the culture substrate by step (4) described later, and the base structure after peeling can be easily removed. Because structures including the cell structure are more likely to remain intact, the resulting cell sheet is physically strong and also highly flexible.

[0053] Furthermore, in order to increase cell adhesion to the culture substrate surface, various extracellular matrices (ECM), serum, or other materials may be used as needed, regardless of whether or not the substrate is treated with a temperature-responsive polymer. These can be used to coat the skin. Examples of ECMs include type I collagen, type IV collagen, laminin, fibronectin, Matrigel, poly-D-lysine, and others. A mixture of these may be used, and the present invention is not particularly limited by these conditions.

[0054] In step (3) of the present invention, the mucosal surface prepared in steps (1) and (2) The skin cells were coated on the surface with a temperature-responsive polymer whose hydration capacity changes in the temperature range of 0 to 80°C. The cells are cultured on a cell culture substrate at a temperature range where the hydration power of the temperature-responsive polymer is weak. This temperature range is typically 37°C, which is the temperature at which cells are cultured. The temperature-responsive polymer used in this invention may be either a homopolymer or a copolymer. Examples of such polymers include those described in Japanese Patent Publication No. 2-211865. Specifically, for example, the temperature-responsive polymer used in this invention is obtained by the homopolymerization or copolymerization of the following monomers. Examples of usable monomers include (meth)acrylamide compounds, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, or vinyl ether derivatives. In the case of copolymers, any two or more of these can be used. Furthermore, monomers other than those listed above may also be used. Copolymerization with polymers, grafting or copolymerization of polymers, or mixtures of polymers and copolymers may be used. Crosslinking is also 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, separation should be performed at 5°C to 50°C. Because this is carried out within a certain range, the temperature-responsive polymer is poly-(n-propyl acrylamine). (Hypopolymer lower critical dissolution temperature 21°C), poly-(Nn-propyl methacrylamide) (27°C), poly-(N-isopropylacrylamide) (32°C), poly-(N-isopropylmethacrylamide) (43°C), poly-(N-cyclopropylacrylamide) (45°C) ), poly-(N-ethoxyethylacrylamide) (at approximately 35°C), poly-(N-ethoxyethyl methacrylamide) (at approximately 45°C), poly-(N-tetrahydrofurfurylacrylamide) (approximately 28°C), poly-(N-tetrahydrofurfuryl methacrylamide) (approximately 35°C), Examples include poly(N,N-ethylmethylacrylamide) (at 56°C) and poly(N,N-diethylacrylamide) (at 32°C). Monomers for copolymerization used to produce the temperature-responsive polymers 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] A method for coating the substrate surface with each of the temperature-responsive polymers described above, used in step (3) of the present invention. The method is not particularly limited, but for example, the substrate and the temperature-responsive polymer 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 surface of the culture substrate 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 coverage is less Furthermore, even when stimulated, the cells on the polymer are difficult to detach, resulting in significantly reduced work efficiency, which is undesirable. Conversely, 2.3 μg / cm³ 2 If the conditions are as described above, cells will have difficulty attaching to that area and will not be able to attach to the cells sufficiently. It becomes difficult to apply. 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 becomes 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 are good, Preferably 8.0 μg / cm³ 2 The following is good: 7.0 μg / cm³2 The following is advantageous: The coating amount of the temperature-responsive polymer is 9.0 μg / cm². 2 If the above conditions are met, further cell adhesion will occur on the temperature-responsive polymer coating layer. Coating with a cell adhesion protein is undesirable because it makes it difficult for cells to adhere. The types of such cell adhesion proteins are not limited in any way, but examples include collagen, laminin, laminin 5, fibronectin, and Matrigel, either individually or in mixtures of two or more. Furthermore, the method of coating with these cell adhesion proteins can be done according to conventional methods, and usually an aqueous solution of the cell adhesion protein is applied to the surface of the substrate, and then the aqueous solution is removed and the substrate is rinsed. Step (3) of the present invention utilizes a cell culture substrate coated with a temperature-responsive polymer. This process utilizes a technology that aims to use the cell sheet itself. Therefore, it is undesirable for the amount of cell adhesion protein coating on the temperature-responsive polymer layer to be excessively large. The amount of coating of the temperature-responsive polymer and the amount of coating of cell adhesion protein can be measured by conventional methods, for example, by directly measuring the cell attachment area using FT-IR-ATR, or by pre-labeling the cells. Methods include immobilizing the polymer in a similar manner and estimating the amount from the amount of labeled polymer immobilized on the cell attachment site, but any of these methods may be used.

[0056] Step (4): Detachment of the cell sheet from the cell culture substrate. In the present invention, the cultured cell sheet prepared on the cell culture substrate by step (3) is used The sample is collected from the nasal mucosal tissue through a process (step (4)) in which it is peeled off and collected while remaining in a sheet-like state. A cultured cell sheet can be obtained using cells as raw material. This step (4) is cell culture When using a cell culture substrate coated with a temperature-responsive polymer as the base material, the process involves changing the temperature of the culture medium to a state where the hydration power of the temperature-responsive polymer is strong, thereby detaching the cultured cell sheet from the cell culture substrate and obtaining the cultured cell sheet while it remains in sheet form; It is characterized by being carried out by [this method].

[0057] In step (4) of the present invention, the cultured cell sheet is coated with a temperature-responsive polymer. To detach and recover the cultured cells from the culture substrate, the temperature of the culture substrate to which the cultured cells are attached must be adjusted. The coating polymer on the material can be removed by raising its upper critical dissolution temperature or lower critical dissolution temperature. This can be done in a culture medium or in other isotonic solutions, and the method can be selected according to the purpose. To remove and collect cells more quickly and efficiently, methods such as lightly tapping or shaking the substrate, stirring the culture medium with a pipette, removing with tweezers, removing with a cell scraper, removing by adding 4°C culture medium, or leaving it on ice before removal can be used. Methods such as peeling, leaving the sheet at 20°C before peeling, or using a carrier for cultured cell sheets for peeling 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, cells collected from nasal mucosal tissue are used as raw materials. When peeling and recovering the cultured cell sheet from the culture substrate, if necessary, a carrier may be attached to the cultured cells at the end of the culture, and the cultured cell sheet, which is made from cells collected from nasal mucosal tissue, may be recovered together with the carrier. Typically, in step (4), the cultured cell sheet When peeling and recovering a cell carrier from a culture substrate coated with a temperature-responsive polymer, after the carrier is firmly attached to the cultured cells at the end of the culture, the cultured cell sheet, which is made from cells collected from nasal mucosal tissue, can be peeled off together with the carrier by raising the temperature above the upper critical dissolution temperature or below the lower critical dissolution temperature of the polymer coating the culture substrate.

[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 stacking them. The method for stacking cultured cell sheets in the present invention is not particularly limited, but for example, it can be obtained by peeling the cultured cell sheets while they are still in sheet form and stacking the peeled cultured cell sheets together using a jig for transferring cultured cells as needed. When stacking cultured cell sheets, the culture can be carried out under the normal culture conditions used when preparing 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 a medium without added serum may be used. Serum culture medium may also be used. If necessary, a jig may be used to move the cell sheet, but such a jig is not limited in material or shape as long as it can capture the detached cell sheet. For example, the material may be polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, polylactic acid, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibrin glue, etc., and the shape may be membrane-like, porous membrane-like, nonwoven fabric-like, or woven fabric-like. It can be used in contact with a cell culture 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 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, if 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 for example, vascular growth factor FGF may be encapsulated in microspheres. The microspheres are implanted and their composition, size, and injection range are varied, allowing them to act on the body for 8-10 days. One method involves cutting polyethylene terephthalate mesh to a desired size to create a bag-like structure, placing FGF dissolved in a high-concentration agarose solution inside the bag, and then removing the bag after 8-10 days to create a space into which blood vessels have been drawn.

[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 interstitial tissue layer was roughly equal to the thickness of the epithelial tissue layer (a thickness ratio of approximately 1.0). The tissue was sectioned into 1-2 mm square sections and, without enzymatic treatment, was subjected to primary (Corning) The four sections were placed on one culture substrate. KCM medium or FAD medium (both DMEM medium) was used. Culture medium containing Ham's F12 in a 3:1 ratio, 10% FBS, and ROCK inhibitor (10 μM Y-27632 (WAKO)) are added. After culturing for 5 days under the conditions (additional substances), the culture is transferred to KCM medium supplemented with 10% FBS and a ROCK inhibitor. The solution is changed, and then on the 8th and 11th days, the culture medium is changed again using the aforementioned medium, and the tissue is collected. Exploration culture was performed to induce cell migration within the sample, and the culture 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 × 10⁻⁶. 4 It was cells / dish.

[0068] Example 2: Preparation of mucosal epithelial cell sheets by serum culture using feeder cells In this embodiment, feeder cells are prepared using the cells obtained in Example 1 as raw materials. Using this method, mucosal epithelial cell sheets were prepared under serum-added culture conditions.

[0069] As feeder cells, mouse-derived 3T3-J2 cells (donated by J-TEC) were used, and ROCK inhibition was performed. Feeder cells were subcultured and grown using KCM medium containing the agent (10 μM Y-27632 (WAKO)). The cultured and grown cells were harvested by trypsin treatment and a cell suspension was prepared.

[0070] Next, 1-2 × 10⁶ cells obtained by the Explant culture method of Example 1 4 cells / cm 2 The feeder cells are mixed with the mixture, and these mixtures are seeded onto a cell culture substrate (Corning primary). The cells were then cultured for 4 days using 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 × 10⁴. 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 used. Seeds were sown in cell inserts coated with the temperature-responsive polymer used in Patent Document 2. In this case as well, the seeding density of nasal mucosal epithelial cells is 3 × 10 in the case of cell culture substrate coated with a 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). The culture was then continued for 8 days in KCM medium without the ROCK inhibitor, with the culture medium being changed on days 3, 5, and 7. The cells were cultured and prepared as a sheet on the culture substrate (Figure 2). Then, the culture medium was brought to 20°C. The nasal mucosal cell sheets were then detached from the culture substrates 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 targets for detection were mouse-derived feeder cells and stem cell / progenitor cell markers of epithelial cells. These are undifferentiated cells, fibroblasts, that express p63. 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, and tissue sections were prepared by vertically oriented thin sections so that the cross-section of the cultured cell sheet was visible. From one cell sheet, locations were randomly selected. For a total of 32 tissue sections per limb / cell sheet, we detected mouse-derived feeder cells, undifferentiated cells expressing p63 (a stem cell / progenitor cell marker for epithelial cells), and fibroblasts.

[0076] Mouse-derived feeder cells are detected using an anti-rabbit PDGFRα antibody (Cell Signaling). The staining was performed using immunohistochemical staining.

[0077] To detect undifferentiated cells expressing p63, an anti-p63 antibody (ab) is used as the primary antibody on tissue sections. The reaction is initiated using a cam, and then color is produced using ENVISION (DAKO) and DAB (DAKO). I did it by inferring.

[0078] Fibroblast detection is performed using platelet-derived growth factor receptor α (PDGFR), a fibroblast marker. This was performed by reacting an antibody against α) (Cell Signaling) as the primary antibody, developing the color 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 proportion of fibroblasts was confirmed by immunohistochemical staining of the obtained cell sheets, only 0 to 1 PDGFRα-positive cell (i.e., fibroblast) was confirmed per field of view 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 cell ratio 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 embodiment, feeder cells are prepared using the cells obtained in Example 1 as raw materials. Without using serum, mucosal epithelial cell sheets were prepared under serum-added culture conditions.

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

[0086] The cultured and proliferated cells were harvested by trypsin treatment, and the cell density was set to 19 × 10⁶. 4 cells / ml Prepare a modified cell suspension and then combine it with a temperature-responsive polymer (poly-N-isopropyl acrylate). Seeds were seeded onto two cell culture substrates (35 mm, manufactured by CellSeed) coated with Luamide. For comparison, cell inserts coated with the temperature-responsive polymer used in Patent Document 2 were used. Cells were seeded in the same manner. In both cases, the seeding density was 10 × 10 for cell culture substrates coated with a degree-responsive polymer. 4 cells / cm 2 , cell inserts coated with temperature-responsive polymer In the case of 20 x 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 culture medium was changed on days 3, 5, and 7 in KCM medium without the ROCK inhibitor, and the culture was carried out as is. The cells were cultured for 8 days to prepare a sheet-like structure on the culture substrate. Then, the culture medium was brought to 20°C. The nasal mucosal cell sheets were then detached from the culture substrates 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 collected from three human subjects (subject IDs: #5, #6, and Cell culture substrate made from nasal mucosal tissue collected from #7) and coated with a temperature-responsive polymer. The cells were prepared using the method described in Example 2. 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 proportion of fibroblasts was confirmed by immunohistochemical staining of the obtained cell sheets, only 0 to 3 PDGFRα-positive cells (i.e., fibroblasts) were 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 = 70.05 cells). The cell ratio 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 the Explant culture method using serum-free medium In this embodiment, mucosal tissue was collected from the inferior turbinate mucosa in the nasal cavity of a surgical patient using an endoscope, and cultured in EpiLife Medium (Thermo Fisher) with Supplement S7 (Thermo Fisher). Raw material cells for producing cultured cell sheets were prepared using the Explant culture method with soil.

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

[0097] Subsequently, the culture medium is replaced with serum-free medium (EpiLife Medium (Thermo Fisher) with Supplement S7 (Thermo Fisher) added), and then the culture medium is replaced with serum-free medium every 1-2 days. While doing so, we performed an Explant culture and continued culturing for 11 days until the migrated 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 treated with recombinant cell dissociation enzymes and then placed in a cell culture medium. The cells were recovered by detaching them from the material. The cell count was 2.7 × 10⁶. 5 It was cells / dish.

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

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

[0101] The nasal mucosal epithelial cells prepared as described above are subjected to a temperature-responsive polymer (poly-N-isopropyl alcohol). The cells were seeded on two cell culture substrates (35 mm, CellSeed) coated with crylamide. The seeding density of nasal mucosal epithelial cells was 3 × 10⁻⁶. 4 cells / cm 2 The culture medium used was serum-free medium (EpiLife Medium (Thermo Fisher) with Supplement S7 (Thermo Fisher) added). The culture was kept in this state for 7 days, changing the medium every 1-2 days until full confluence was reached. To induce differentiation, the culture medium was changed to KCM medium or FAD medium (both containing DMEM medium without ROCK inhibitors: Ham's F12 = 3:1, with 10% FBS added), the medium was changed again after 3 days, and on the 4th day... By raising the temperature to 20°C, the nasal mucosal cell sheet (Figure 6, right) is coated with a temperature-responsive polymer and cultured. It detached from the substrate.

[0102] The resulting cell sheet maintains its shape (Figure 7), and can be grasped with tweezers. We were able to detach them. Therefore, we used the feeder cells obtained in Example 2. It was shown that the cells possessed similar strength to mucosal epithelial cell sheets cultured in serum.

[0103] Next, the cell composition of the obtained cell sheets was examined by immunohistochemical staining. The target cells for detection were undifferentiated cells expressing p63, a stem cell / progenitor cell marker for epithelial cells. The cells were fibroblasts. Cell sheets were prepared using nasal mucosal tissue collected from three human subjects (subject IDs: #8, #9, #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. Tissue sections were then prepared by vertically oriented thin sections so that the cross-section of the cultured cell sheet was visible. Undifferentiated cells and fibroblasts expressing p63, a stem cell / progenitor cell marker for epithelial cells, were detected.

[0104] To detect undifferentiated cells expressing p63, tissue sections are reacted with an anti-p63 antibody (Abcam) as the primary antibody, and the resulting tissue is color-developed using ENVISION (DAKO) and DAB (DAKO). I did it by inferring.

[0105] Immunohistochemical staining revealed that p63 expression was observed in cell culture substrates coated with a responsive polymer. The cell sheets prepared using this method (Figure 8) showed an undifferentiated cell content in the range of 88-89%.

[0106] [Table 3]

[0107] On the other hand, immunohistochemical staining for PDGFRα was performed on the obtained cell sheet, revealing a line When the proportion of fibroblasts was examined (Figure 8), the cell culture substrate coated with a temperature-responsive polymer was found to be... The cell sheets prepared using this method had a fibroblast ratio ranging from 0% to 2.9%.

[0108] Based on the above, nasal mucosal cell sheets prepared using serum-free culture media 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 of the middle ear cavity of rabbits from which the middle ear mucosa had been removed beforehand. .

[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, a human nasal mucosal tissue sample of approximately 10 × 10 mm was collected endoscopically in an outpatient setting, and the tissue was cultured in KCM (Keratinocyte Culture Medium) containing the patient's own serum according to the methods of Example 1 and Example 3. Using this method, and in accordance with the pre-prepared manufacturing instructions and manufacturing records, human nasal mucosal epithelial cell sheets were prepared at the cell processing facility (CPF) of Tokyo Jikei University School of Medicine. The manufacturing process was carried out.

[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 were observed. It hasn't been done. [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

[Claim 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.

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