JP4874514B2 - Method for stratifying culture of epithelial cells, stratified epithelial cell sheet obtained therefrom, and method of use thereof - Google Patents

Description

  The present invention relates to a stratified culture method for epithelial cells in fields such as biology and medicine, a stratified epithelial cell sheet obtained therefrom, and a method for using the same.

  Due to the remarkable development of medical technology, in recent years, organ transplants that attempt to replace difficult-to-treat organs with other organs have become common. The target organs are very diverse, such as skin, cornea, kidney, liver, heart, etc., and the progress after surgery has improved remarkably, and has already been established as a medical technology. Taking corneal transplantation as an example, an eye bank was established in Japan about 40 years ago and transplantation activities began. However, the number of donors is still small, and there are about 20,000 patients who need corneal transplantation in Japan alone, whereas only about 1/10 patients who can actually undergo transplantation treatment are about 2000. It is said. Despite the almost established technology of corneal transplantation, the current situation is that the next medical technology is required due to the shortage of donors.

  Against this background, techniques for transplanting artificial substitutes or cells that have been cultured and organized have attracted attention. Representative examples thereof include artificial skin and cultured skin. However, artificial skin using a synthetic polymer may cause rejection and the like, and is not preferable as skin for transplantation. On the other hand, since the cultured skin is obtained by culturing a part of the normal skin of the person to a desired size, even if it is used, there is no concern about rejection or the like, and it can be said to be the most natural masking agent.

  Japanese Patent Publication No. 2-23191 discloses culturing epidermis keratinocytes derived from human newborns under conditions in which a keratinous tissue film is formed on the surface of a container, and peeling off the produced keratinous tissue membrane using an enzyme. A method for producing transplantable cultured cell membranes is described. Specifically, by using 3T3 cells as a feeder layer, the seeded epidermal cells proliferate and stratify as they are. This method has now become the mainstream method for culturing epidermal keratinocytes. However, it is often pointed out that this method has drawbacks, that is, the 3T3 cells are mouse-derived cells. In general, it is said that the 3T3 cells disappear while culturing epidermal keratinocytes, but it is still impossible to prove that they have disappeared 100%.

  In order to solve this problem, various studies have been made so far. For example, 3T3 cells are cultured on another culture substrate, a substance effective for epidermal keratinocytes is put into the medium, and only the supernatant is transferred to a system in which epidermal keratinocytes are cultured. (JP-A-9-313172, JP-A-2001-149070). However, even in this method, even though the contamination of the cells of the heterologous animal itself is prevented, the various proteins produced by the heterologous animal cells are not divided, and basically the same problem remains. In addition, attempts have been made to use human-derived cells as a feeder layer. However, cells having the same activity as 3T3 cells have not been obtained yet, and an effective technique to replace 3T3 cells is strongly desired. It was rare.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel stratified culture method of epithelial cells from a completely different idea from the prior art and a stratified epithelial cell sheet obtained therefrom. Moreover, an object of this invention is to provide the utilization method.

  In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, surprisingly, epithelial cells are cultured on the porous membrane, and both the upper layer and the lower layer are filled with the medium with the porous membrane as a boundary. It was found that epithelial cells are stratified if the medium is supplied from the lower layer. Further, it has been found that even when an extracellular matrix layer is provided on the culture bed of the lower layer of the porous membrane, and feeder cells are cultured in or on the inside, the epithelial cells are stratified. Further, the surface of the porous membrane is coated with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C., and epithelial cells are cultured and layered thereon, and then the temperature of the culture solution is set. It has been found that if the cultured corneal epithelial cell sheet or its stratified sheet is brought into close contact with the carrier, it can be peeled off as it is together with the carrier. The present invention has been completed based on such findings.

That is, when culturing epithelial cells on a porous membrane, the present invention fills both the upper layer portion and the lower layer portion with the medium with the porous membrane as a boundary, and always passes through the porous membrane while the cells are cultured. Provided is a method for stratifying culture of epithelial cells in which a medium is supplied to cells from the lower layer.
The present invention also provides an epithelial cell stratification culture method characterized in that an extracellular matrix layer is provided on a culture bed of a porous membrane lower layer portion, and feeder cells are cultured inside, on or both of them. To do.
Furthermore, the present invention covers the surface of the porous membrane with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water, on which epithelial cells are cultured and layered,
(1) The culture solution temperature is set to the upper critical solution temperature or higher or the lower critical solution temperature or lower,
(2) Adhering the cultured corneal epithelial cell sheet or its multilayered sheet to a carrier,
(3) A layered culture method for epithelial cells, characterized in that it is peeled off together with the carrier, is proposed.
In addition, the present invention provides a stratified epithelial cell sheet obtained by the above method.
In addition, the present invention provides a treatment method characterized by transplanting the above-mentioned stratified epithelial cell sheet to an affected part in which a part or all of a tissue is damaged or lost.

  According to the method described in the present invention, epithelial cells are cultured on a porous membrane to be layered without using feeder cells. In addition, by culturing feeder cells with an extracellular matrix, epithelial cells can be stratified without any degeneration of the feeder cells. Furthermore, if a temperature-responsive polymer is coated on the porous membrane, the epithelial cells that have been stratified only by cooling can be detached, and therefore, a highly encapsulated stratification that has extremely high engraftment to living tissue. The epithelial cell sheet can be obtained.

  In the present invention, when epithelial cells are cultured on a porous membrane, both the upper layer and the lower layer are filled with the medium with the porous membrane as a boundary, and the medium always passes through the porous membrane while the cells are cultured. An epithelial cell stratification culture method characterized by being supplied to cells from a lower layer. At that time, it is not particularly limited whether feeder cells for differentiating or maintaining epithelial cells in the lower layer are cultured or not, but according to the method of the present invention, feeder cells are used. It is preferable that the cells can be cultured without manufacturing, and manufacturing efficiency or contamination of feeder cells can be prevented.

  Suitable epithelial cells used in the present invention include, for example, corneal epithelial cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, or a mixture of two or more, but the type is not limited. Is not to be done. The origin of the epithelial cells is not particularly limited, and examples thereof include humans, dogs, cats, rabbits, rats, pigs, sheep and the like. When using for treatment, it is desirable to use human-derived cells.

  The culture medium for cell culture in the present invention is not particularly limited as long as it is a commonly used medium for cells to be cultured, but is used when the obtained stratified epithelial cell sheet is used for human therapy. It is desirable that the medium components have a clear origin or are recognized as pharmaceuticals. In the present invention, this medium is filled with the upper and lower layers through a porous membrane in which epithelial cells are cultured. In this method, epithelial cells on the porous membrane are supplied not only with the medium in the upper layer but also with the medium in the lower layer. Although the detailed reason has not been clarified at this time, it is considered that supply of a sufficient medium from the lower layer is useful for stratification of epithelial cells.

  The present invention also provides an epithelial cell stratification culture method in which an extracellular matrix layer is provided on a culture bed in a lower layer of a porous membrane, and feeder cells are cultured inside, on or both. According to this method, it has been found that the feeder cells do not proliferate without any modification of the feeder cells, and continue to produce useful substances when the epithelial cells cultured on the porous membrane are layered. This method does not require mitomycin C treatment, X-ray, or gamma ray treatment, which has been conventionally performed in order to suppress the proliferation of feeder cells, simplifies the operability during cultivation, and originally cultivates cell culture such as mitomycin C. This is preferable because there is no contamination of drugs that are not used in the field, and large-scale operations such as X-ray and gamma ray processing are unnecessary. Examples of the extracellular matrix used in the present invention include any one of collagen, laminin, fibronectin, or a mixture of two or more, but the type is not limited. The coating amount of the extracellular matrix and the coating method used in the present invention are not particularly limited, and may be gelled or the extracellular matrix solution may be solidified according to a conventional method.

  Examples of feeder cells used in the present invention include fibroblasts, tissue stem cells, and embryonic stem cells, but are not particularly limited. In addition, epithelial cells and feeder cells may be derived from the same species of animal, but when the obtained stratified epithelial cell sheet is used for transplantation, epithelial cells and feeder cells are derived from the same species of animal. It is more desirable. In addition, when this stratified epithelial cell sheet is used for human therapy, it is desirable to use human-derived cells for epithelial cells and feeder cells.

  The temperature-responsive polymer used for coating on the surface of the porous membrane has an upper critical solution temperature or a lower critical solution temperature of 0 ° C to 80 ° C, more preferably 20 ° C to 50 ° C in an aqueous solution. If the upper critical lysis temperature or the lower critical lysis temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, when the upper critical lysis temperature or the lower critical lysis temperature is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed, which is also not preferable.

  The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer.

The method for coating the porous film with the temperature-responsive polymer is not particularly limited, and for example, a method described in JP-A-2-21865 may be used. That is, the coating is performed by applying the substrate and the monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, coating, kneading, etc. It can be performed by physical adsorption or the like. The coverage of the temperature responsive polymer may have a range of 0.4~4.5μg / cm ^2, preferably 0.7~3.5μg / cm ^2, more preferably 0.9~3.0μg / Cm ² . When the coating amount is less than 0.2 μg / cm ² , the cells on the polymer are difficult to peel off even when a stimulus is applied, and the working efficiency is remarkably deteriorated. On the other hand, if it is 4.5 μg / cm ² or more, it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells.

  The form of the porous film in the present invention is not particularly limited, and examples thereof include a form like a cell insert or a flat film form. At that time, epithelial cells need to be cultured in the upper layer of the porous membrane.

  In the present invention, epithelial cells are cultured on the porous membrane produced as described above. The medium temperature is not particularly limited as long as the polymer coated on the porous membrane surface has an upper critical solution temperature or lower, and when the polymer has a lower critical solution temperature or higher, the medium temperature is not particularly limited. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. The culture conditions other than the temperature may be in accordance with conventional methods and are not particularly limited. For example, the medium to be used may be a medium to which serum such as known fetal calf serum (FCS) is added, or a serum-free medium to which such serum is not added.

  In the method of the present invention, in order to peel and collect the stratified epithelial cells from the porous membrane, the cultured epithelial cell sheet is brought into close contact with the carrier, and the temperature of the support material to which the cells are attached is adjusted to the temperature of the support substrate. By making the temperature higher than the upper critical solution temperature or lower than the lower critical solution temperature of the coating polymer, it can be peeled off together with the carrier. Note that peeling of the sheet can be performed in a culture solution in which cells are cultured or in another isotonic solution, and can be selected according to the purpose. In addition, for the purpose of peeling and collecting the multi-layered sheet at a high yield, a method of gently tapping or shaking the cell culture support, a method of stirring the medium using a pipette, etc. alone, or You may use together. In addition, the cultured cells may be separated and recovered by washing with an isotonic solution or the like as necessary.

  As the carrier, for example, a polymer film, a structure molded from the polymer film, a metallic jig, or the like can be used. For example, when a polymer is used as the carrier material, specific examples of the material include polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and derivatives thereof, papers, chitin, chitosan, collagen, urethane, and the like. Can be mentioned.

  The shape of the carrier is not particularly limited. For example, when the obtained stratified epithelial cell sheet is transplanted, a part of the carrier that is cut to the same extent as the transplant site or larger than the transplant site is used. When used, the stratified epithelial cell sheet is conveniently fixed only to the peripheral part of the cut-out, and it is only necessary to apply the cell sheet in the cut-out part to the transplantation site.

  The stratified epithelial cell sheet in the present invention is not damaged by proteolytic enzymes represented by dispase, trypsin and the like at the time of culture. For this reason, the stratified epithelial cell sheet peeled from the base material has a high strength because the cell-cell desmosome structure is maintained, there are few structural defects. In addition, in the sheet of the present invention, the basement membrane-like protein between the cell and the substrate formed during the culture is not damaged by the enzyme. As a result, it is possible to adhere well to the affected tissue at the time of transplantation, and an efficient treatment can be performed. Specifically, when using a normal proteolytic enzyme such as trypsin, the cell-cell desmosome structure and the cell, the basement membrane-like protein between the substrates is hardly retained, Therefore, the cells are separated and separated in an individual state. Among them, dispase, a proteolytic enzyme, is known to be able to be detached in a state where the cell-cell desmosome structure is maintained at 10 to 60%. The obtained cell sheet is weak because it almost destroys the protein. In contrast, the cell sheet of the present invention is in a state where 80% or more of the desmosome structure and the basement membrane-like protein remain, and can obtain various effects as described above.

  In the present invention, the cell sheet may be peeled off from the carrier after applying the stratified epithelial cell sheet to the affected area. The method of peeling is not limited at all, but for example, a method of wetting the carrier to weaken the adhesion between the carrier and the cell sheet, or using a knife such as a knife, scissors, laser light, plasma wave, etc. Also, a method of cutting may be used. For example, when using a cell sheet that is in close contact with a carrier that has been partially cut out as described above, cutting along the boundary line of the affected area using laser light or the like avoids attachment of the cell sheet to an extra area other than the affected area. It is convenient.

  The fixing method of the stratified epithelial cell sheet and the biological tissue shown in the present invention is not particularly limited, and the cell sheet and the biological tissue may be sutured, or the stratified epithelium as shown in the present invention. Since the cell sheet adheres quickly to the living tissue, the cell sheet attached to the affected part does not need to be sutured to the living body side.

  The use of the stratified epithelial cell sheet shown in the present invention is not limited at all, but for example, treatment of burns, scars, bruises, corneal erosions, corneal ulcers, etc., or excimer laser irradiation to the central part. The PRK method, which corrects myopia by reducing the refractive power of the cornea by reducing the refractive power of the cornea, cutting the corneal stroma layer with a thickness of 160 μm with a microkeratome, and then turning the flap, excimer laser With the LASIK method, the surface of the corneal layer is trimmed, the flap is returned to its original position, alcohol is applied to soften the corneal surface, and the thickness of the corneal epithelium is reduced to 50 μm without using a microkeratome. Cut the flap to create a flap, scrape off the corneal stroma with an excimer laser, trim the surface, and finally put the flap back in place This is effective for refractive correction such as the returning LASEC method.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

Examples 1 and 2

Corneal epithelial tissue is collected from a rabbit corneal limbus under deep anesthesia according to a conventional method, and cells obtained by 0.05% trypsin treatment are treated with a temperature-responsive polymer as shown in FIG. It was plated in cm ² coated cell culture on the insert. The cell seeding number and the medium composition used are shown in FIG. 1 (37 ° C., 5% CO ₂ ). In Example 1, NIH-3T3 cells treated with mitomycin C were seeded as feeder cells in the lower layer of the insert. In Example 2, no feeder cells were used. The appearance of rabbit stratified corneal epithelial cells after 13 days of culture is shown in FIG. 2 (Example 1) and FIG. 3 (Example 2), respectively. In either case, rabbit corneal epithelial cells proliferated and reached stratification until they reached confluence. Next, the cell insert was cooled at 20 ° C. for 30 minutes, and an attempt was made to peel the rabbit stratified corneal epithelial cell sheet on the cell culture insert using a ring-shaped carrier. did it. The appearance when the rabbit stratified corneal epithelial cell sheet is peeled is shown in FIG. 4 (Example 1) and FIG. 5 (Example 2), respectively. Furthermore, in order to confirm that the detached cell sheet was stratified, the obtained rabbit stratified corneal epithelial cell sheet section was stained according to a conventional method (H & E staining). The dyeing results are shown in FIG. 6 (Example 1) and FIG. 7 (Example 2), respectively. In any case, it was confirmed that the layers were multi-layered.

Comparative Example 1

Corneal epithelial tissue is collected from a rabbit corneal ring under deep anesthesia according to a conventional method, and cells obtained by 0.05% trypsin treatment are cultured with a temperature-responsive polymer coated at 2.0 μg / cm ^2. The same procedure as in Example 1 was performed except that seeding was performed on a dish. At that time, feeder cells were not used. After 13 days of culture, the appearance of rabbit stratified corneal epithelial cells was confirmed, but the rabbit stratified corneal epithelial cells proliferated until they reached confluence but were not stratified.

Examples 3 and 4

A corneal epithelial tissue is collected from a human corneal limbus according to a conventional method and treated with 0.05% trypsin, and a temperature-responsive polymer as shown in FIG. 8 is coated with 1.9 μg / cm ^2. Seeded on the cultured cell culture inserts. The cell seeding number and the medium composition used are shown in FIG. 8 (37 ° C., 5% CO ₂ ). In Example 3, NIH-3T3 cells treated with mitomycin C were seeded as feeder cells in the lower layer of the insert. In Example 4, no feeder cells were used. The appearance of human stratified corneal epithelial cells after 14 days of culture is shown in FIG. 9 (Example 3) and FIG. 10 (Example 4), respectively. In either case, human corneal epithelial cells proliferated and reached stratification until they reached confluence. Next, the cell insert was cooled at 20 ° C. for 30 minutes, and an attempt was made to peel off the human stratified corneal epithelial cell sheet on the cell culture insert using a ring-shaped carrier. did it. The appearance when the human stratified corneal epithelial cell sheet is peeled is shown in FIG. 11 (Example 4). Furthermore, in order to confirm that the detached cell sheet was stratified, the obtained human stratified corneal epithelial cell sheet section was stained according to a conventional method (H & E staining). The dyed results are shown in FIG. 12 (Example 3) and FIG. 13 (Example 4), respectively. In any case, it was confirmed that the layers were multi-layered.

Comparative Example 2

Corneal epithelial tissue is collected from the donated human corneal limbus according to a conventional method, and cells obtained by 0.05% trypsin treatment are placed on a culture dish coated with a temperature-responsive polymer of 1.9 μg / cm ^2. The same procedure as in Example 3 was performed except that seeding. At that time, feeder cells were not used. After 14 days of culture, the appearance of human stratified corneal epithelial cells was confirmed, but the human stratified corneal epithelial cells proliferated until they reached confluence but were not stratified.

  According to the method described in the present invention, epithelial cells are cultured on a porous membrane to be layered without using feeder cells. In addition, by culturing feeder cells with an extracellular matrix, epithelial cells can be stratified without any degeneration of the feeder cells. Furthermore, if a temperature-responsive polymer is coated on the porous membrane, the epithelial cells that have been stratified only by cooling can be detached, and therefore, a highly encapsulated stratification that has extremely high engraftment to living tissue. The epithelial cell sheet can be obtained. The stratified epithelial cell sheet obtained by this method is strongly expected to have clinical applications such as corneal transplantation, skin transplantation, corneal disease treatment, and myopia treatment. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.

  The implementation method shown in Example 1, 2 is shown.   2 is a photograph showing the appearance of rabbit stratified corneal epithelial cells after 13 days of culture in Example 1. FIG.   It is a photograph showing the appearance of rabbit stratified corneal epithelial cells after 13 days of culture in the examples.   2 is a photograph showing a state in which the rabbit stratified corneal epithelial cell sheet of Example 1 is peeled off.   2 is a photograph showing a state where the rabbit stratified corneal epithelial cell sheet of Example 2 is peeled off.   It is a photograph which shows the appearance when the rabbit stratified corneal epithelial cell sheet section of Example 1 is stained with H & E.   It is a photograph which shows the appearance when the rabbit stratified corneal epithelial cell sheet section of Example 2 is stained with H & E.   The implementation method shown in Example 3 and 4 is shown.   2 is a photograph showing the appearance of human stratified corneal epithelial cells after 14 days of culture in Example 3. FIG.   2 is a photograph showing the appearance of human stratified corneal epithelial cells after 14 days of culture in Example 4. FIG.   It is a photograph which shows the state where the human stratified corneal epithelial cell sheet of Example 4 was peeled off.   It is a photograph which shows the appearance when the human stratified corneal epithelial cell sheet section of Example 3 is stained with H & E.   It is a photograph which shows the appearance when the human stratified corneal epithelial cell sheet section of Example 4 is stained with H & E.

Claims (16)

When culturing epithelial cells without using feeder cells on the porous membrane , both the upper layer and the lower layer are filled with the medium with the porous membrane as a boundary, and the cells are always passed through the porous membrane while the cells are being cultured. A layered culture method for epithelial cells, wherein the medium is supplied to the cells from the lower layer , and the medium contains serum and epidermal growth factor . The method for stratifying culture of epithelial cells according to claim 1 , wherein the epithelial cells are corneal epithelial cells, epidermal keratinocytes, oral mucosal cells, and conjunctival epithelial cells. The method for stratifying culture of epithelial cells according to claim 1 or 2 , wherein the epithelial cells are derived from human. After coating the surface of the porous membrane with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water, epithelial cells are cultured and layered,
(1) The culture solution temperature is set to the upper critical solution temperature or higher or the lower critical solution temperature or lower,
(2) Adhering the cultured corneal epithelial cell sheet or its multilayered sheet to a carrier,
(3) The method for stratifying culture of epithelial cells according to any one of claims 1 to 3, wherein the cells are peeled off together with the carrier. The layered culture method of epithelial cells according to claim 4 , wherein the temperature-responsive polymer is poly (N-isopropylacrylamide). The method for stratifying culture of epithelial cells according to claim 4 or 5 , wherein the carrier has a ring shape with the center cut out. The layered culture method of epithelial cells according to any one of claims 4 to 6, wherein the detachment is not treated with a proteolytic enzyme. The layered culture method for epithelial cells according to any one of claims 1 to 7 , wherein the porous membrane is a cell insert. A stratified epithelial cell sheet containing no feeder cells obtained by the method according to any one of claims 1 to 8 . The stratified epithelial cell sheet according to claim 9 for treating an affected part in which a part or all of the tissue is damaged or missing. The stratified epithelial cell sheet according to claim 10 , wherein the tissue is an anterior ocular tissue, skin tissue, or mucosal tissue. The stratified epithelial cell sheet according to claim 10 or 11 , which is cut along the size and shape of the affected part when covering the affected part. The stratified epithelial cell sheet according to any one of claims 10 to 12, wherein the treatment is a bilateral intractable keratoconjunctival disease. The stratified epithelial cell sheet according to any one of claims 10 to 12 , wherein the treatment is corneal erosion or corneal ulcer. The stratified epithelial cell sheet according to any one of claims 10 to 12, wherein the treatment is refractive correction by an RK method, a PRK method, or a LASIK method. The stratified epithelial cell sheet according to any one of claims 10 to 12, wherein the treatment is refractive correction by the LASEK method.

Images