WO2007029676A1

WO2007029676A1 - Biological tissue sheet and method for preparation thereof

Info

Publication number: WO2007029676A1
Application number: PCT/JP2006/317510
Authority: WO
Inventors: Kouji Hashimoto; Yuuji Shirakata; Junji Hamuro; Wakana Ito
Original assignee: Arblast Co., Ltd.
Priority date: 2005-09-07
Filing date: 2006-09-05
Publication date: 2007-03-15

Abstract

Disclosed is a biological tissue sheet which can show a high therapeutic effect and shows a high safety in transplantation. A method for preparation of a biological tissue sheet comprising the steps of: (a) providing a human fibroblast; (b) placing a collagen sheet on the human fibroblast which has been seeded in a culture vessel; (c) preparing a biologically derived cell and seeding the cell on the collagen sheet; and (d) culturing the biologically derived cell in the absence of a cell derived from a different animal to grow the cell.

Description

Specification

Biological tissue sheet and method for producing the same

Technical field

[0001] The present invention relates to a biological tissue sheet. Specifically, the present invention relates to a biological tissue sheet containing cells derived from keratoconjunctival epithelial cells, skin epidermal cells, hair follicle epithelial cells, oral mucosal epithelial cells, airway mucosal epithelial cells, intestinal mucosal epithelial cells, and the like. It relates to the production method of the sheet and the use (transplantation method etc.) of the sheet.

Background art

[0002] The skin is an organ that covers the outermost layer of a living body, and is a kind of barrier that protects a living body from an external force. The skin is composed of the epidermis, dermis, and subcutaneous tissue. The epidermis is mainly keratinocyte power, which contains a small number of pigment cells, Langerno cells, and cells. The cells that make up the epidermis are mainly keratinocytes, (1) keratinocytes that have lost their nuclei, which occupy the outermost layer, (2) cells that have nuclei beneath them (granular cells, spiny cells, basal cells) The epidermis basement membrane exists between the basal cells in the lowermost layer and the dermis. The basal layer is a single cell layer, which is the mother cell layer of epidermal keratinocytes, and it is thought that the only cells with the ability to divide are basal cells.

[0003] A state in which the epidermis has been lost for some reason is an ulcer. At the site where the epidermis is missing, the epidermis is regenerated and the ulcer surface becomes epithelialized by the proliferation of keratinocytes from the periphery or by the proliferation of keratinocytes derived from some hair follicles. However, in the case of burns that cause extensive epidermal defects at once, or intractable ulcers that lack hair follicles, epithelialization takes time only by regeneration of the epidermis from the surrounding area.

[0004] The cell cycle of the basal layer is about 450 hours. Daughter cells born by division change morphologically and functionally as they move to the spinous layer, and then keratinize through the granular layer to become the stratum corneum and eventually fall out of the body. The time until dropout is called turnover time, which is estimated to be 47 to 48 days.

[0005] In the epidermis regeneration model that is generally accepted, epidermal keratinocytes are divided into stem cells, transit-amplifying cells, post-mitotic cells based on their ability to divide. It is classified into three types of cells (post-dividing cells). A stem cell is a cell that has infinite self-renewal ability and generates a transit-amplifying cell by division. Transit-amplifying cells have a certain division ability and become transit-amplifying cells after division, but eventually lose division ability and become post-mitotic cells.

[0006] Stem cell of epidermis is considered to have the following characteristics. (L) The stem cell itself has a long cell cycle (slow cycling) o (2) The location varies depending on the site. (3) It exists as a set. (4) It strongly expresses α 2 | 8 1 and α 3 | 8 1 integrins. However, about 40% of the cell strength S integrin is strongly expressed in basal cells, and in fact stem cells are estimated to be about 10% of basal cells. In other words, there are transit-amplifying cells and post-mitotic cells in the basal layer.

[0007] When the epidermis is damaged, proliferation stimulation occurs, and the cells actively start to proliferate and start to regenerate. At this time, a series of epidermal growth factor groups plays the most important role. Epidermal growth factor (EGF) family Eko, EGF, transforming growth factor (TGF)-a, heparin binding EGF like growth factor, HB—EGF), betacellulin (be tacellulin), amphiregulin, neuregulins, and other forces that are actually involved in epidermal keratinocyte proliferation are TGF— _α , HB—EGF, and amphiregulin. It is thought to be regrin, and these factors have been shown to act autoproliferatively on epidermal keratinocytes. On the other hand, TGF-β, vitamin D, retinoic acid, etc. are known as factors that suppress the growth of epidermal keratinocytes.

Three

Yes.

[0008] In the field of regenerative medicine, development of a graft for transplanting a damaged site in which all layers of the dermis and epithelium are deficient in human skin is in progress. For example, JP-A-10-277143 (Patent Document 1) describes a graft for treating a full-thickness defect such as human skin and a method for producing the same. The graft disclosed in this document is made by embedding fibroblasts derived from dermal tissue in human fibrin sheet and attaching the epidermal tissue to the surface of this sheet.

Conventionally, for skin defect repair, full-thickness skin grafts that transfer the entire skin, and the epidermis and upper dermis are transferred. Divided skin grafting is carried out, but the size of the collection site is limited. In that respect, the cultured epidermis has the advantage that a small skin piece can be obtained several thousand times just by collecting a small piece of skin once, and it can be repeatedly stored because it can be cryopreserved, compared to conventional skin grafting. It seems to be the biggest advantage.

The epidermis sheet is divided into an autologous cultured epidermis sheet and an autologous cultured epidermis sheet depending on the origin of the cells used. In many cases, autologous skin sheet transplantation is mainly intended to cover and engraft the epidermal defect site, while transplantation of other culture skin sheet is effective as a biological dressing. Many people expect From basic research on epidermal cells, it has been clarified that epidermal keratinocytes produce various cell growth factors and cytokines, and the effectiveness of other-cultured epidermal sheets has been revealed.

[0009] If the technology for supplying the cultured epidermis safely and stably is completed, regenerative medicine will be put into practical use in conjunction with the advancement of transplantation technology, and the gospel to patients cannot be measured. When considering epidermal regeneration, it is very reasonable to separate cultivated and proliferative basal cells' incubate, proliferate in large quantities, regenerate epidermis, and use for transplantation. . However, the basic horticules include \, transit-amplifying cells, and post-mitotic cells, which are not stem cells, and techniques for selectively culturing epidermal stem cells to produce more efficient cultured skin. The establishment of a technique is desired.

So far, many researchers have attempted to research and develop technologies for culturing epidermal cells, and the results have brought tremendous benefits in the field of skin biology. Specifically, by using cultured keratinocytes, the characteristics of epidermal cells, regeneration, differentiation, and proliferation mechanisms of epidermis are rapidly becoming clear. On the other hand, small skin pieces are collected from patients, keratinocytes are subcultured, and can be proliferated in large quantities, and can be cryopreserved. Cultured skin is being used for treatment.

[0010] One of the tissues expected to contribute to regenerative medicine alongside the skin is the cornea. Cornea

It is the outermost layer of the optical system that constitutes the eyeball, and is a transparent bloodless tissue that contributes to obtaining good visual acuity by forming a smooth surface together with tears. In addition, the keratoconjunctival epithelial cells are always in contact with the outside world, and have a protective action to protect the eyeball with foreign substances such as microorganisms in the outside world and light rays such as ultraviolet rays. That is, keratoconjunctival epithelial cells are transparent to the cornea It plays a vital role in protecting the sex and the entire eyeball and maintaining homeostasis.

[0011] The cornea may become turbid and lose its transparency due to pathological conditions such as keratitis, corneal ulcer, and perforation. For such a permanent decrease in visual acuity due to turbidity of the cornea, treatment by cornea transplantation using the cornea provided by the eyeball donor Kas et al. Has been performed. In the corneal transplantation, the transparent cornea is transplanted after removing the cornea from which the transparency of the patient has been lost, and the transparency is restored by this transplantation, and visual acuity can be restored again.

While such corneal transplantation can be expected to have an effective therapeutic effect, there are epilepsy diseases that cannot be addressed by corneal transplantation alone. Examples include Stevens-Johnson syndrome, pemphigoid, chemical trauma, and burns. Normally, keratoconjunctival epithelial cells repeat division every day, old cells peel off, and new cells are regenerated from stem cell tissue. However, it has been found that in the above pathological conditions, the stem cell tissue that regenerates the cornea is damaged.

[0012] Stem cell tissue that regenerates corneal epithelium is called “corneal limbal tissue”, and is limited to the boundary between black eyes and white eyes, and is in a special environment exposed to the outside world. In the above-mentioned pathological condition, it is considered that this stem cell tissue itself is eradicated by some kind of disorder. And by eradicating this stem cell tissue, the defective part is covered with surrounding conjunctival epithelium, lacking transparency, and exerting an extremely low visual acuity. Thus, in the above-mentioned pathological condition, the corneal limbus is withered, so that the transplanted cornea cannot be maintained for a long time simply by transplanting the cornea. Therefore, in order to achieve permanent ocular surface reconstruction, it is also necessary to transplant the corneal annulus. As one method for transplanting the limbus, an amniotic membrane transplantation method has been developed (Medical Asahi, September 1999 issue: p62-65, N Engl J Med340: 1697-1703, 1999: Non-patent document 1 ). The amniotic membrane used in this transplantation method is obtained from the placenta of pregnant women who have undergone cesarean section. Since the amniotic membrane has a thick basement membrane, it acts as a substrate for the proliferation and differentiation of keratoconjunctival epithelial cells when transplanted. In addition, since the amniotic membrane has both anti-inflammatory and scarring suppression effects that are almost immunogenic, the keratoconjunctival epithelium and these stem cell tissues transplanted onto the amniotic membrane are rejected by the transplant recipient (recipient). Etc. will be protected.

Patent Document 1: Japanese Patent Laid-Open No. 10-277143 Non-Patent Document 1: Medical Asahi, September 1999 issue: p62-65, N Engl J Med340: 1697-1703, 1999

Disclosure of the invention

Problems to be solved by the invention

[0014] Culture of epidermal keratinocytes has been considered difficult in the past. In 1975, Rheinwald and Green announced the mouse 3T3 feeder-layer method, making it possible to culture epidermal keratinocytes for the first time. . This method was not easy to culture because mouse 3T3 cells were used as a feeder layer, and there were differences between lots due to the use of fetal calf serum. In the 1980s, Hennings and Yuspa showed that epidermal keratinocytes became undifferentiated and increased in proliferation when the Ca ²⁺ concentration in the culture medium was lowered. Furthermore, Boyce and Ham developed MCDB153 medium, which is a low Ca ²⁺ concentration medium, and showed that serum-free culture of human epidermal keratinocytes is possible by adding calf pituitary extract. In 1986, Pittelkow et al. Cultivated epidermal keratinocytes using a serum-free culture method, and then changed to a fetal calf serum-supplemented high calcium medium to produce a cultured epidermal sheet and transplant it to a burn patient. I got a grade. Currently, this serum-free culture method is the mainstream, but 3T3 cells derived from different animals are used as feeder layers. In transplants obtained by culturing in the presence of cells derived from different species, there is a risk of contamination with products derived from different species. Therefore, transplantation using the graft is regarded as xenotransplantation or equivalent, and is considered to have a serious problem in ethics and safety. In fact, there are no examples of xenotransplantation being put to practical use in the medical field!

[0015] Transplantation of cultured epidermis sheets made it possible to cover a wide range of wound surfaces by subculturing keratinocytes from stamp-sized skin. In addition, since it can be stored frozen, it can be applied to the treatment of intractable and recurrent skin ulcers that require repeated transplantation. It is not always easy to satisfactorily engraft culture sheets produced by conventional culture methods, for example, transplanted cultured skin sheets. This is because the cultured epidermis sheet lacks the components of the basement membrane and the stratified epidermis does not form a strong stratum corneum. In this regard, the 3D cultured skin developed by Bell is also the closest to the skin at present, with both the stratum corneum and the granule layer being recognized, but due to the complexity of the procedure and the need for special techniques, it is still in Japan. Not popular . Three-dimensional cultured skin has already been demonstrated overseas and has already been commercialized. However, it is not possible to expect permanent engraftment with the ability to promote epithelialization in allogeneic transplantation. In Japan, there is little prospect of widespread transplantation in terms of ethics and safety, and there has been a focus on improving autotransplantation.

[0016] On the other hand, as a surgical treatment for an ocular surface disease in which the cornea is covered with the conjunctival epithelium, corneal epithelial transplantation is currently performed, but refractory keratoconjunctival disease with strong inflammation ( For Stevens-Johnson syndrome, pemphigoid, corneal corrosion, etc., the prognosis is extremely poor. The biggest reason is that the foreign corneal epithelium with strong antigenicity is recognized and rejected by the host immune system. In addition, complications caused by systemic and local administration of large amounts of immunosuppressive agents after surgery to prevent rejection are a major factor in the poor prognosis. On the other hand, when the corneal epithelium of the mouth is used, there is a problem of donor shortage. Achieving technology that can produce dozens of corneal epithelial sheets using corneas that can be obtained with a single eye force will solve the problem of insufficient donors.

Means for solving the problem

[0017] The present invention has been made in view of the above background and problems, and an object thereof is to provide a biological tissue sheet that is expected to have a high therapeutic effect and has high safety at the time of transplantation. For the purpose of encouraging, the present inventors first tried to produce a cultured skin sheet. Specifically, in consideration of safety, the condition that the cells derived from different animals (feeder cells) are not used when culturing epidermal keratinocytes is used, and good growth and tissue formation of epidermal keratinocytes are achieved. We looked for possible culture methods. As a result, they have found a new culture method of culturing epidermal keratinocytes on a collagen sheet placed on human fibroblasts. An attempt was made to produce a cultured skin sheet using human amniotic membrane as a collagen sheet to verify the effectiveness of the culture method, and it was equipped with a cell layer with a structure approximating that of a normal epidermis and excellent in compatibility with living organisms. Succeeded in building a cultured skin sheet. In other words, according to the culture method, it is possible to create an environment suitable for the proliferation and tissue proliferation of epidermal keratinocytes without using heterogeneous animal cells as feeder cells. It has been found that a cultured skin sheet can be obtained. By the way, when the structure of the obtained cultured skin sheet was examined in detail, it was found that fibroblasts infiltrated into the amniotic membrane. This or Furthermore, the fibroblasts interact with collagen in the amniotic membrane to ensure good growth of the fibroblasts themselves, and the nutritional components necessary for the survival, proliferation and organization of epidermal keratinocytes It was speculated that a state of being supplied from fibroblasts was created through this, and as a result, a high-quality epidermal cell layer could be constructed.

On the other hand, in order to investigate the versatility of the above-described culture method that was found to be effective for the construction of cultured skin sheets, the same experiment was conducted by replacing the cells used for culture with cells derived from other tissues in terms of epidermal keratinocyte strength. did. As a result, in an experimental system using corneal epithelial cells, it was possible to achieve good cell growth and organization similar to epidermal keratinocytes, and succeeded in constructing a high-quality cultured corneal epithelial sheet. As a result, it has been found that the above culture method is not only suitable for the construction of the epidermal cell layer but is highly versatile and extremely useful as a technique in the field of regenerative medicine.

As described above, the present inventors have succeeded in producing a safe and practical biological tissue sheet that does not use cells derived from different animals.

The present invention has been completed on the basis of the above results and knowledge, and provides the following configuration.

That is, the present invention provides a biological tissue sheet containing biological cells grown on a collagen sheet placed on human fibroblasts. It is preferable to use amniotic membrane as the collagen sheet here. Amniotic membrane is not only immunogenic but also has anti-inflammation and anti-scarring effects. As the collagen sheet, it is particularly preferable to use amniotic membrane from which the epithelium (epithelial cell layer) has been removed. This is because the absence of epithelium improves the growth of living cells seeded thereon. This is also because a further reduction in immunogenicity is achieved. On the other hand, in one embodiment of the present invention, amniotic membrane added with trehalose is used as a collagen sheet. Such amniotic membrane is excellent in flexibility and functions better as a substrate for cell culture.

As a medium for growing cells derived from living organisms, (1) a serum-free medium, or (2) a medium containing only serum derived from the recipient as a serum component can be used.

The biological cell is preferably a cell derived from the corneal epithelium, conjunctival epithelium, skin epidermis, hair follicle epithelium, oral mucosa, airway mucosa or intestinal mucosa. In one embodiment of the present invention, in addition to the proliferated cells, the human fibroblast-derived cells used infiltrate and contain a collagen sheet to form a living tissue sheet. A biological tissue sheet according to another embodiment of the present invention includes a layered cell layer formed by expanded cells.

The biological tissue sheet of the present invention is, for example, directly or amniotic (amniotic membrane different from the collagen sheet used as a culture substrate. For the sake of distinction from the amniotic membrane used as the culture substrate in one embodiment of the present invention, the “second amniotic membrane is used for convenience. And also transplanted into a tissue defect. In the latter case, typically, after transplanting the second amniotic membrane to the tissue defect, a biological tissue sheet is transplanted on the amniotic membrane.

[0020] The present invention further provides a method for producing a biological tissue sheet. The production method of the present invention includes the following steps. That is, (a) preparing human fibroblasts; (b) placing a collagen sheet on the human fibroblasts seeded in a culture vessel; (c) preparing living-derived cells; Inoculating a biological cell on the collagen sheet; and (d) culturing and proliferating the biological cell in the absence of a heterologous animal cell.In one aspect of the invention, in step (b), A collagen sheet is placed after a predetermined time has elapsed since the seeding of human fibroblasts. This step promotes the adhesion and monolayering of human fibroblasts to the culture container, thereby achieving a high quality sheet of the biological tissue sheet.

In one aspect of the present invention, the following steps are performed between step (b) and step (c). That is, (b) ′ is a step of culturing the human fibroblast for a predetermined time. By this step, the infiltration of human fibroblasts into the collagen sheet is promoted, and the high quality of the living yarn and woven sheet can be achieved.

In another aspect of the present invention, (e) after the living body-derived cells have grown, the step of bringing the outermost layer into contact with air is further performed. This step promotes keratinization (epithelialization) of the cell layer.

Preferably, amniotic membrane is used as the collagen sheet in step (b). It is particularly preferable to use amniotic membrane from which the epithelium (epithelial cell layer) has been removed as the collagen sheet. . On the other hand, in one embodiment of the present invention, amniotic membrane added with trehalose is used as a collagen sheet.

As a medium for carrying out step (d), (1) a serum-free medium, or (2) a medium containing only serum derived from a recipient as a serum component can be used.

The biological cell is preferably a cell derived from the corneal epithelium, conjunctival epithelium, skin epidermis, hair follicle epithelium, oral mucosa, airway mucosa or intestinal mucosa.

Brief Description of Drawings

FIG. 1 is a flowchart showing an amnion epithelial removal procedure.

FIG. 2 is a diagram for explaining an amniotic membrane fixing method. In (a), the amniotic membrane is sandwiched between a pair of frames, and in (b), the amniotic membrane is sandwiched between the frame and a flat plate member.

FIG. 3 is a HE-stained image showing the state of the cultured skin sheet on day 17 after transplantation.

FIG. 4 is a diagram showing the results of immunohistochemical staining for cultured skin sheets.

FIG. 5 shows the results of immunohistochemical staining of cultured skin sheets.

[Fig. 6] Experimental method (upper) and experimental results (lower) regarding the relationship between the seeding time of epidermal keratinocytes and the quality of the cultured skin sheet to be constructed. The lower left is the HE-stained image of the cultured skin sheet constructed under condition 1 (time after amnion placement, time to seed the epidermis keratinocytes), the lower right is condition 2 (after amnion placement, It is an HE-stained image of a cultured skin sheet constructed under the condition of seeding epidermal keratinocytes without taking time.

[Fig. 7] Experimental method (upper) and experimental result (lower) regarding the relationship between the time of placing (attaching) the amniotic membrane and the quality of the cultured skin sheet to be constructed. The lower left is an HE-stained image of a cultured skin sheet constructed under Condition 1 (conditions where the fibroblast seeding power is short in the time interval until placement on the amniotic membrane), and the lower right is Condition 2 (fibroblast seeding power is up to the amnion placement) This is a HE-stained image of a cultured skin sheet constructed with long time intervals and conditions.

FIG. 8 is an HE-stained image of a cultured corneal epithelial sheet constructed using amniotic membrane and human fibroblasts.

FIG. 9 is an immunostained image (confocal laser microscope, 100 × magnification) of a cultured corneal epithelial sheet constructed using amniotic membrane and human fibroblasts. Upper row (A) shows immunostaining results for keratin 3. The lower panel (B) shows the result of immunostaining for keratin 12. Left side: Green color Display primary After staining with antibody, stained with secondary antibody Al _eX a488. Center: Red coloration Colored with DNA staining reagent PI. Right: Superimposed left and center photos.

Explanation of symbols

[0022] 1, 2, 3 frames

4 Plate member

10 Amniotic membrane

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] The present invention provides a biological tissue sheet having the following constitution. That is, it is a living tissue sheet containing living body-derived cells grown on a collagen sheet placed on human fibroblasts. Living body-derived cells form a cell layer. The features of the biological tissue sheet of the present invention and its production method will be described in detail below.

[0024] The biological tissue sheet of the present invention comprises (a) a step of preparing human fibroblasts, (b) a step of placing a collagen sheet on the human fibroblasts seeded in a culture vessel, (c) Preparing a cell derived from a living body and seeding the living body-derived cell on the collagen sheet; and (d) culturing and proliferating the living body-derived cell in the absence of a heterologous animal cell. Can do. Hereinafter, each step will be described.

[0025] 1. Step (a)

In step (a), human fibroblasts are prepared. The origin of human fibroblasts is not particularly limited. For example, human fibroblasts derived from skin tissue, ocular tissue (cornea, sclera, conjunctiva, etc.), or oral mucosal tissue can be used. Although human fibroblasts provided in the form of cell lines can be used, it is preferable to use recipient-derived fibroblasts from the viewpoint of biocompatibility. In addition, human fibroblasts may be selected in consideration of the type of cells derived from a living body to be cultured on a collagen sheet (in other words, the type of living tissue sheet to be produced). Specifically, human fibroblasts derived from the same tissue as the living body cell to be used (or a tissue adjacent to the living body) may be employed. With such a combination of biologically derived cells and human fibroblasts, the proliferation of the biologically derived cells and the expected tissue properties are improved. An example of a preferred combination of biological cells and human fibroblasts is a combination of epidermal keratinocytes (biological cells) and human fibroblasts derived from skin tissue. Can be mentioned.

Human fibroblasts can be cultured by a conventional method. For example, human fibroblasts can be cultured in an appropriate medium in the presence of 37 ° C and 5% CO. Used for human fibroblast culture

2

The medium to be used is not particularly limited as long as the cells can grow, and a medium usually used for fibroblast culture may be used. For example, a DMEM medium containing serum (such as fetal calf serum) can be used. As serum added to the medium, human serum, fetal bovine serum, sheep serum, and the like can be used. Among them, it is preferable to use a serum that is derived from the same species (human serum) and autologous serum (that is, the serum of the recipient itself). Of course, if possible, it is most preferable to use autologous serum that eliminates the risk of causing immune rejection.

Human fibroblasts may be cultured using a medium supplemented with growth factors and antibiotics. By using growth factors and antibiotics, cell proliferation rate and survival rate can be improved and contamination can be prevented.

Human fibroblasts may be cultured using a medium that is serum-free and does not contain proteins derived from different animals. That is, a serum-free culture method may be employed as the culture method in the present invention. In such an embodiment, problems such as immune rejection due to contamination of serum-derived components can be avoided.

It should be noted that the above-described culture conditions can also be employed for human fibroblast seeding (step (b)) and culture after seeding (step (b) ′) described below.

[0026] 2. Step (b)

In step (b), first, the prepared human fibroblasts are seeded in a culture vessel. Considering the size of the biological tissue sheet finally obtained, use a culture container of an appropriate size. For example, a commercially available culture insert or petri dish with a moderate force can be selected and used. The material of the culture vessel and the presence or absence of surface coating are not particularly limited.

[0027] Human fibroblasts, for example, plated at a cell density of 10,000 to 50,000 pieces / cm ^2. If the cell density at the time of seeding is too small, one of the effects of the present invention that the growth of living cells is improved by using human fibroblasts is not sufficiently exhibited. On the other hand, sowing If the cell density at the time is too high, the nutrients supplied to the culture solution are insufficient, which is not preferable.

[0028] A collagen sheet is placed on the human fibroblasts seeded in the culture vessel. Preferably, the collagen sheet is placed after a predetermined time has passed for the seeding force of human fibroblasts. Ensuring sufficient time after seeding of human fibroblasts promotes adhesion and monolayering of human fibroblasts to the culture container, and is effective in producing high-quality biological tissue sheets. The predetermined time here is, for example, 1 hour or more, preferably 2 hours or more, more preferably 4 hours or more, and even more preferably 1 day or more (eg, 1 day, 2 days, 3 days). is there.

[0029] The "collagen sheet" functions as a culture substrate for living cells. The type of collagen used as a raw material for the collagen sheet is not particularly limited, and type I collagen, type III collagen, type IV collagen and the like can be used. A mixture of a plurality of types of collagen can also be used. These collagens can be extracted and purified from the skins of animals such as pigs, sushi, and hedges, and connective tissues such as soft bones by acid solubilization, alkali solubilization, enzyme solubilization, etc. it can. For the purpose of reducing antigenicity, it is preferable to use a so-called atherocollagen in which the telopeptide is removed by treatment with a degrading enzyme such as pepsin or trypsin. Collagen derived from amnion, particularly human amnion, may be used as a material for the collagen sheet. Here, the term “derived from amniotic membrane” means that it is widely obtained using amnion as a starting material.

It is preferable to use amniotic membrane as the collagen sheet. The “amniotic membrane” is a membrane that covers the outermost layer of the uterus and placenta in mammals, and is composed of a basement membrane and an epithelial layer formed on a collagen-rich parenchyma. Amniotic membranes such as humans, monkeys, chimpanzees, pigs, horses, and ushi can be used. Among them, it is preferable to use human amniotic membrane. It also has advantages in terms of safety, including immunogenicity and virus infection. For example, human amnion can also collect force such as human fetal membrane and placenta obtained as a postpartum at delivery. Specifically, human amniotic membrane can be prepared by treating and purifying an integral body consisting of human fetal membrane, placenta and umbilical cord obtained immediately after delivery. As a treatment and purification method, a known method such as a method described in JP-A-5-56987 can be adopted. That is, the amniotic membrane is peeled off from the fetal membrane obtained at the time of delivery Then, the remaining tissue is removed by physical treatment such as ultrasonic washing and enzyme treatment, and human amniotic membrane can be prepared through a washing step as appropriate. The human amniotic membrane thus prepared can be stored frozen until use. Freezing of the human amniotic membrane can be performed, for example, at −80 ° C. in a liquid in which DMEM (Dulbecco's modified Eagle's medium) and glycerol are mixed in an equal volume ratio. Cryopreservation is expected to improve operability as well as decrease antigenicity.

[0030] Although the amniotic membrane can be used as it is, it is preferable to use a material obtained by removing the epithelium from the amniotic membrane by reptile treatment or the like. When using human amniotic membrane from which the epithelium has been removed, it is preferable to seed biologically-derived cells on the surface side (ie, the basement membrane side) where the epithelium has been removed in step (c) described later. This is because this surface side is rich in type IV collagen, and it is considered that the seeded living cells are proliferated and stratified well.

For example, after thawing the human amniotic membrane cryopreserved as described above, treatment with EDTA or a proteolytic enzyme relaxes the adhesion between cells, and the epithelium is replated using a cell scraper or the like. A removed human amniotic membrane can be prepared. It is preferable to use human amniotic membrane from which the epithelium has been removed by a method including the following steps.

(1) A step of preparing an amniotic membrane separated from a living body.

(2) A step of subjecting the amniotic membrane to a freeze-thaw treatment.

(3) A step of applying a trypsin treatment to the amniotic membrane after the freeze-thaw treatment.

(4) A step of washing the amniotic membrane after the trypsin treatment.

[0031] According to the epithelial removal method, it is possible to completely remove the epithelium while suppressing damage to the basement membrane as much as the conventional manual epithelial removal method. That is, according to the epithelial removal method, an amniotic membrane having a basement membrane in which the epithelium is completely removed and the original structure is well maintained can be obtained. Such an amniotic membrane functions well as, for example, a cell culture substrate (base material). On the other hand, the following epithelial removal method is very easy to operate and requires less operation time than conventional manual methods. It is also easy to process a large number of amniotic membranes at the same time. Furthermore, since skilled technology is not particularly required, it is easy to automate. Hereinafter, each step of the epithelial removal method will be described in detail (see FIG. 1).

[0032] (1) Preparation of amniotic membrane

In step (1), prepare the amniotic membrane. The amniotic membrane here is preferably human amniotic membrane. For example, human amnion can be collected by force such as human fetal membrane or placenta obtained as a postpartum at the time of delivery. Specifically, the human amniotic membrane can be prepared by treating and purifying an integral product such as human fetal membrane, placenta and umbilical cord obtained immediately after delivery. As a method for preparing such human amniotic membrane, known methods such as the method described in JP-A-5-56987 can be employed. Typically, this step is performed as follows.

(1-1) Collection of amniotic membrane

A part of the placenta tissue is collected at the time of delivery, and then the amniotic tissue is manually detached from the placenta tissue. You may freeze once at this stage.

(1-2) Removal of blood cell components

Wash and remove blood cell components adhering to the amniotic membrane with physiological saline. Also, the chorion is manually peeled off and removed. Thus, it is preferable to use amniotic membrane from which blood cell components and chorion are removed at this stage, but after the freeze-thaw treatment (step 2) described later, removal of blood cell components and peeling of Z or chorion are performed. You can also.

The human amniotic membrane thus prepared can be stored frozen until the next treatment. Freezing of the human amniotic membrane can be performed, for example, at −80 ° C. in a solution in which DMEM (Dulbecco's modified Eagle's medium) and glycerol are mixed in an equal volume ratio. Cryopreservation is expected to improve operability as well as decrease antigenicity.

[0033] It is preferable that the amniotic membrane prepared by the above procedure is fixed to a frame and then subjected to the subsequent treatment. It becomes easy to handle by fixing the amniotic membrane with a frame.

Figure 2 shows a specific example of how to fix the amniotic membrane. In the example of Figure 2a, two frames (1, 2) of the same shape are used. The amniotic membrane 10 is fixed by holding the edges of these two frames. Fix the amniotic membrane in an expanded state. In the example of FIG. 2 b, the amniotic membrane 10 is fixed using the frame 3 and the plate-like member 4. First, the amniotic membrane 10 is spread on the plate-like member 4. At this time, the epithelial side of the amniotic membrane 10 is turned up. Subsequently, the upper force of the amniotic membrane 10 is also put on the frame 3, and the edge of the amniotic membrane 10 is sandwiched between the plate-like member 4 and the frame 3. As a result, only the epithelial side of the amniotic membrane 10 is exposed. Therefore, When the trypsin treatment described below is performed, the trypsin solution can be brought into contact only with the epithelial side of the amniotic membrane (for example, a trypsin solution is added inside the frame 3). As a result, trypsin treatment can be performed without affecting the parts other than the epithelium (the amnion dense layer and the basement membrane). In other words, while the trypsin acts on the epithelium of the amniotic membrane, it is possible to protect the denseness of the amniotic membrane, etc.

[0034] (2) Freezing and thawing treatment

In this step, the amniotic membrane is once frozen and then thawed. This freezing and thawing process makes it easier for the amniotic epithelial layer to peel off during subsequent trypsin treatment. This is thought to be due to the loosening of the adhesive state (bonded state) between the amniotic epithelial layer and the basement membrane.

A freezing temperature of about 20 ° C to about 80 ° C can be used. In consideration of the fact that a sufficient frozen state can be obtained and a general-purpose freezer can be used, it is preferable to freeze at about 80 ° C. On the other hand, a melting temperature of about 4 ° C to about 50 ° C can be employed. Preferably the melting temperature is about 37 ° C.

It is preferable to repeat the freeze-thaw treatment. By repeatedly performing the treatment, the effect of the freeze-thaw treatment that the epithelium is easily detached in the subsequent trypsin treatment is enhanced. However, if it is repeated more than necessary, it is also expected to adversely affect parts other than the epithelium. Therefore, for example, it is preferable to repeatedly perform freeze-thaw treatment in the range of 2 to 4 times. As a result of investigations by the present inventors, it was found that necessary and sufficient effects can be obtained by performing freeze-thaw treatment twice at a freezing temperature of −80 ° C. and a thawing temperature of 37 ° C. From this knowledge, it can be said that the freeze-thaw treatment is preferably performed twice under the conditions of a freezing temperature of -80 ° C and a thawing temperature of 37 ° C.

The conditions (freezing temperature and thawing temperature) for each time when the freeze-thaw treatment is repeatedly performed may be the same, partly different, or different from each other. However, from the viewpoint of operability, it is preferable that the conditions are the same each time.

[0035] (3) Trypsin treatment

In this step, the amniotic membrane after the freeze-thaw treatment is treated with trypsin. Trypsinization is performed by bringing a trypsin solution into contact with the amniotic membrane. For example, use a trypsin solution with a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v). Can do. Preferably, a trypsin solution having a trypsin concentration of about 0.02% (w / v) is used. If the trypsin concentration of the trypsin solution is too low, the action of trypsin will not be fully exerted. On the other hand, if the trypsin concentration is too high, trypsin can act well on the amniotic epithelium, while trypsin also acts on the amnion dense layer and the basement membrane, which may damage the part.

Many trypsins are commercially available such as those derived from ushi, porcine, and human. For example, Trypsin-EDTA (Invitrogen) and trypsin 1: 250 (Sigma) can be preferably used.

[0036] A chelating agent is usually added to the trypsin solution, but the chelating agent is not essential.

As a chelating agent, EDTA, NTA, DTPA, HEDTA, GLDA, etc. can be used. Any combination of these may be used. The chelating agent is added, for example, to a concentration of about O.lmM to about 0.6 mM.

[0037] It is preferable to carry out trypsin treatment under conditions where only the amnion epithelial side is in contact with the trypsin solution. This is to protect the action force of trypsin on parts other than the amniotic epithelium. For example, immerse only the amnion epithelium side in a trypsin solution, do not add trypsin solution to the amnion epithelium side, apply it, and block the amnion chorion side to avoid contact with the solution. Thereafter, only the amnion epithelial side can be brought into contact with the trypsin solution by, for example, immersing it completely in a trypsin solution. As described above, if the amniotic membrane (frame-fixed amniotic membrane) fixed in advance to the frame as shown in Fig. 2b is used, only the epithelial side of the amniotic membrane is exposed, so the frame-fixed amniotic membrane is immersed in a trypsin solution, for example. It is possible to contact only the amnion epithelium side with the trypsin solution. This method also has the advantage that the trypsin treatment can be performed by a simple operation of immersing the frame-fixed amniotic membrane. Even when the amniotic membrane fixed to the frame is used in this way, if the entire frame is immersed in a trypsin solution, only the epithelial side portion of the amniotic membrane is immersed in the trypsin solution (for example, the epithelium of the amniotic membrane). (You can dip it in a trypsin solution with the side down), add the trypsin solution in the frame, or apply the trypsin solution to the amnion epithelial side to infect only the epithelial side of the amniotic membrane with the trypsin solution. .

[0038] The trypsin treatment time (contact time of the trypsin solution) is, for example, about 5 minutes to about 60 minutes. Good It is preferably about 10 minutes to about 20 minutes, more preferably about 15 minutes. If the treatment time is too short, trypsin cannot be sufficiently exerted, resulting in insufficient removal of the amniotic epithelium. On the other hand, if the treatment time is too long, trypsin may also act on the basement membrane and dense layer of the amniotic membrane to damage the part.

The temperature condition of the trypsin treatment is, for example, about 25 ° C. to about 42 ° C. so that trypsin works well.

It is preferred to keep the amniotic membrane stationary while contacting the trypsin solution. It is also the force that the trypsin solution is thought to penetrate through the basement membrane and the dense layer.

The trypsin treatment can be performed in a plurality of times.

[0039] (4) Cleaning

After contacting the trypsin solution by the above method, the amniotic membrane is washed. This washing removes the attached trypsin solution and simultaneously removes the amniotic epithelium (epithelial cells). For example, leave it in a liquid with an appropriate flow (for example, flowing water), shake it in a suitable liquid (for example, shake up and down), or apply ultrasonic waves while immersed in a suitable liquid. The amniotic membrane after trypsinization is washed by adding. Examples of the liquid used for washing include physiological saline, phosphate buffer, pure water, and DMEM.

[0040] The washed amniotic membrane may be refrigerated or frozen until use. For example, it can be stored in a state of being immersed in a storage solution containing glyceride (for example, 50% glycerol-containing DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL)).

[0041] (Use of cocoon amniotic membrane with trehalose)

As the collagen sheet, amniotic membrane to which trehalose is added can also be used. In this case, it is preferable that the epithelium of the amniotic membrane is removed.

The addition of trehalose increases the flexibility of the amniotic membrane, particularly when it is lyophilized (see Japanese Patent Application 2005-214339 for details). Amniotic membrane with trehalose works well as a substrate for cell culture (see Japanese Patent Application No. 2005-214339 for details).

Here, if the matrix protein inside the amniotic membrane is weakened, the strength of the amniotic membrane is reduced and it becomes susceptible to damage. Also, moisture cannot be firmly held inside, and it becomes brittle and loses elasticity. G Rehalose acts on loosened bonds in matrix proteins to strengthen the bonds between the proteins, thereby normalizing the water retention function inside the amniotic membrane, maintaining the original moisture, cohesion, and elasticity of the amniotic membrane. Expected. By adding trehalose, it is considered that the matrix protein inside the amniotic membrane becomes soft with freezing and drying treatment, and in particular, it can effectively prevent swelling and weakening in water.

Trenorose (substance name, generic name) is a compound represented by (X-D-Glucopyranosyl (1,1) —a—D—Glucopyranosi de.

Attachment of trehalose to the amniotic membrane can be performed, for example, by treating the amniotic membrane with a trehalose solution. For example, the amniotic membrane is immersed in a solution obtained by dissolving trehalose in distilled water or phosphate buffered saline (PBS (-)) so that the concentration is 5% (w / v) to 20% (w / v). To do. The temperature during immersion is, for example, in the range of 4 ° C to 37 ° C. The immersion time is, for example, in the range of 1 hour to 1 day. As for trehalose, for example, Treha (registered trademark) provided by Hayashibara Shoji Co., Ltd. or HI Plus Plus Life Science Co., Ltd. can be used.

As a method of adding trehalose to the amniotic membrane, a method of applying a trehalose solution to the surface of the amniotic membrane, a method of spraying the trehalose solution to the surface of the amniotic membrane, a method of adding trehalose directly to the surface of the amniotic membrane, etc. may be adopted.

[0042] (Use of reconstructed amniotic membrane)

A reconstructed amniotic membrane can also be used as the collagen sheet. Specifically, for example, amniotic membrane that has been once decomposed by homogenizer, ultrasonic wave, or enzymatic treatment and reconstructed into a membrane shape can be used. The treatment method is preferably a homogenizer. This is because it is expected to keep the minute structure of the basement membrane relatively high. The conditions (rotation speed) of the homogenization treatment are, for example, 3000 rpm to 50000 rpm, preferably 10000 rpm to 40000 rpm, and more preferably <30000 rpm.

[0043] (Use of amniotic membrane reinforced with bioabsorbable material)

An amnion in which the chorionic side of the amniotic membrane is coated with a bioabsorbable material can also be used as the collagen sheet. By using the amniotic membrane thus reinforced, the operability is improved. As a bioabsorbable material used for such purposes, it is earlier than amniotic membrane. Decomposition 'It is preferable to adopt a material that is absorbed. For example, polydaractin 910, gelatin, collagen, polylactic acid and the like can be suitably used as the bioabsorbable material here. The form of the bioabsorbable material used for reinforcement is not particularly limited. For example, the amniotic membrane is reinforced by covering the amnion chorion side with a bioabsorbable material formed into a mesh or sheet. The amniotic membrane can be either wet or dry during the reinforcement process.

[0044] 3. Step (b) '

Immediately after placing the collagen sheet on human fibroblasts, seeding and culturing of biological cells (step (c)) can be performed, but preferably human fibroblasts are inoculated before seeding the biological cells. Incubate for a predetermined time (step (b) '). Performing step (b) ′ is effective for producing a high-quality biological tissue sheet. In other words, the implementation of this step promotes the infiltration of human fibroblasts into the collagen sheet, and living body-derived cells can receive the supply of nutrient components with the strength of human fibroblasts from the initial stage of culture. . As a result, the growth and organization of the cells derived from the living body are improved, which is advantageous for the construction of a high-quality cell layer. The predetermined time here is, for example, 2 hours or more, preferably 1 day or more, more preferably 3 days or more, and even more preferably 5 days or more (f, 5 days, 6 days, 7 days).

[0045] 4. Step (c)

In the step, living body-derived cells are prepared, and the living body-derived cells are seeded on a collagen sheet. As the cells derived from the living body, use cells that are suitable for the purpose of the finally obtained living tissue sheet. For example, when preparing a sheet for regeneration of skin epidermal tissue, skin epidermal cells (including stem cells and precursor cells) and hair follicle epithelial cells (including stem cells and precursor cells) are preferably used. The Similarly, corneal epithelial cells (including stem cells and progenitor cells) are preferably used for the purpose of regenerating corneal epithelial tissue, and mucosal epithelial cells (for the purpose of regenerating mucosal epithelial tissue ( Their stem cells and progenitor cells) are preferably used. Examples of mucosal epithelial cells include oral mucosal epithelial cells, intestinal mucosal epithelial cells, airway mucosal epithelial cells and the like.

The preparation method of living body-derived cells will be described taking skin epidermal cells, corneal epithelial cells, oral mucosal epithelial cells, intestinal mucosal epithelial cells and airway mucosal epithelial cells as examples. [0046] (Skin epidermal cells)

First of all, when collecting the skin, preparatively disinfect the collection site with a disinfectant such as povidone, apply an antifungal agent externally, and then apply a small skin piece to the skin biopsy. Collect. When cultivating epidermal keratinocytes, remove as much of the skin as possible with a pair of scissors, adipose tissue and dermis, and wash several times with Dulbecco's phosphate buffer (PBS). Sterilize in 70% ethanol for 1 minute. Cut into strips, soak in dispase solution, and stand at 4 ° C. The epidermis is then peeled off from the dermis. After washing the peeled epidermis, loosen the epidermis and adjust the epidermal keratinocyte suspension. Suspend cells in serum-free medium, inoculate on collagen-coated share, and perform subculture.

[0047] (corneal epithelial cells)

Corneal epithelial cells can be obtained from corneal limbal tissue force. For example, endothelium cells such as corneal limbal tissue force are detached and removed, and the conjunctiva is excised to produce a single cell suspension. This is stored in a nitrogen tank and then rapidly thawed at 37 ° C to prepare the corneal epithelial cell suspension. Subculture if necessary. Moreover, you may use for subculture without cryopreserving. For subculture, for example, EpiLife ™ (Cascade), which is a serum-free medium, MCDB153 medium (Nissui Pharmaceutical Co., Ltd.), media prepared by modifying the amino acid composition of these media, etc. should be used. Can do.

[0048] (Oral mucosal epithelial cells)

Oral mucosal epithelial cells include cells present in the root of the tooth (inner oral mucosal epithelial cells), lip cells, palate cells, buttocks cells, and the like. Of these, the intraoral mucosal epithelial cells are particularly preferred because of their high proliferative ability and low antigenicity. Oral mucosal epithelial cells can be collected by excising the area where the target cells are present with a scalpel or by cleaving. In the oral marginal mucosal epithelial cells, the oral mucosal epithelium adhering to the tooth extraction can be separated from the enamel cement transition and collected. In order to remove impurities such as connective tissue, treatment with enzymes such as dispase or trypsin or filter treatment is preferred!

[0049] (Intestinal mucosal epithelial cells)

Intestinal mucosal epithelial cells are collected from colonoscopic intestinal epithelial biopsy tissue or open surgery Sometimes collected in the usual way. In addition, epidermal cells can be excised from the tissue by lazer capture microdissection. The technique of the present invention is also applied to a biological tissue sheet produced using all human digestive tract epithelial cells of the esophagus, stomach, duodenum, small intestine, and large intestine. When human gastrointestinal epithelium is damaged by ulcers or inflammation, bone marrow-derived cells play a rescue role in response to emergency situations, and the epithelium is repaired. Gastrointestinal epithelial cells, though some, are made by bone marrow. In this sense, the significance of the present invention can be regarded as equivalent to that using corneal epithelial cells. The number of epithelial cells made from bone marrow, which is usually only about a few thousand, increases from 50 times to 100 times in the process of healing ulcers (wounds) inside the gastrointestinal tract due to gastric ulcer, colitis, etc. It has been found that 1 in 10 gastrointestinal epithelial cells are derived from the bone marrow. Gastrointestinal mucosal epithelial cell-derived tissue tissue prepared here is used for intractable ulcers and inflammation of enteric diseases such as severe intestinal infections, ulcerative colitis, Crohn's disease, Behcet's disease, etc. In the sense of promoting the regeneration of the intestinal epithelium, it is considered to be extremely meaningful. Expected to be useful for intestinal allergy

[0050] (Airway mucosal epithelial cells)

Airway mucosal epithelial cells are easily obtained from biopsy tissue of the airway mucosa, and in the same way as described above, treatment with an enzyme such as dispase trypsin or filtering is performed to remove impurities such as connective tissue. Is preferred. Airway mucosal epithelial cells play an important role in the pathogenesis of various infectious diseases through the biosynthesis and release of j8 defensin. The role of airway mucosal epithelium is high in asthma and allergic diseases! Providing a biological tissue sheet produced from the airway mucosal epithelial cells according to the present invention to the airway mucosa that has undergone tissue damage goes beyond the emergency response and leads to the provision of an artificial airway. In particular, the immunosuppressive action of the sheet prepared on the amniotic membrane is beneficial.

[0051] In particular, oral mucosal epithelial cells and intestinal mucosal epithelial cells are preferably treated with an enzyme such as dispase trypsin or filtered to remove impurities such as connective tissue after collection of the tissue. Better ,.

[0052] It is preferable that the living body-derived cells also have a person (recipient) ability to receive transplantation of the living tissue sheet. In other words, the donor of the living cell and the recipient of the living transplant sheet are the same A person is preferred. By using such autologous cells, the problem of immune rejection is solved.

[0053] The prepared biological cells are seeded on a collagen sheet and then subjected to culture (step (d)). Biological cells, such as cell density of about 1 X 10 ³ cells ZCM ² or more, preferably rather about 1 X 10 ³ cells / cm ² ~ about 1 X 10 ⁷ cells / cm ^2, more preferably about 1 X 10 ⁴ pieces / cm ² to about 1 × 10 ⁶ pieces Zcm ² is seeded on the amniotic membrane.

[0054] Here, two or more types of biological cells may be used in combination. In the present specification, the formation of a cell layer by two or more types of cells is also referred to as “hybridization”. Here, for convenience of explanation, one cell type used when constructing a hybrid cell layer is defined as a first cell, and a cell type different from this is defined as a second cell.

The types of cells used in the construction of the hybridized cell layer will be described in detail below, taking as an example the case where the biological tissue sheet of the present invention is prepared for corneal epithelial reconstruction. First, oral mucosal epithelial cells, conjunctival epithelial cells, nasal mucosal epithelial cells, other mucosal epithelial cells, or any one of these mucosal epithelia is used as one of the cell types (first cells) used to form the cell layer. An undifferentiated cell that can be constructed is preferably used. As a rule, autologous cells are used for the first cell. In the present specification, “self” refers to a subject to whom the biological tissue sheet of the present invention is applied, that is, a person (recipient) who receives a transplant. On the other hand, those other than “self” are called “others”.

On the other hand, corneal epithelial cells, conjunctival epithelial cells, or amniotic epithelial cells are preferably used as the cell type (second cell) used for forming the cell layer together with the first cells. These cells are collected from the living tissue in which they are present. Specifically, for example, a part of the tissue in which the target cells are present is collected with a scalpel, etc., and then processed into a cell suspension (suspension) after removal of connective tissue, separation of cells, etc. To do. Two or more different types of cells may be used as the first cell. Similarly, two or more different types of cells may be used for the second cell.

[0055] The presence of stem cells has been suggested in the oral mucosal epithelium suitable as a collection source for the first cells, and it is considered that differentiation induction into cells forming an epithelial cell layer is easy. In addition, using oral mucosal epithelial cells is easy to collect, can collect a large amount of cells, Has advantages such as the ability to prepare transplanted material using its own cells even when treating patients with binocular disease. In particular, the advantage that the transplant material derived from one's own cells can be applied to patients who cannot collect corneal epithelial cells is expected to greatly eliminate the clinically important rejection problem.

Oral mucosal epithelial cells collected from the oral cavity other than the patient to be transplanted with the sheet-like composition prepared according to the present invention can also be used, but considering immune rejection, oral mucosal epithelial cells from the patient's own oral cavity Is preferably collected and subjected to culture. The oral mucosa has a high proliferative capacity, and usually wounds are healed by taking antibiotics for a few days after surgery, disinfecting with isodine, and so on.

On the other hand, the corneal epithelial cells of another person (a mouth) can be preferably used as the second cells. Such corneal epithelial cells can be obtained from, for example, an infectious disease-free donor eyeball from an eye bank (Northwest Lions eye bank, etc.). Cells usable as the second cell are not limited to corneal epithelial cells, and conjunctival epithelial cells, amniotic epithelial cells, and the like may be used. However, if a corneal epithelial cell, which is a cell constituting the corneal epithelium in a living body, or a conjunctival epithelial cell existing in the vicinity thereof is employed, a sheet-like composition that better reproduces the characteristics of the corneal epithelium can be constructed. it is conceivable that. As a result of the study by the present inventors, it was confirmed that when a corneal epithelial cell was used as the second cell, a cell layer similar to the corneal epithelium could be constructed. This fact supports the above-mentioned expectation and confirms that corneal epithelial cells are particularly suitable as the second cells. On the other hand, even when amniotic epithelial cells are used as the second cells, it is possible to form a cell layer that well reproduces the characteristics required for the cornea. It has been certified. This fact indicates that amniotic epithelial cells can be suitably used as the second cells.

[0057] The ability to use own cells as the second cells If other cells are used, the cells can be obtained more easily. For example, even when a sheet-like composition is prepared for treatment of a binocular patient, corneal epithelial cells as second cells can be obtained.

[0058] The first cells and the second cells (hereinafter collectively referred to as "first cells etc.") prepared respectively are seeded on the amniotic membrane and subjected to culture. Usually, the first and second cells prepared in the form of a cell suspension are dropped onto the amniotic membrane and cultured.

Typically, the seeding of the first cell and the seeding of the second cell are performed at the same time (the term "simultaneous" here is literally the same time as well as placing a substantial time interval after one seeding. If the other cells are seeded at different timings, for example, the second cells are seeded after several minutes to several hours after the seeding of the first cells. Good. By shifting the seeding time in this way, for example, a region rich in cells derived from the first cell is localized and a cell layer is constructed, so that a homogeneous cell layer can be constructed. It is possible.

[0059] The ratio of the first cells and the like to be seeded is not particularly limited, but typically, approximately the same number of first cells and second cells are seeded. In the experiment using oral mucosal epithelial cells as the first cells and corneal epithelial cells as the second cells, the number of the first cells: the number of the second cells S3: 7, 5: 5, and Comparison of the cases of 7: 3 showed that there was no clear difference between them in terms of cell proliferation and stratification (data not shown).

[0060] 5. Step (d)

In this step, living body-derived cells are cultured and grown in the absence of heterologous animal cells. In the present invention, “in the absence of heterologous animal cells” means that animal cells that are heterologous to the living body-derived cells are not used as a condition for culturing the living body-derived cells. Specifically, when human cells (for example, human epidermal keratinocytes or human corneal epithelial cells) are used as living cells, cells of animal species other than humans, such as mice and rats, are present in the culture medium (coexisting) This is a condition that you do not want. By carrying out the culture under such conditions, There is no possibility that foreign-derived components (including heterologous cells themselves) will be mixed into the finally obtained transplantation material (ie, biological tissue sheet).

Culture of cells derived from living organisms can be performed by a conventional method. The medium used for culturing living body-derived cells is not particularly limited as long as the cells can be grown. For example, M CDB153 medium (Nissui Pharmaceutical Co., Ltd.), EpiLife ™ (Cascade), medium prepared by modifying the amino acid composition of these mediums, DMEM (D A medium in which HSM medium and DMEM medium are mixed at a predetermined ratio, such as a medium in which ulbecco's modified Eagle's medium) and Ham's F12 medium are mixed at a predetermined ratio, can be used. As serum added to the medium, human serum, fetal bovine serum, sheep serum, and the like can be used. Among them, it is preferable to use serum derived from the same species (human serum) or autologous serum (that is, the serum of the recipient itself). Of course, if possible, it is most preferable to use autologous serum that eliminates the risk of causing an immune rejection reaction. Human fibroblasts may be cultured using a medium supplemented with growth factors and antibiotics. By using growth factors and antibiotics, cell proliferation rate and survival rate can be improved and contamination can be prevented.

Biological cells may be cultured using a medium that is serum-free and does not contain a protein derived from a different animal. That is, a serum-free culture method may be employed as the culture method in the present invention. In such an embodiment, problems such as immune rejection due to contamination of serum-derived components can be avoided.

This can be done by changing the culture conditions in the middle of the culture step for the purpose of good growth of living cells.

As a result of step (d), living body-derived cells grow on the collagen sheet. When it is necessary to keratinize the surface layer of the cell layer thus obtained (for example, when a cultured skin sheet or a cultured epidermal sheet is prepared using epidermal keratinocytes, or corneal epithelium is used using corneal epithelial cells) In the case of producing a sheet), a step (step (e)) in which the surface layer of the cell layer is brought into contact with air is performed. This step is also referred to as air lifting in this specification. This step (e) is performed for the differentiation of the cells forming the cell layer and the induction of the barrier function. In this step, the surface of the culture solution is lowered by temporarily removing a part of the culture solution using a dropper, pipette, etc., thereby exposing the outermost layer of the cell layer to the outside of the culture solution. It can be carried out. Alternatively, the cell layer can be lifted together with the amniotic membrane, and the outermost layer can be temporarily exposed from the culture medium surface. Furthermore, air may be sent into the culture solution using a tube or the like, and air may be brought into contact with the uppermost layer of the cell layer. From the viewpoint of ease of operation, it is preferable to carry out the method by lowering the surface of the culture solution and exposing the outermost layer of the cell layer.

The time for performing this step (e), that is, the time for bringing the outermost layer of the cell layer into contact with air is a force that varies depending on the type of cell layer, the state of the cell, the culture conditions, etc. For the purpose of constructing an epidermal cell layer, it is preferably 5 days to 2 weeks, more preferably about 1 week. On the other hand, when it is intended to construct corneal epithelial cells, it is preferably 2 days to 1 week, more preferably 2 days to 4 days.

6. Use of second amniotic membrane

Here, it has been clarified by the present inventors that the epidermis keratinocytes proliferate well on the amniotic membrane and that the cell migration ability is well demonstrated (for details, see PCT ZJP2005Z2334). reference). That is, it is clear that amniotic membrane is extremely excellent as a culture substrate for epidermal keratinocytes. Considering this knowledge, two types of transplantation are performed: (1) Epidermal keratinocytes cultured on amniotic membrane placed on human fibroblasts are collected together with amniotic membrane, and then placed on another amniotic membrane. A sheet-like construct obtained by culturing (an amniotic membrane (first amniotic membrane) is adhered to one side of another amniotic membrane (second amniotic membrane), and a part of the first amniotic membrane is covered with the other one. In this part, a structure having a cell layer covering the second amniotic membrane) is transplanted into the epidermal defect part, and (2) epidermal keratinocytes cultured on the amniotic membrane placed on human fibroblasts are collected together with the amniotic membrane. However, it was considered that an excellent method for reconstructing the epidermis was to transplant it onto another amniotic membrane that had previously been transplanted into the skin defect. Here, it is very difficult to conserve epithelium in conservative treatments for defects up to the entire dermis and subcutaneous tissue, so it is necessary to prepare the transplant bed first. Conventionally, artificial dermis has been used for dermal defects, and a certain degree of therapeutic effect has been obtained. In the artificial dermis, vascular regeneration is observed, but when the cultured epidermis is transplanted on the artificial dermis, it is pointed out that engraftment is poor due to insufficient basement membrane composition. It is. On the other hand, if the transplantation method described in (1) or (2) above is used in combination with artificial dermis transplantation, the cultured epidermis is transplanted through the amniotic membrane having the basement membrane constituents. You can expect rates. In addition, since the cultured epidermis is formed on the amniotic membrane, after transplantation, the cells that make up the cultured epidermis are promoted to proliferate and migrate to the surrounding area, and as a result, the cultured epidermis can rapidly expand. A high therapeutic effect can be obtained. Therefore, it is expected that an excellent therapeutic method can be established even for skin defects extending to a wide range of subcutaneous tissues by combining artificial dermal transplantation with the above transplantation technique.

Based on the above findings, in one aspect of the present invention, after step (d) (cultivation step), (f) a step of collecting the living body-derived cells together with the amniotic membrane as the collagen sheet; A step of culturing and proliferating the living body-derived cell after the living body-derived cell and the amniotic membrane are placed on another amniotic membrane (second amniotic membrane) with the amnion side down is further performed. By performing step (D and step (g), another amniotic membrane (second amniotic membrane) is adhered to one side of the amniotic membrane, and the amniotic membrane is partly covered and the other part is the second amniotic membrane. A construct having a cell layer covering the amniotic membrane is obtained, wherein the entire surface of the second amniotic membrane is covered with the cell layer by appropriately adjusting the size of the second amniotic membrane, culture conditions, etc. And, a part of the surface of the second amniotic membrane is covered with a cell layer, so that one can obtain!

[0063] In step (£), the cells derived from the living body after the culture and the amniotic membrane used as the culture substrate are collected. For example, when living cells are cultured on an amniotic membrane placed in a culture dish, the proliferating living cells and amniotic membrane can be recovered by peeling off the amniotic membrane after culturing.

[0064] In step (g), the collected biological cells and the amniotic membrane are placed on another amniotic membrane, and then the biological cells are cultured again. Thus, in this embodiment, two-stage culture (step (d) and step)) is performed.

In step (g), first, a construct comprising the collected biological cells and amniotic membrane (hereinafter referred to as “cell-amniotic membrane construct”) is placed on a separately prepared amniotic membrane (second amniotic membrane) with the amnion side down. Placed. Multiple cell-amniotic constructs can also be placed on the second amniotic membrane. For example, the cell-amniotic construct recovered after culturing in step (d) can be duplicated by cutting. Several cell-amniotic constructs can be obtained. Alternatively, it is possible to prepare a plurality of cell-amniotic constructs by preparing a plurality of culture systems independent of each other and carrying out step (d) in parallel.

When a plurality of cell-amniotic constructs are placed on the second amniotic membrane, it is preferable to arrange the cell-amniotic constructs so that they are uniformly dispersed, that is, the intervals are constant. In subsequent cultures (and after transplantation into the living body), when the cells proliferate and the cell layer extends to the surroundings, the cell layer is quickly and efficiently removed from the area where the initial cell layer is not formed in the second amniotic membrane. It is because it can coat | cover with. That is, if the above method is adopted, a cell layer having a large surface area can be formed in a short time on the second amniotic membrane. In this way, a more efficient cell layer can be produced. On the other hand, before the cell layer covering the entire surface of the second amniotic membrane is formed, the cell layer together with the second amniotic membrane can be transplanted into the tissue defect part of the living body. Formation of the layer is promoted and a high therapeutic effect is obtained.

Since a cell layer having a large surface area can be obtained in a short time, the above method is particularly suitable for producing a cultured skin sheet (or a cultured skin sheet). If it is necessary to keratinize the surface layer of the cell layer, use the same method as above before step (1), between step (1) and step (g), or after step (g). Perform air lifting (step (e))

[0065] The culture in step (g) can be performed under the same conditions as the culture conditions in step (d). That is, it is preferable to use a medium that is serum-free and does not contain a protein derived from a heterologous animal as a medium that is preferably cultured in the absence of the heterologous animal cell. When using a medium containing serum, it is preferable to use autologous serum, the ability to use serum derived from the same species (human-derived serum when culturing human biological cells). Similarly to step (d), in step (g), the culture conditions may be changed during the culture step for the purpose of good growth of the cells derived from living organisms.

[0066] The biological tissue sheet of the present invention is used for regeneration (reconstruction) of skin epidermis, hair follicle epithelium, corneal epithelium, oral mucosal epithelium, intestinal mucosal epithelium, and airway mucosal epithelium. For example, the living tissue sheet of the present invention can be directly transplanted into a tissue defect part of a living body. "Nao The “grafting” means transplantation without substantially interposing another substance between the tissue defect and the biological tissue sheet. On the other hand, transplant the living tissue sheet of the present invention (except for those using the second amniotic membrane in the production process) into the tissue defect part of the living body so that other substances are interposed between them. You can also. For example, a biological tissue sheet can be transplanted into a tissue defect through an amniotic membrane (second amniotic membrane) different from the collagen sheet used as a culture substrate. Specifically, after transplanting amniotic membrane (second amniotic membrane) to the tissue defect part, a biological tissue sheet prepared in advance can be transplanted onto the amniotic membrane. According to such a transplantation technique, the presence of amniotic membrane as a base is expected to allow the cells contained in the biological tissue sheet to proliferate efficiently and migrate well to the surroundings. That is, rapid extension of the cell layer constituting the biological tissue sheet can be expected, and a high therapeutic effect can be obtained. On the other hand, the external defect is protected by covering the tissue defect with amniotic membrane. This also contributes to the improvement of the therapeutic effect.

[0067] Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1

[0068] Preparation of cultured skin sheet using amniotic membrane and human fibroblasts

1. Preparation of amniotic membrane

1-1. Collection of amniotic membrane

After a thorough informed consent was obtained with a gynecologist in advance for a pregnant woman scheduled for cesarean section with no systemic complications, amniotic membrane was collected at the cesarean section in the operating room. The operation was careful of cleanliness, and a special gown was worn after hand washing according to the surgical operation. Before delivery, a clean bat for collecting amnion and physiological saline for washing were prepared. After delivery, the placenta tissue was transferred to a vat and the amnion tissue was manually detached from the placenta. The adhesion between the amniotic membrane and the placenta was strong! The part was excised with scissors.

[0069] 1-2. Treatment of amniotic membrane

The amniotic treatment was performed in the order of (1) washing and chorionic membrane removal, (2) trimming, and (3) storage. In all processes, sterilize all containers and equipment that should be used in a clean fume hood and sterilize disposable dishes (dish, etc.). (Sportable) type was used. Remove blood components adhering to the collected amniotic membrane while rinsing with physiological saline, then rinsing with a sufficient amount of physiological saline, with the chorion facing upward, and visually form a pale white to white membrane. The observed chorion was detached. After this, the area near the placenta, which was uneven and opaque by hand touch and visual observation, was cut and removed with a scalpel. Next, the amniotic membrane was divided into sizes of about 4 × 4 cm using a scalpel. After division, amniotic membranes in good condition were selected based on shape and thickness. Subsequently, the selected amnion was washed a total of 2 times with 5 g / mL gentamicin-added phosphate buffer (PBS).

[0070] 1-3. Preservation of amniotic membrane

After collecting, washing and sorting the amniotic membrane into the preservation solution, place it in a 2 mL sterile cryotube containing 1 mL of preservation solution, label it one by one, and place it in a deep freezer at 80 ° C. saved. DMEM (Dulbecco's modified MEM medium, Invitrogen) containing 50% sterilized glycerol was used as the stock solution. The stored amniotic membrane was used for 3 months, and was incinerated when the expiration date passed. Note that the following epithelial processing may be performed without performing such storage processing.

[0071] 1 -4. Treatment of amniotic epithelium

— The amniotic membrane stored at 80 ° C was thawed at room temperature, and then washed twice with 5 g / mL gentamicin-added phosphate buffer (PBS). Immerse the washed amniotic membrane in a 0.02% EDTA solution (Cambrex) (100mm Petri dish), 37 ° C in a CO incubator under 5% CO conditions, 2-6

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Reacted for hours. After the reaction, the amniotic membrane was washed twice with a sufficient amount of PBS, and the epithelium was manually cleaved (removed) using a cell scraper (Nunc USA) under a stereomicroscope. The amniotic membrane with epithelial epithelium was placed in a 2 mL sterile cryotube supplemented with 1 mL of amniotic membrane preservation solution and stored at −80 ° C. in an ultra-low temperature freezer until use. In addition, it was confirmed by optical and electron microscopic operations (scanning electron microscope) that no more amniotic epithelial cells were removed by this treatment method (data not shown).

[0072] 2. Preparation of human epidermal keratinocytes

2- 1. Collection of skin

Pre-collection sites for about 3 days in a preventive manner with povidone After application, small skin pieces were collected according to skin biopsy.

[0073] 2- 2. Serum-free culture method of epidermal keratinocytes

Adipose tissue and dermis were removed as much as possible with scissors and washed several times with Dulbecco's phosphate buffer (PBS). Subsequently, it was sterilized by immersion in 70% ethanol for 1 minute. After washing with PBS, cut into strips 3mm wide and 10mm long and soaked in Dispase Solution (Dispase II, Godo Shusei, 250 units / ml Dulbecco's Modified MEM Medium; DMEM) at 4 ° C (18-24 hours). The next day, the epidermis was peeled from the dermis using tweezers. The peeled dermis is subjected to fibroblast culture (3. below). _{0 The} peeled epidermis is washed with DMEM, then washed with PB S, and then immersed in a 0.25% trypsin solution at 37 ° C for 10 minutes. A trypsin treatment was performed. The treated epidermis was transferred to a plastic petri dish containing a trypsin neutralizing solution, and then the epidermis pieces were loosened with tweezers. The resulting cell suspension was transferred to a 50 ml sterile tube. PBS was added to prepare the epidermal keratinocyte suspension. After counting the number of cells, the cells were centrifuged at lOOOOrpm for 5 minutes to precipitate the cells. After removing the supernatant by aspiration, the cells were suspended in MCDB153 medium, which is a serum-free medium. The cell suspension thus obtained was seeded at a rate of 2 to 3 × 10 ⁶ cells / ΙΟπύ culture medium per 100 mm collagen coated petri dish (Asahi Techno Glass, type I collagen coated dish; 4010-010). The culture solution was changed the next day, and thereafter the culture solution was changed every other day. Subculture was performed when the cell density reached about 70-80%.

[0074] 3. Preparation of human fibroblasts

2-2 The dermis peeled off in 2 was washed with DMEM, and then cut into pieces with a knife on one side of 1-2mm. Shredded dermis pieces were closely attached to a type I collagen coat dish at an interval of about 1 cm. The dish was left for 30 minutes in a CO incubator. This removes the dermis fragments.

2

It was in close contact with it. Thereafter, about 5 mL of DMEM medium containing 10% fetal calf serum was added to the dish, and then allowed to stand for 7 days. On the 7th day, the first culture broth was changed. We confirmed that fibroblasts migrated from the dermis. Passage was performed when the cells proliferated and migrated to 5 mm around the dermis. After washing with PBS, 3 mL of a solution containing 0.125% trypsin and 0.05% EDT A was added and treated at 37 ° C for 3 minutes. After confirming that the cells detached from the bottom of the dish with a microscope, 3 mL of trypsin inhibitor was added, and then the cells were collected in a 50 mL tube. The remaining cells were collected using PBS. 10 centrifuge tubes The cells were centrifuged at 00 rpm for 5 minutes to precipitate the cells, and the supernatant was removed by aspiration. DMEM medium containing 10% fetal bovine serum was added to the centrifuge tube to suspend the cells. The fibroblast suspension thus obtained was seeded in a cell culture dish. Subculture was performed when the cell density reached 90-100%. The cells were stored frozen as appropriate. The cryopreservation solution was stored in liquid nitrogen using 10% glycerol, 20% FCS, and 70% DMEM.

[0075] 4. Seeding of human fibroblasts on culture insert and placing amnion (attachment)

Collagen I coat 100 mm dish (or 60 mm date) from CO incubator

2

The fibroblasts cultured in (1) were taken out and confirmed to be confluent with a phase-contrast microscope, and then loaded into a safety cabinet. Remove medium in 100 mm dish (or 60 mm dish) containing fibroblasts, wash with PBS (-), add 0.05% trypsin-EDTA, 37 ° C, 5% CO Let sit for 2 minutes under. Phase contrast after 2 minutes

2

After confirming that the cells were detached with a microscope, trypsin reaction was stopped by adding DMEM supplemented with 10% FBS. The cell suspension was collected in a 15 mL tube. The supernatant was removed by centrifuging at 1000 rpm for 5 minutes at room temperature in a desktop multi-centrifuge. During centrifugation, 5 mL of DMEM supplemented with 10% FBS was injected into a 100 mm dish, and then a culture insert (manufactured by Coaster) was placed. A part of the cell suspension prepared by suspending the cell precipitate after centrifugation in DMEM supplemented with 10% FBS was mixed with an equal amount of 0.4% trypan blue, and the number of cells and the cell viability were measured with a hemocytometer. Based on the measurement results, the cell suspension was diluted with DMEM supplemented with 10% FBS to 2.1xl0 ⁵ cells / mL. 10 mL of diluted cell suspension was added to the culture insert. In order to adhere the cells, dish (37 ° C, 5% CO in a CO incubator)

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And culture insert) were allowed to stand for about 4 hours.

[0076] On the other hand, while the dish is left in the CO incubator, the amniotic membrane is treated as follows.

2

Prepared. First, the amniotic membrane (freeze-stored) prepared in 1 above was thawed at room temperature and transferred to a 100 mm diameter Petri dish. Subsequently, the amniotic membrane was washed in two 100 mm diameter Petri dishes containing PBS (-) containing 5 g / mL gentamicin, and then immersed in a 60 mm diameter Petri dish containing 10% FBS-added DMEM.

[0077] From the CO incubator, the dish seeded with human fibroblasts was taken out and the phase difference was removed.

2

Make sure that the cells adhere to the culture insert with a microscope, and then inside the safety cabinet. It was carried in. After removing the dish insert from the dish, place it in another container (eg, 100 mm diameter Petri dish), and add 10% FBS added to the amniotic membrane without DMEM. The amniotic membrane was placed in the culture insert (on the fibroblast layer). While observing the monitor under a stereomicroscope at a magnification of 7 to 16 times, use a hairless set so that air bubbles do not enter between the amniotic membrane and the culture insert and that no wrinkles occur in the amniotic membrane. Amnion was attached to the culture insert. After applying the amniotic membrane, a 0-ring was placed on the amniotic membrane using an insulative scissors to prevent the amniotic membrane from floating. Place the culture insert with amniotic membrane attached on a 100mm dish according to the above procedure, then inject 1 mL of 10% FBS-added DMEM into the culture insert, and then in a CO incubator at 37 ° C and 5% CO. Left to stand.

2

5. Seeding of human epidermal keratinocytes

On the 5th day after the amnion sticking operation, human epidermal keratinocytes were seeded on the amnion. First, the human epidermal keratinocytes prepared in 2. were peeled off using trypsin 'EDTA and collected. An epidermis keratinocyte suspension adjusted to a concentration of 2 million cells / 0.25 ml by centrifugation was obtained. The dish with the culture insert seeded with human fibroblasts and amnion was removed from the CO incubator and loaded into a safety cabinet. Kartya

2

The culture medium in one insert was removed, and the outside of the culture insert was removed leaving about 5 mL of culture medium. After 0.5 ml of epidermal keratinocyte suspension was seeded on the amniotic membrane in the culture insert, it was transferred into a CO incubator. 1.5 so that epidermal keratinocytes adhere to the amniotic membrane

2

It was allowed to stand in the incubator for ˜2.0 hours, and then 5 ml of the submerged culture medium was gently added to the inside of the culture insert and further 5 ml was added to the outside of the culture insert. The next day, the medium for submerging was gently added to the inside of the culture insert, and 5 ml was added to the outside of the culture insert.

After that, until the start of Air-lifting, in principle, observe with a phase contrast microscope once every two days, and replace the culture medium so that the culture medium for submerging is 15 mL inside the culture insert and 15 mL outside the culture insert. It was.

The submerged culture solution was prepared as follows.

Serum-free medium for keratinocytes (HSM medium) 3 doses plus 1 dose of DMEM Medium containing 0.3% tuss fetal serum

[0079] 6. Cultivation under gas phase

Air-lifting was performed about 2 days after seeding of epidermal keratinocytes. The culture medium inside and outside the culture insert was carefully removed, and 1.6 mL of the culture medium for stratification was added outside the culture insert. The culture medium was changed twice a day in the morning and evening. The cultured skin sheet was completed after 7 days of air exposure.

The culture solution for stratification was prepared as follows.

Serum-free medium for keratinocytes (HSM medium) 1 dose of DMEM plus 1% culture medium containing fetus serum

[0080] 7. Evaluation of cultured skin sheet

7- 1. Histological evaluation of cultured skin sheet (after transplantation)

The cultured skin sheet constructed by the above culture method was transplanted with the amnion side as the contact surface to the skin defect formed on the back of nude mice. On the 17th day after transplantation, a part of the tissue at the transplant site was excised to prepare a specimen and subjected to HE staining. In addition, frozen tissue sections were prepared and subjected to immunohistochemical staining. Immunohistochemical staining was performed using Chile's Histofine SAB-AP kit and Eufuxin was used as a substrate. The antibodies used for immunohistochemical staining are as follows. Monoclonal antibodies: E-cadherin (HECD-1, Tak ara), keratin 10 (LHP1, NeoMarkers), j8 4 integrin (3E1, Chemicon), α2 integrin (MAB1950, Chemicon), keratin 14 (LL002, NeoMarkers), laminin 5 (GB1, Sera-Lab), type IV collagen (2311C3, Chemicon), type VII collagen (LH7.2, NeoMar kers), polyclonal antibody; desmoglein 3 ( ⁵ G11, Zymed).

Fig. 3 shows the HE-stained image. The magnification is increased in the order of upper left, upper right, lower left, and lower right. As is clear from the HE-stained image, a skin layer of about 10 m is formed. From the results of HE staining, the grafts were well engrafted and the morphology of the epidermis was close to that of normal skin. That is, it consisted of a more compact stratum corneum than the upper layer, 1 to 2 granular layers, 5 to 8 spiny layers, and 1 basal layer. Strongly enlarged findings showed dense adhesion at the epidermis / dermis junction, and many images of fibroblasts entering the amnion. In addition, many luminal structures are seen in the dermis directly under the amniotic membrane, resulting in the formation of capillaries. It became clear that it was done actively.

From the above results, it was confirmed that if the cultured skin sheet prepared by the above method was used, the skin defect part could be reconstructed to a normal state in a short period of time, that is, the effectiveness of the cultured skin sheet prepared by the above method was confirmed.

[0081] On the other hand, as a result of immunohistochemical staining, laminin 5, type IV collagen, and type VII collagen, which are the main constituents of the basement membrane, are expressed in the epidermis / dermis junction, and the pattern is normal skin. And almost the same.

E-cadherin, a major component of epidermal cell junction, is expressed between cells in all layers, and oc 2 intedarin and j8 4 integrin are expressed between cells, mainly in the basal layer, and are distributed almost the same as normal skin. , Expression. Desmoglein 3, a constituent protein of desmosome, was expressed between cells from the basal layer to the parabasal layer, and the expression pattern was similar to that of normal skin. Keratin 10 was expressed from the parabasal layer to the upper spiny layer, and was expressed in the same manner as in normal skin. Keratin 14 was expressed in the basal layer and the parabasal layer, and the expression pattern was slightly different from the expression of only the basal layer in normal skin (Figs. 4 and 5).

[0082] As described above, it has been clarified that the cultured skin sheet produced by the above method has properties similar to normal epidermis and functions effectively as a skin reconstruction transplant material.

[0083] 7- 2. Relationship between seeding time of epidermal keratinocytes and quality of 3D cultured skin sheet

The cultured skin sheet that is finally constructed after seeding the epidermis keratinocytes, that is, the time until the epidermis keratinocytes are seeded after the amnion is placed (attached) on the monolayer fibroblasts The effect on the quality of food was examined. The outline of the experimental method is shown in the upper part of Fig. 6. In condition 1 (conditions for seeding epidermis keratinocytes after placing on the amniotic membrane, the experimental procedure on the upper left side of Fig. 6), the epidermal keratinocytes were removed 5 days after placing the amniotic membrane on fibroblasts. Sowing. On the other hand, in condition 2 (the condition in which epidermal keratinocytes are seeded without placing time after placing the amniotic membrane, the experimental procedure on the upper right side of FIG. 6), the epidermal keratinocytes immediately after placing the amniotic membrane on the fibroblasts. Sowing. In order to match the total culture time, amniotic membrane should be placed 4 hours after seeding of fibroblasts in condition 1, and amniotic membrane should be placed 5 days after seeding of fibroblasts in condition 2. I rubbed.

The constructed cultured skin sheets were each stained with HE and evaluated histologically. Condition 1 The HE-stained image of the cultured skin sheet constructed in Fig. 6 is shown in the lower left of Fig. 6, and the HE-stained image of the cultured skin sheet constructed in Condition 2 is shown in the lower right of the figure. In the cultured skin sheet constructed in condition 1, keratinocytes are more regularly aligned in the epidermal layer formed on the amniotic membrane than in the cultured skin sheet constructed in condition 2. In addition, the skin layer of the cultured skin sheet constructed under Condition 1 is thicker, and a gradual layer structure is clearly recognized. Furthermore, the flatness and denseness of the stratum corneum located at the uppermost part of the skin layer are also good. On the other hand, focusing on the amniotic membrane part used as the culture substrate, the cultured skin sheet constructed under condition 1 shows infiltration of many fibroblasts. Therefore, in the culture method under Condition 1, many fibroblasts infiltrate into the amniotic membrane before the seeding of the amniotic membrane placement force epidermal keratinocytes, and this causes growth of the epidermal keratinocytes. It is presumed that a high-quality skin layer was formed as a result of preparing a suitable environment.

As described above, after placing the amniotic membrane on the fibroblasts (affixing), securing sufficient time to promote infiltration of fibroblasts into the amniotic membrane is necessary for the production of high-quality cultured skin sheets. It turned out to be effective.

7- 3. Relationship between the time of placing (attaching) the amniotic membrane and the quality of the three-dimensional cultured skin sheet

The effect of the time of placing the amniotic membrane, that is, the time until the amniotic membrane was placed after the seeding of fibroblasts, on the quality of the finally constructed cultured skin sheet was examined. An outline of the experimental method is shown in the upper part of Fig. 7. Under condition 1 (conditions for disseminating fibroblasts, the time interval until placement of the amniotic membrane was short), the amniotic membrane was placed on the fibroblasts 4 hours after the seeding of fibroblasts. On the other hand, in condition 2 (the time interval from seeding of fibroblasts to placement of amniotic membrane was long), the amniotic membrane was placed on fibroblasts one day after seeding of fibroblasts. Subsequent culture conditions and operation procedures were the same.

The constructed cultured skin sheets were each stained with HE and evaluated histologically. The HE-stained image of the cultured skin sheet constructed in Condition 1 is shown in the lower left of Fig. 7, and the HE-stained image of the cultured skin sheet constructed in Condition 2 is shown in the lower right of the figure. The cultured skin sheet constructed under condition 2 has a thicker epithelial layer and a clear stratum corneum than the cultured skin sheet constructed under condition 1. Shikaso also has good flatness and denseness of the stratum corneum. On the other hand, when focusing on the amniotic membrane and fibroblasts used as the culture substrate, the amniotic membrane between conditions 1 and 2 Although the number of fibroblasts that infiltrate into the cells does not differ greatly, a fibroblast layer that uniformly covers the bottom of the amniotic membrane is observed in the cultured skin sheet constructed under condition 2. From this, under condition 2, after fibroblasts are seeded, a sufficient amount of time is applied to place the amniotic membrane for good adhesion of fibroblasts to the surface of the culture insert and fibroblasts. As a result, the growth promoting effect of fibroblasts through the amniotic membrane on epidermal keratinocytes was demonstrated well and uniformly, and high quality with an epidermis layer and a stratum corneum that approximated normal conditions. It is presumed that a cultured skin sheet was formed.

As described above, it was found that securing sufficient time after fibroblast seeding to promote adhesion and monolayer formation of fibroblasts was effective for the production of high-quality cultured skin sheets.

Example 2

[0085] Preparation of three-dimensional cultured corneal epithelial sheet using amniotic membrane and human fibroblasts

1. Preparation of amniotic membrane

The amniotic membrane from which the epithelium was removed was prepared in the same manner as in the case of producing a cultured skin sheet

[0086] 2. Preparation of human corneal epithelial cells

2- 1. Procurement of cornea

The cornea was purchased from Northwest Lions Eye Bank (Seattle, USA) with a grade equivalent to that for transplantation.

[0087] 2- 2. Serum-free culture method of corneal epithelial cells

(1) Single cell formation of corneal epithelial cells

The central part of the cornea was punched and removed with a 7.0 mm trepan blade, the conjunctiva was removed with a scissors under the stereomicroscope, the sclera was removed with a scalpel, and the substantial part was removed with a scissors. The corneal epithelial tissue is washed with PBS (-), then immersed in 1.2 U / mL Dispase solution (Roche, Dispasell, 2.4 U / mL), in a CO incubator under 5% CO condition at 37 ° C for 1 hour.

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, Let it stand. Subsequently, the corneal epithelial tissue was immersed in 0.02% EDTA-added PBS (-) and PBS (-) in this order for 2 minutes at room temperature. With a corneal epithelial tissue immersed in 2.4 mL of DMEM / F12 or HSM medium under a stereomicroscope, the epithelial cells are detached using a pestle and the cell mass A corneal epithelial cell suspension containing a large amount of was prepared. The corneal epithelial cell suspension was divided into two aliquots of 1.2 mL, and 0.3 mL of 0.25% trypsin EDTA (Invitrogen) was added to each suspension and mixed.

In a CO incubator under 5% CO condition, leave at 37 ° C for about 10 minutes, and under 40x magnification

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While observing with a phase contrast microscope, pipetting was performed with a micropipettor until the cell mass disappeared. The corneal epithelial cell suspension prepared in this manner was transferred to a 15 mL centrifuge tube. After suspension, a part of the cell suspension was mixed with an equal volume of 0.4% trypan blue, and the cell viability was measured with a hemocytometer. The 15 mL centrifuge tube containing the cell suspension was centrifuged at 1000 rpm for 5 minutes at room temperature using a desktop multi-centrifuge, and the supernatant was removed. The HSM medium was added to the tube after centrifugation so that the total volume was 1.0 mL, and the cells were suspended. CO 2 incubator at 37 ° C, 5% CO.

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Static culture was performed in one.

(2) Cell culture and passage

Every day after the start of culture, the cell growth rate and cell morphology of the cells were observed using a phase contrast microscope under a magnification of 100 times. Once grown to approximately 70% subconfluent, the cells were passaged as follows. The subculture was repeated until the number of cells necessary for production of the cultured corneal epithelial sheet was obtained.

(a) Collagen-coated dish force also removed the culture solution.

(b) 2 mL of PBS (-) was added to the collagen coat dish and then removed (washed).

(c) Add 0.05 mL trypsin-EDTA (1 mL), and in a CO incubator, the cells

2

It was allowed to stand until it became spherical (10 minutes), and a cell suspension was prepared by pipetting with a micropipette.

(d) The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant.

(e) The HSM culture solution was placed in a centrifuge tube to make 1.0 mL, and the cells were suspended.

(f) A part of the cell suspension was mixed with an equal amount of 0.4% trypan blue, and the cell count and cell viability were measured with a hemocytometer.

(g) The whole cell suspension was seeded on a 60 mm or 100 mm collagen-coated dish and statically cultured in a CO incubator under conditions of 37 ° C and 5% CO. [0089] 3. Preparation of human fibroblasts

Human fibroblasts were prepared in the same manner as in the production of cultured skin sheets.

[0090] 4. Seeding of human fibroblasts on culture insert and placing amnion (attachment)

Human fibroblasts were seeded on the culture insert and amnion was placed in the same manner as in the production of the cultured skin sheet. However, a 6-well plate was used in place of the 100 mm dish, and a culture insert (Coaster Co., Ltd.) suitable for the 6-well plate was used.

[0091] 5. Seeding of human corneal epithelial cells

Corneal epithelial cells were seeded on the amniotic membrane 5 days after the amnion sticking operation. First, the corneal epithelial cells prepared in 2 were observed with a phase contrast microscope, and it was confirmed that they were about 70-80% subconfluent. The culture solution was removed, washed with PBS (−), added with 2 mL of 0.05% trypsin-EDTA, and allowed to stand at 37 ° C., 5% CO for 5 minutes. Then trypsin inhibitor

2

1 mL was added, and the cell suspension was pipetted and collected in a 50 mL centrifuge tube. The 50 mL centrifuge tube containing the cell suspension was centrifuged at 1000 rpm for 5 minutes at room temperature in a desktop multi-centrifuge. After centrifugation, the supernatant was removed, and 10 mL of submerged A culture solution was added. The cell suspension obtained by pipetting well until the cell mass disappeared was transferred to a 15 mL centrifuge tube. A part of the cell suspension was mixed with an equal amount of 0.4% trypan blue, and the cell count and cell viability were measured with a hemocytometer. The 15 mL centrifuge tube containing the cell suspension was centrifuged at 1000 rpm for 5 minutes at room temperature using a desktop multi-centrifuge. After centrifugation, the supernatant was removed, and the submerged A culture solution was added to the centrifuge tube so that the cell count was 1 × 10 ³ cells / l based on the cell count results, and the cells were suspended.

[0092] The 6-well plate on which the culture insert on which human fibroblasts were seeded and amnion was affixed was taken out of the CO incubator and carried into a safety cabinet.

2

The culture medium inside and outside the culture insert was removed, and about 1 mL of submerged A culture medium was added outside the culture insert. Inoculate the corneal epithelial cell suspension prepared at 1 X 10 ³ cells / l on the amniotic membrane in the culture insert, and use the submerged A medium so that the volume of the solution in the culture insert is 1 mL. I made a mess up. A 6-well plate on which a cell-seeded cell insert is placed is loaded into a CO incubator, 37 ° C, 5% Static culture was performed under CO conditions.

2

Thereafter, until the start of Air-lifting, 1 every two days in principle, performs observation by phase contrast microscope, so that by submerged for A broth culture insert within 1 mL, and culture I concert outside _{2 m} L The culture medium was replaced.

A submerged A culture was prepared as follows. HSM medium: Dulbecco's modified MEM medium = 3: 1, FCS; 0.3%, 2.5 μg / mL gentamicin

[0093] 6. Submerged culture

About 10 days after seeding of corneal epithelial cells, air-lifting was performed. The culture medium outside and inside the culture insert was carefully removed, and 1.6 mL of the layer A culture medium was added outside the culture insert. The culture medium was changed twice a day in the morning and evening, and the cultured corneal epithelial sheet was completed after 3 days of air exposure.

The A culture solution for stratification was prepared as follows. HSM medium: Dulbecco's modified MEM medium = 1: 1, FCS; 2%, 2.5 μg / mL gentamicin

[0094] 7. Evaluation of three-dimensional cultured corneal epithelial sheet

7- 1. Photography with a stereomicroscope

Remove the 6 well plate containing the cultured corneal epithelial sheet from the CO incubator.

2

After observing the degree of stratification and cell morphology with a phase contrast microscope, an external photograph was taken with a stereomicroscope. Observations using phase contrast and a stereomicroscope did not confirm any noticeable changes such as cell loss.

[0095] 7— 2. Sheet division and tissue embedding

Next, the inner and bottom surfaces of the culture insert were washed with PBS (-) containing 5 g / mL gentamicin, and the culture insert was placed on a silicone sheet in a 100 mm diameter Petri dish with the culture insert mark on top. The insert was placed and the sheet was punched out with an 18 mm trepan. It was placed on a 9mm graph paper (OHP) with a silicone mat, taking care not to lose power in the top, bottom, left and right. The top, bottom, left, and right edges were on the grid line, and the sheet was cut with a force razor along the line passing through the center, and divided into four equal parts. Two sheets on the diagonal of this equally divided sheet were embedded using OCT compound.

[0096] 7- 3. Number of cells · Measurement of cell viability 7-2 Divide the two sheets that were left unembedded, and immerse them in a 35 mm dish in a mixture of 1.5 mL of PBS (-) and 1.5 mL of 1.2 U / mL dispase. Then, it was allowed to stand in a CO incubator at 37 ° C for 30 minutes under 5% CO conditions. 30 minutes later

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The corneal epithelial cell layer was peeled off from the amniotic membrane in a sheet form, immersed in 2.5 mL of 0.05% trypsin-EDTA solution, and allowed to stand in a CO incubator under 5% CO conditions at 37 ° C for 5 minutes. So

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After that, the cell suspension suspended by pipetting and converted into a single cell was transferred to a 15 mL centrifuge tube. The supernatant was removed by centrifugation at 1500 rpm for 5 minutes at room temperature in a table-top multi-centrifuge. SHEM was placed in a centrifuge tube so that the total volume was 1.0 mL, and the cells were suspended. A portion of the cell suspension was taken and mixed with an equal volume of 0.4% trypan blue, and the cell count and cell viability were measured with a hemocytometer. The number of cells per cell and the survival rate of these sheets were 1.4 xlO ⁶ cells and 96.8%, respectively.

7-4. Verification of histological characteristics of cultured corneal epithelial sheet

(1) HE staining

The cultured corneal epithelial sheet embedded in 7-2 above was sliced with a cryostat to prepare slide sections. This slide section was subjected to HE staining as described below. The following operations were performed at room temperature.

First, the slide sections were fixed with mild form for 5 minutes, and then washed with running water for about 10 minutes. After washing, the cells were stained with hematoxylin for 5 to 10 seconds and washed with running water for 5 to 10 minutes. Next, it was stained with eosin for 3 to 5 seconds and washed with running water for 5 to 15 minutes. After washing, dehydration was performed for several seconds in order of 70, 90, 95%, and absolute ethanol. After dehydration with absolute ethanol, it was immersed in xylene for 5 minutes and then immersed in fresh xylene for 30 minutes or more. After that, the mounting medium (Yanteran-Yu) was dropped, the cover glass was placed, the slide was sealed, and when the mounting medium was dry, the coating was applied around the cover glass. When the cure was dry, it was observed with an optical microscope.

As can be seen in the HE-stained image power, the prepared cultured corneal epithelial sheet had an epithelial layer stratified into 4-6 layers with a thickness of about 50 m (Fig. 8). On the basal side (amniotic membrane side) of this epithelial layer, a group of cells similar to basal cells having a comparative columnar shape existed. In addition, the outermost cell has a flat core that is flat, and unlike the skin, its surface is not keratinized. Was confirmed. From the above, it was shown by optical microscope observation that an epithelial layer similar to normal cornea was formed on the amniotic membrane.

[0098] (2) Confirmation of cornea-specific protein expression by immunostaining

Immunostaining was performed to examine the immunohistological characteristics of the prepared cultured corneal epithelial sheet.

That is, keratin-specific keratins 3 and 12 were examined. The cultured corneal epithelial sheet embedded in 7-2 above was sliced with a cryostat to prepare a slide section. The slide sections were subjected to immunostaining as described below. First, the slide sections were washed with PBS (−) and then blocked with 1% BSA to suppress nonspecific antibody reaction. Thereafter, an antibody against each keratin (primary antibody) was reacted at room temperature for 1 hour. After the reaction, it was washed with a blocking solution containing Triton X-100 for 15 minutes under the conditions of 3 times, and then a fluorescently labeled antibody (secondary antibody) was reacted at room temperature for 1 hour. After the reaction, the plate was washed with PBS (-) for 15 minutes under three conditions, PI was added dropwise, sealed with a slide glass, and the tissue was observed with a confocal or fluorescent microscope. The antibodies used are as follows. Primary antibody: Keratin 3 (cytokeratin3 / 12, PROGEN ゝ Cat.No.61807), Keratin 12 (Anti—Keratinl2, Transgenic Inc ゝ Cat.No. KR074) Secondary antibody: Alexa488 anti mouse (Molecular probes ゝ Cat.No. .A- 11029, used for keratin 3), Alexa488 anti rabbit (Molecular probes, Cat. No. A-11034, used for keratin 12)

As a result of immunostaining, the expression of keratin 3 and keratin 12 was confirmed in all layers of the cultured corneal epithelial sheet (FIG. 9). This expression was almost the same as that of normal corneal epithelial tissue.

Example 3

[0099] Preparation of biological tissue sheet using trehalose-treated amniotic membrane

1. Trehalose treatment 'Preparation of freeze-dried amniotic membrane

The amnion from which the epithelium was removed, prepared by the procedure described in 1. of Example 1, was immersed in a 10% (w / v) trehalose solution at 37 ° C. for 2 hours. The trehalose solution was prepared by diluting trehalose (Toreno Life Co., Ltd., Life Plus, Life Sciences, Hayashibara) with distilled water. The pH of the solution was kept in the range of 7-10. The amniotic membrane was clamped with a pair of sterilized plastic frames and then fixed with clips. 80 ° C deep free per frame After confirming that the amniotic membrane was frozen, freeze drying (-110 ° C, about 1 hour) was performed using a vacuum freeze dryer (Yamato, NEOCO OL). Conditions were set according to the instruction manual so that a sufficiently dry product could be obtained. The amniotic membrane after freeze-drying was removed from the frame force, transferred to a two-layered bag with polyamide nylon on the outside and polyethylene on the inside, and vacuum packed using a household vacuum pack device (frame nova, magic pack). Thus the amnion vacuum packed state obtained by by irradiating _Ί line (approximately 25 kGy) and sterilized. The amnion after sterilization was stored at room temperature in a vacuum packed state until just before use. In addition, the storage starting power was maintained at the state immediately after freeze-drying even after 12 months.

[0100] 2. Production of biological tissue sheet

(1) Cultured skin sheet

A cultured skin sheet is obtained in the same manner as in 2. to 6. of Example 1 except that the trehalose-treated lyophilized amniotic membrane obtained in 1. is used as the amniotic membrane used for the culture substrate.

(2) Cultured corneal epithelial sheet

A cultured corneal epithelial sheet is obtained in the same manner as in 2. to 6. of Example 2, except that the trehalose-treated lyophilized amniotic membrane obtained in 1. is used as the amniotic membrane used as the culture substrate.

Industrial applicability

[0101] The use of the biological tissue sheet provided by the present invention can be used for regeneration (reconstruction) of wide skin epidermis, corneal epithelium, oral mucosal epithelium, airway mucosal epithelium and intestinal mucosal epithelium. Among these, it can be suitably used for regeneration of the skin epidermis or corneal epithelium.

In addition, the biological tissue sheet of the present invention can be used for gene therapy. There are two types of gene therapy, in vivo and ex vivo. The former is a method in which a gene is directly introduced into a living body, and the latter is a method in which a cell is taken out and the gene is introduced and then returned to the body. In view of the current state of the art of gene therapy, an ex vivo method is immediately developed as long as a gene is introduced into cultured skin, cultured corneal epithelium, or cultured intestinal mucosal epithelial sheet. The effectiveness of gene transfer into keratinocytes by various viral vectors has been shown, and in particular, when an adenovirus vector is used, it can be introduced into almost 100% of keratinocytes. In gene therapy in the dermatological field, abnormalities such as type VII collagen and laminin 5 For the treatment of genetic diseases such as congenital epidermolysis bullosa, the introduction of normal genes into autologous cultured epidermal sheets is expected to be an extremely effective treatment. In addition, there is a possibility of cultured skin as a so-called delivery system that introduces genes into keratinocytes to produce and supplement deficient molecules in systemic diseases caused by deficiencies in the body's molecules such as diabetes and hemophilia. It is expected that more and more applications of cultured biological tissue will be developed.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

The contents of papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

Claims

The scope of the claims

[I] A biological tissue sheet containing biological cells grown on a collagen sheet placed on human fibroblasts.

[2] The living tissue sheet according to claim 1, wherein the collagen sheet is amniotic membrane.

3. The biological tissue sheet according to claim 2, wherein the amniotic membrane is an amniotic membrane from which the epithelium has been removed.

4. The biological tissue sheet according to claim 2 or 3, wherein the amniotic membrane is an amniotic membrane to which trehalose is added.

[5] The biologically derived cells are grown using a serum-free medium.

The biological tissue sheet according to any one of 4 to 4.

[6] The living tissue sheet according to any one of claims 1 to 4, wherein the living body-derived cells are grown using a medium containing only serum derived from a recipient as a serum component.

[7] The cell according to any one of claims 1 to 6, wherein the living body-derived cell is a cell derived from a corneal epithelium, a conjunctival epithelium, a skin epidermis, a hair follicle epithelium, an oral mucosa, an airway mucosa, or an intestinal mucosa. Living tissue sheet.

[8] The collagen sheet according to any one of claims 1 to 7, further comprising the collagen sheet infiltrated with the cells derived from the human fibroblasts in addition to the cells derived from the living body. Biological tissue sheet.

[9] The biological tissue sheet according to any one of [1] to [8], further comprising a multilayered cell layer formed by the proliferated cells derived from the living body.

[10] A biological tissue sheet in which a biological cell layer is formed on an amniotic membrane infiltrated with human fibroblasts.

[II] A method for producing a biological tissue sheet comprising the following steps:

(a) providing human fibroblasts;

(b) a step of placing a collagen sheet on the human fibroblasts seeded in a culture vessel; (c) preparing a living body-derived cell and seeding the living body-derived cell on the collagen sheet;

(d) A step of culturing and proliferating the living body-derived cells in the absence of heterologous animal cells.

12. The production method according to claim 11, wherein in the step (b), the collagen sheet is placed after a predetermined time has elapsed since the seeding of the human fibroblasts.

[13] The manufacturing method according to claim 11 or 12, wherein the following steps are performed between step (b) and step (c):

(b) 'culturing the human fibroblast for a predetermined time.

[14] The production method according to any one of claims 11 to 13, further comprising the following steps:

(e) A step of bringing the outermost layer into contact with air after the biological cells are grown.

[15] The production method according to any one of [11] to [14], wherein the collagen sheet is amniotic membrane.

16. The production method according to claim 15, wherein the amniotic membrane is an amniotic membrane from which an epithelium has been removed.

17. The production method according to claim 15 or 16, wherein the amniotic membrane is an amniotic membrane added with trehalose.

[18] The step (d) is performed using a serum-free medium.

The production method according to any one of ˜17.

[19] The method according to any one of claims 11 to 17, wherein the step (d) is performed using a medium containing only serum derived from a recipient as a serum component. Law.

[20] The cell according to any one of claims 11 to 19, wherein the biological cell is a cell derived from a corneal epithelium, a conjunctival epithelium, a skin epidermis, a hair follicle epithelium, an oral mucosa, an airway mucosa, or an intestinal mucosa. Manufacturing method.

[21] A transplantation method using the biological tissue sheet of any one of claims 1 to 10 as a transplant material.