Cell composition for treatment of uterine tissue and method for producing same

Abstract

An object of the present invention is to provide a cell composition for treatment of uterine tissue that heals damage occurring in uterine tissue, including intrauterine adhesion, to a state that enables pregnancy. In addition, an object of the present invention is to provide a method for producing this cell composition for treatment of uterine tissue, and a cell composition for treatment of uterine tissue produced according to that method. The cell composition, which has a first cell layer containing epithelial cells and a second cell layer containing stromal cells, and the first cell layer is laminated on the second cell layer, has the ability to heal damage occurring in uterine tissue, including intrauterine adhesion, and restore it to a state that enables pregnancy.

Claims

1. A method for producing a cell composition for treatment of uterine tissue, the method comprising: (1) culturing a cell group containing epithelial cells on a first cell culture support to obtain a first cell layer, and then recovering the first cell layer from the first cell culture support; (2) culturing a cell group containing stromal cells on a second cell culture support to obtain a second cell layer, and then recovering the second cell layer from the second culture support; and (3) laminating the first cell layer on the second cell layer, wherein: the first cell culture support and the second cell culture support have a polymer that undergoes a change in hydration force over a temperature range of 0 C. to 80 C. coated on the surface thereof; and the epithelial cells are derived from uterine tissue.

2. The production method according to claim 1, wherein the first cell culture support is a porous cell culture insert.

3. The production method according to claim 1, wherein the epithelial cells are epithelial cells contained in a cell group obtained by: (1) a cell separation step for mincing endometrial tissue followed by treating with a cell separation enzyme to obtain an endometrial tissue cell group, (2) a culturing step for seeding the endometrial tissue cell group on a cell culture vessel and culturing for 30 minutes to 4 hours, and (3) a step for collecting cells that do not adhere to the cell culture vessel after the culturing step.

4. The production method according to claim 1, wherein the stromal cells are endometrial stromal cells.

5. The production method according to claim 1, wherein the stromal cells are endometrial stromal cells contained in a cell group obtained by: (1) a cell separation step for mincing endometrial tissue followed by treating with a cell separation enzyme to obtain an endometrial tissue cell group, (2) a culturing step for seeding the endometrial tissue cell group on a cell culture vessel and culturing for 30 minutes to 4 hours, and (3) a step for collecting the cells that adhere to the cell culture Vessel after the culturing step.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a drawing indicating the procedure for fabricating an endometrial cell sheet of the present invention.

(2) FIG. 2 indicates tissue sections of an endometrial cell sheet obtained in Example 1. FIG. 2(A) indicates an image obtained by HE staining of the tissue section. FIG. 2(B) indicates an image obtained by immunostaining of the tissue section with a fluorescent-labeled antibody. Red represents vimentin, green represents CK18 and blue represents the location of the cell nucleus.

(3) FIG. 3 indicates images of the uterine tissue of nude rats transplanted with an endometrial cell sheet fabricated in Example 2. The two upper left images indicate the uterus of a normal nude rat, the lower images and upper right image indicate the uterus of a nude rat obtained by transplantation of an endometrial cell sheet into an endometrium full-thickness defect nude rat model, and the tissue sections were stained by HE staining and GFP staining. Arrows indicate the structure of reconstructed endometrial glands.

(4) FIG. 4 is a drawing indicating the flow of an experiment for evaluating pregnancy in the present invention.

(5) FIG. 5 indicates the fertility restorative effect of an endometrial cell sheet of Example 3. FIG. 5(A) depicts the pregnant uterus of a nude rat transplanted the endometrial cell sheet. The endometrial cell sheet was transplanted after causing a full-thickness defect in the endometrium of one of the horns of a bicornuate uterus. The GFP-positive area indicates the transplanted cell sheet, and the gestational sac was able to be confirmed to have been formed at that site. FIG. 5(B) depicts the status of the fetus as determined by ultrasonography. FIG. 5(C) depicts the uterus of an individual not transplanted with a cell sheet after causing a full-thickness defect in the endometrium of one of the horns of a bicornuate uterus.

(6) FIG. 6 indicates cross-sections of pregnant uteri and fetuses of nude rats transplanted with an endometrial cell sheet of Example 3. The three images indicate the respective uterine cross-section of each individual. The areas emitting green fluorescence are the transplanted endometrial cell sheets. Arrows indicate fetuses.

(7) FIG. 7 indicates tissue sections of the uterus and fetus of a nude rat transplanted with an endometrial cell sheet of Example 3. FIG. 7(A) depicts a tissue cross-section of the uterus and fetus following HE staining. FIG. 7(B) depicts the cross-section following GFP and -SMA immunostaining. The GFP-positive sites indicate the transplanted endometrial cell sheet.

(8) FIG. 8 indicates uterine tissue sections of a nude rat transplanted with an endometrial cell sheet of Example 2. The upper row indicates images obtained by HE staining, while the lower row indicates images obtained by GFP immunostaining. FIG. 8(A) depicts cross-sections of the uterus. FIG. 8(B) depicts partially enlarged views of FIG. 8(A). FIG. 8(C) depicts a site of the endometrium where the cell sheet was transplanted while enlarging a site that is not pregnant.

(9) FIG. 9 indicates the fertility restorative effect of transplanting an endometrial cell sheet of Example 4. FIG. 9(A) depicts the uterus of an SD rat transplanted with the endometrial cell sheet. FIG. 9(B) depicts an image observed following irradiation of the same uterus as (A) with a light source that excites GFP. The site emitting green fluorescence is the location of the transplanted cell sheet. FIG. (C) depicts an image observed during ultrasonography of the uterus on the side transplanted with the cell sheet.

(10) FIG. 10 indicates uterine tissue of a nude rat transplanted with an endometrial cell sheet of Comparative Example 1. FIG. 10(A) depicts an image obtained by HE staining of a uterine tissue section. FIG. 10(B) depicts a horizontal cross-section following GFP immunostaining. The green color indicates the transplanted cell sheet. The blue color indicates the cell nucleus. FIG. 10(C) depicts a coronal cross-section following GFP immunostaining.

(11) FIG. 11 indicates uterine tissue of a nude rat transplanted with an endometrial cell sheet (stromal sheet) of Comparative Example 2. A silicone tube was placed to prevent adhesion of the uterine cavity. FIG. 11(A) depicts the transplanted cell sheet (green fluorescent area). The area indicated with the black circle indicates the silicone tube. FIG. 11(B) depicts the silicone tube immobilized with sutures (arrow).

(12) FIG. 12 indicates the uterine tissue of a nude rat transplanted with an endometrial cell sheet (stromal sheet) of Comparative Example 2. FIG. 12(A) depicts a tissue section following HE staining. In FIG. 12(B), green color indicates the transplanted cell sheet, red color indicates smooth muscle stained with -SMA antibody, and blue color indicates the cell nucleus. In FIG. 12(C), red color indicates progesterone receptors and blue color indicates the cell nucleus.

BEST MODE FOR CARRYING OUT THE INVENTION

(13) The present invention relates to a cell composition for treatment of uterine tissue. In the present invention, a cell composition includes all compositions that contain cells. For example, the cell composition may be a composition to which has been added cells and a protein that composes an extracellular matrix, or may be a composition that contains cells and protein that composes an extracellular matrix produced by the cells, and there are no particular limitations thereon. In addition, the protein that composes the extracellular matrix may be a recombinant protein, or a protein produced from cells incorporated with a gene that encodes that protein or cells that have been transfected with that gene by a vector and the like, and there are no particular limitations thereon.

(14) Uterine tissue refers to tissue of the female reproductive organ of a mammal that serves as an organ for containing a fetus when the fetus develops in the body during pregnancy. In mammals and in humans in particular, uterine tissue has a structure that forms a box-like cavity. The formation of fertilized eggs and the development of the fetus take place in the uterine body. The uterine body is composed of the endometrium, myometrium and parametrium.

(15) The endometrium can be classified into two components based on its tissue structure, and can be divided into the endometrial epithelial layer and endometrial stromal layer. The endometrial epithelial layer is composed of single layer columnar cells, contains secretory cells and ciliated cells, and constitutes the cell layer closest to the uterine cavity. The endometrial stromal layer is a structure that has a large number of endometrial glands. The entrance to the endometrial glands is connected to epithelium facing the uterine cavity, and the innermost portion extends to the deepest portion of the endometrium. Mucus is secreted from the endometrial glands, covers the surface of the endometrium, and assists in the development of fertilized eggs and embryos.

(16) The endometrium can be classified into two layers according to function. These two layers consist of the basal layer, which is located in the deeper portion of the endometrium that does not undergo a change during the estrus cycle, and the functional layer, which undergoes changes in thickness and structure during the estrus cycle and contains the endometrial glands and the coiled artery.

(17) The human endometrium is dependent on female hormones in the form of estrogen and progesterone, and undergoes changes in thickness corresponding to the estrus cycle. Estrogen levels increase during the period from the end of menstruation to ovulation, resulting in proliferation of the endometrium, and particularly the functional layer (proliferative phase). Once ovulation has ended, although estrogen levels temporarily decrease, estrogen levels and progesterone levels subsequently increase again resulting in thickening of the endometrium (secretory phase). At this time, the uterine glands of the endometrium develop and secretion is promoted resulting in the occurrence of edema of the endometrial stromal layer. An environment that is suitable for implantation of a fertilized egg is created in this manner. Subsequently, the menstrual period repeats on a 28 day cycle and exfoliation of the functional layer occurs. At this time, both estrogen and progesterone levels decrease. This thickening and exfoliation of the endometrium according to the estrus cycle fulfills an important role in women for a normal pregnancy.

(18) The cell composition for treatment of uterine tissue of the present invention is a cell composition that has a first cell layer containing epithelial cells and a second cell layer containing stromal cells, and the first cell layer is laminated on the second cell layer. Although epithelial cells include epithelium that covers the body surface, epithelium that composes the mucous membranes of hollow organs, acinar cells that compose exocrine glands and glandular cells that compose endocrine glands, the epithelial cells used in the present invention are preferably cells that compose mucous membranes. Examples thereof include epithelial cells contained in oral mucosa, nasal mucosa, esophageal mucosa and uterine mucosa (endometrium). In particular, epithelial cells derived from uterine tissue are preferable, while the use of epithelial cells derived from the endometrium is more preferable, since they are able to prevent adhesion caused by curettage of uterine tissue and restore uterine tissue to that which undergoes changes corresponding to the estrus cycle or restores uterine tissue to a stage that enables pregnancy. In addition, these epithelial cells may be a single type or may be a mixture of a plurality of types of cells, and there are no particular limitations thereon. In addition, they may also be epithelial cells derived from pluripotent stem cells such as ES cells, iPS cells or Muse cells. The proportion of epithelial cells contained in the first cell layer is 60% or more, preferably 70% or more, more preferably 80% or more and even more preferably 90% or more (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). The first cell layer may also contain cells other than epithelial cells, and there are no particular limitations on the types of those cells.

(19) Stromal cells refer to cells that compose the supporting tissue of epithelial cells, and include fibroblasts, vascular endothelial cells, smooth muscle cells and uterine stromal cells. Stromal cells are involved in inflammatory reactions and wound healing reactions, and fulfill an important role in maintaining normal tissue. In the present invention, stromal cells preferably include uterine stromal cells since they allow the obtaining of the effect of preventing adhesion caused by curettage of uterine tissue and restoration of uterine tissue to that which undergoes changes corresponding to the estrus cycle or restoration of uterine tissue to a state that enables pregnancy. In addition, the stromal cells may also be derived from pluripotent stem cells such as ES cells, iPS cells or Muse cells. The proportion of epithelial cells contained in the second cell layer is 60% or more, preferably 70% or more and more preferably 80% or more (such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%). The second cell layer may also contain cells other than stromal cells, and there are no particular limitations on the types of those cells. In addition, these epithelial cells may be cells of a single type or may be a mixture of a plurality of types of cells.

(20) There are no particular restrictions on the origin of the animal species of the cells used in the present invention, and examples thereof include humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees and immunodeficient types thereof. It is preferable to use cells derived from humans in the case of using the cell composition of the present invention for the treatment of humans, it is preferable to use cells derived from pigs in the case of using for the treatment of pigs, it is preferable to use cells derived from monkeys in the case of using for the treatment of monkeys, and it is preferable to use cells derived from chimpanzees in the case of using for the treatment of chimpanzees. In addition, in the case the patient undergoing treatment is a human, the cells may be collected from the patient (autologous transplantation), cells may be used that have been collected from another person (allogenic transplantation), or a commercially available cell line may be used.

(21) The cell layers of the present invention refer to a cell group in the form of a sheet composed of one layer or a plurality of layers (such as 2 to 6 layers) obtained by culturing on a cell culture vessel. Although there are no particular limitations on the method used to obtain the cell layers, examples thereof include a method consisting of culturing cells on a cell culture vessel coated with a polymer that undergoes a change in molecular structure due to a stimulus such as temperature, pH or light followed by separating the cells from the surface of the cell culture vessel in the form of a cell layer while maintaining the adhered state between cells by inducing a change in the surface of the cell culture vessel by changing conditions such as the temperature, pH or light, and a method consisting of culturing the cells in an arbitrary cell culture vessel and physically separating the cells from the edge of the cell culture vessel with tweezers and the like. A particularly preferable method consists of culturing cells on a cell culture vessel, which has a polymer coated on the surface thereof that undergoes a change in hydration force over a temperature range of 0 C. to 80 C., within a temperature range at which the hydration force of the polymer is weak, followed by culturing while changing the temperature of the culture broth to a state in which the hydration force of the polymer is strong to separate the cells in the form of a sheet. At that time, the cells are cultured on a cell culture vessel, which is coated on the surface thereof with a polymer that undergoes a change in hydration force over a temperature range of 0 C. to 80 C., within a temperature range in which the hydration force of the polymer is weak. The temperature is preferably a temperature of 37 C. at which cells are normally cultured. The temperature-responsive polymer used in the present invention may be a homopolymer or copolymer. An example of such a polymer is the polymer described in Japanese Unexamined Patent Publication No. H02-211865. More specifically, the polymer is obtained by, for example, homopolymerization or copolymerization of the monomers indicated below. Examples of monomers that can be used include (meth)acrylamide compounds, N- (or N,N-di)alkyl-substituted (meth)acrylamide derivatives and vinyl ether derivatives, and in the case of a copolymer, any two or more of these types can be used. Moreover, copolymerization of monomer types other than those indicated above, graft polymerization or copolymerization of multiple polymers, or a mixture of polymers and copolymers may also be used. In addition, the polymer can also be crosslinked within a range that does not impair the inherent properties of the polymer. At that time, since the target of culturing and separation consists of cells and separation is performed over a temperature range of 5 C. to 50 C., examples of the temperature-responsive polymer include poly(N-n-propylacrylamide) (lower limit critical solution temperature of homopolymer: 21 C.), poly(N-n-propylmethacrylamide) (ditto 27 C.), poly(N-isopropylacrylamide) (ditto 32 C.), poly(N-isopropylmethacrylamide) (ditto 43 C.), poly(N-cyclopropylacrylamide) (ditto 45 C.), poly(N-ethoxyethylacrylamide) (ditto approx. 35 C.), poly(N-ethoxyethylmethacrylamide) (ditto approx. 45 C.), poly(N-tetrahydrofurfurylacrylamide) (ditto approx. 28 C.), poly(N-tetrahydrofurfurylmethacrylamide) (ditto approx. 35 C.), poly(N,N-ethylmethylacrylamide) (ditto 56 C.) and poly(N,N-diethylacrylamide) (ditto 32 C.). Examples of monomers used for copolymerization in the present invention include, but are not limited to, water-containing polymers such as polyacrylamide, poly(N,N-diethylacrylamide), poly(N,N-dimethylacrylamide), polyethylene oxide, polyacrylic acid and salts thereof, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), polyvinyl alcohol, polyvinylpyrrolidone, cellulose or carboxymethyl cellulose.

(22) There are no particular limitations on the method used in the present invention to coat the culture vessel surface with each of the polymers as previously described, and examples thereof consist of physically adsorbing by coating or kneading and the like the aforementioned monomers or polymers to the vessel by any of electron beam irradiation (EB), -ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment or an organic polymerization reaction. The amount of temperature-responsive polymer coated onto the surface of the culture vessel is within the range of 1.2 g/cm.sup.2 to 2.3 g/cm.sup.2, preferably 1.4 g/cm.sup.2 to 1.9 g/cm.sup.2, and more preferably 1.5 g/cm.sup.2 to 1.8 g/cm.sup.2. When the coated amount is less than 1.1 g/cm.sup.2, the cells have difficulty in separating from the polymer even when imparted with a stimulus resulting in a considerable decrease in work efficiency, thereby making this undesirable. Conversely, if the coated amount is 2.3 g/cm.sup.2 or more, cells have difficulty adhering within this range and adequate adhesion of the cells becomes difficult. In such cases, if a cell adhesive protein is further coated onto the temperature-responsive polymer coating layer, the amount of temperature-responsive polymer coated on the vessel surface may be 2.3 g/cm.sup.2 or more, and the coated amount of the temperature-responsive polymer at that time is 9.0 g/cm.sup.2 or less, preferably 8.0 g/cm.sup.2 or less and more preferably 7.0 g/cm.sup.2 or less. If the coated amount of the temperature-responsive polymer is 9.0 g/cm.sup.2 or more, cell adhesion becomes difficult even if a cell adhesive protein is further coated on the temperature-responsive protein coating layer, thereby making this undesirable. There are no particular limitations on the type of cell adhesive protein used, and examples thereof include collagen, laminin, laminin 5, fibronectin and Matrigel either alone or as a mixture of two or more types thereof. In addition, the method used to coat these cell adhesive proteins may be in accordance with a normal method, and a method is normally used in which an aqueous solution of the cell adhesive protein is coated onto the surface of the vessel followed by removing the aqueous solution and rinsing. The present invention constitutes a technology that attempts to use a cell sheet per se that utilizes a temperature-responsive culture dish. Thus, the coated amount of cell adhesive protein on the temperature-responsive polymer layer is preferably not excessively large. The coated amount of the temperature-responsive polymer and the coated amount of the cell adhesive protein may be measured in accordance with normal methods, and examples of methods that may be used include any of a method consisting of measuring a portion where cells are adhered directly using FT-IR-ATR, and a method consisting of immobilizing a preliminarily labeled polymer using a similar method and estimating the coated amount from the amount of labeled polymer immobilized by a portion where cells are adhered.

(23) In the method of the present invention, although varying according to the animal species of the cells used, the number of cells disseminated during culturing is typically 0.210.sup.6 to 1010.sup.6 cells/cm.sup.2, preferably 0.310.sup.6 to 910.sup.6 cells/cm.sup.2, and more preferably 0.410.sup.6 to 810.sup.6 cells/cm.sup.2. In the present invention, in order to separate and recover the cultured cell sheet from the temperature-responsive culture vessel, the cultured cell sheet can be separated by making the temperature of the culture vessel adhered with cultured cells to be equal to or higher than the upper limit critical solution temperature or equal to lower than the lower limit critical solution temperature of the coated polymer. At that time, the cultured cell sheet can be separated in the culture broth or other isotonic solution, and can be selected according to the objective. A method consisting of gently tapping or shaking the culture vessel, stirring the medium using a pipette, or using a tweezers may be used alone or in combination for the purpose of separating and recovering the cells more quickly and with greater efficiency. Culturing conditions other than temperature may be in accordance with ordinary methods and there are no particular limitations thereon. For example, the medium used may be a medium containing a known serum such as fetal bovine serum (FBS), or may be a serum-free medium to which such serum has not been added.

(24) The following provides an explanation of the preceding matters using the example of poly(N-isopropylacrylamide) for the temperature-responsive polymer. Poly(N-isopropylacrylamide) is known to be a polymer that has a lower limit critical solution temperature at 31 C., and if put into the free state, undergoes dehydration at a temperature of 31 C. or higher in water resulting in coagulation of the polymer chain that causes it to become turbid. Conversely, at a temperature of 31 C. or lower, the polymer chain is hydrated and enters a state in which it is dissolved in water. In the present invention, this polymer is coated and immobilized on the surface of a Petri dish or other culture vessel. Thus, if the temperature is equal to higher than 31 C., although the polymer on the surface of the culture vessel similarly undergoes dehydration, since the polymer chain is coated and immobilized on the surface of the culture vessel, the surface of the culture vessel becomes hydrophobic. Conversely, if the temperature is equal to or lower than 31 C., although the polymer on the surface of the culture vessel is hydrated, since the polymer chain is coated and immobilized on the surface of the culture vessel, the surface of the culture vessel becomes hydrophilic. The hydrophobic surface at this time is a surface that is suitable for the adhesion and proliferation of cells, while the hydrophilic surface prevents cells from adhering thereto, thereby resulting in cultured cells or a cell sheet being separated simply by cooling.

(25) Glass, modified glass and compounds such as polystyrene or polymethacrylate used in ordinary cell culturing, as well as substances typically capable of imparting a shape, such as polymer compounds other than those listed above and ceramics, can all be used for the culture vessel that is subjected to coating.

(26) There are no particular limitations on the form of the cell culture vessel used in the present invention, and examples thereof include a dish, multi-plate, flask or cell insert cultured on a porous membrane as well as that having a flat shape. In the case the cultured cells are epithelial cells, the use of a cell insert enables culture broth to make contact above and below the cells resulting in layering of the cells, thereby making this preferable. Examples of the cell culture vessel that is subjected to coating include glass, modified glass and compounds such as polystyrene or polymethacrylate used in ordinary cell culturing, as well as substances typically capable of imparting a shape, such as polymer compounds other than those listed above and ceramics.

(27) The cell sheet used in the present invention is not subjected to damage by proteases represented by dispase or trypsin during culturing. Consequently, the cell sheet separated from the cell culture vessel has adhesive protein, and when cells have been separated in the form of a sheet, the desmosome structures between cells are maintained to a certain degree. As a result, the cells can be favorably adhered when placing on vascular bed and are able to take efficiently. Although the typical protease, dispase, is known to be able to cause separation in a state in which 10% to 40% of the desmosome structures between cells are maintained, since nearly all basal membrane-like proteins between the cells and culture vessel end up being destroyed, the resulting cell sheet has weak strength. In contrast, the cell sheet of the present invention is able to obtain the various effects as previously described as a result of being in a state in which 60% or more of the desmosome structures and basal membrane-like proteins remain intact.

(28) There are no particular limitations on the method used to fabricate the cell composition having a plurality of cell layers in the present invention, and examples of methods for obtaining the cell composition include a method consisting of disseminating cells in a cell culture vessel and coating a gel containing a protein composing an extracellular matrix protein (such as laminin, collagen, gelatin, cadherin, hyaluronic acid, fibronectin, fibrin, elastin, chitin, chitosan or hydronectin) thereon, followed by further seeding cells to obtain a layered cell composition, and a method consisting of separating cultured cells in the form of a sheet and layering a plurality of cultured cell sheets using a cultured cell transfer tool as necessary. There are no particular limitations on the temperature at that time provided that, in the case the aforementioned polymer coated on the surface of the culture vessel has an upper limit critical solution temperature, it is equal to or lower than that temperature, or in the case the aforementioned polymer has a lower limit critical solution temperature, it is equal to or higher than that temperature. However, it goes without saying that culturing at an excessively low temperature range that prevents the cultured cells from proliferating, or an excessively high temperature range that causes the cultured cells to die, is unsuitable. Culturing conditions at a temperature other than those temperatures may be performed in accordance with ordinary methods and there are no particular limitations thereon. For example, the medium used may be a known medium to which serum has been added such as fetal bovine serum, or may be a serum-free medium to which serum has not been added. In addition, a tool may be used as necessary to transfer the cell sheet. Although there are no particular limitations whatsoever on the material or shape of the tool provided it is able to grasp the separated cell sheet, examples of materials of the tool include polyvinylidene fluoride (PVDF), silicon, polyvinyl alcohol, urethane, cellulose and derivatives thereof, chitin, chitosan, collagen, gelatin and fibrin sealant, and the tool is used by contacting the cell sheet while in the form of a film, porous film, nonwoven fabric or woven fabric.

(29) The cell composition obtained in the present invention can be transplanted to a prescribed site in the body. At that time, vascularization may be preliminarily induced at the transplant site, and there are no particular limitations thereon. Here, although there are no particular limitations on the method used to induce vascularization, and examples of methods thereof include a method consisting of embedding a vascular growth factor in the form of FGF in a microsphere, and allowing to act in the body for 8 to 10 days while changing the composition, size and injection range of the microsphere, and a method consisting of cutting a polyethylene terephthalate mesh to an arbitrary size, forming the mesh into the shape of a bag, placing FGF dissolved in a highly concentrated agarose solution inside the bag and removing the bag after 8 to 10 days to create a space in which vascularization has been induced.

EXAMPLES

(30) Although the following provides a more detailed explanation of the present invention based on examples thereof, these examples do not limit the present invention in any way. Furthermore, the experimental protocols using rats of the examples were approved by the ethics committee relating to animal experiments of the Tokyo Women's Medical University, and were implemented in accordance with the Guide for the Care and Use of Laboratory Animals (1996 edition) published by the U.S. National Institute of Health (NIH).

(31) (Animals)

(32) Fabrication of cell sheets: Three-week-old GFP rats (strain: SD-Tg (CAG-EGFP), Japan SLC, Inc.) and SD rats (strain: S1c:SD, Japan SLC, Inc.) were purchased and euthanized with CO.sub.2.

(33) Transplanted animals: Nine-week-old nude rats (strain: F344/NJcl-rnu/rnu, CLEA Japan, Inc.) were purchased and used for transplant at age 10 weeks.

(34) The animals were anesthetized with 4% isoflurane and maintained at about 1.8%. Hypothermia was prevented by using a hot plate during the procedure. The regenerated uterus and damaged uterus rats were sacrificed by exsanguination following deep anesthesia. When extracting fetuses from pregnant individuals, the mothers were sacrificed by placing under deep anesthesia, and the fetuses and mothers were sacrificed by the same method. At that time, a longer duration of anesthesia than normal was used for the fetuses in consideration of their higher tolerance to hypoxia.

(35) (Fabrication of Cell Sheets)

(36) The method used to fabricate cell sheets is indicated in FIG. 1. More specifically, 3-week-old SD rats or GFP rats were laparotomized to confirm the uterus. The ligament connecting the ovaries on both sides with the pelvis was dissected and the cervix was resected after spreading open the mesometrium followed by extracting the uterus. Subsequently, the extracted uterus was separated into the myometrium and endometrium under a microscope. The separated endometrial tissue was treated for 20 minutes in a mixture of 0.25% trypsin and 0.1% EDTA at 37 C. while shaking at 150 bpm. Following treatment, the mixture was pipetted about 70 to 100 times with a 5 mL pipette and then about 200 times with a 1000 m Pipetman. This was performed until the mixture was visually confirmed to be free of aggregates as an indicator of completion. After terminating the reaction with an equal amount of medium containing 10% FBS, the mixture was filtered with 100 m and 40 m cell strainers. The acquired cells were completely disseminated into a 100 mm dish and allowed to stand undisturbed for 2 hours (to be referred to as the cell sorting step). Two hours later, the supernatant was collected and washed three times with PBS. The PBS used during washing was also collected. This supernatant and the washed cells were combined and designated as (1). After washing, the cells remaining in the dish were allowed to react for 5 minutes at 37 C. in a mixture of 0.25% trypsin and 0.1% EDTA and then collected. The collected cells were designated as (2). The cells of (1) were seeded on an insert immobilized with a temperature-responsive polymer at 5.010.sup.6 cells/dish, and the cells of (2) were seeded on a 35 mm temperature-responsive culture dish (Upcell, CellSeed Inc.) at 2.510.sup.6 cells/dish. The cells of (1) were cultured using DMEM/F12 medium containing 10 g/mL of insulin, 10 g/mL of transferrin, 0.038 M selenite, 100 g/mL of hydrocortisone, 2.5 nM retinoic acid, 100 M ascorbic acid and 10 ng/mL of EGF. The cells of (2) were cultured using DMEM/F12 medium (1:1) containing 10% FBS, 1% penicillin, 1% streptomycin and 1% Hepes. Four days after seeding, the cells seeded in the insert were separated from the wall surfaces by tracing the periphery of the insert over the entire circumference using a 200 m chip. The cells seeded in the culture dish were separated from the wall surfaces by tracing the periphery of the dish over the entire circumference about 6 times using a 1000 m chip. Both cells were subsequently harvested as cell sheets by incubation for 1 hour at 20 C. The cell sheet of (1) was separated with tweezers in cases in case of difficult to separate. The separated cell sheets were suspended in medium and PBS. The sheet of (1) was transferred to an ordinary culture dish and suspended therein. Subsequently, a plastic sheet measuring about 1 cm1 cm was first placed under the cell sheet of (2) in the culture dish in which the cell sheet of (2) was suspended, followed by spreading the cell sheet over the plastic sheet by removing the medium. The spread cell sheet was placed in a culture dish, in which another cell sheet was suspended, together with the plastic sheet, and placed under the suspended second cell sheet. The second sheet was then spread over the plastic sheet so as to form a two-layer laminated state by removing the medium in the same manner as the first sheet. A three-layer laminated sheet was then fabricated by spreading the cell sheet of (2) over the cell sheet of (1) on the two-layer laminate using the same method (resulting in a laminate having three cell sheets in the order of (1), (2) and (2) from the top).

Example 1

(37) (In Vitro Evaluation of Layered Sheets)

(38) The layered cell sheets were placed on an ordinary insert (Falcon) and spread out by removing the medium. 2 mL of medium (DMEM/F12 (1:1) containing 10% FBS, 1% penicillin, 1% streptomycin and 1% Hepes) were placed in a 6-well plate. The layered cell sheets were then incubated for 3 days at 37 C. and 5% CO.sub.2 while in that state. Subsequently, the cell sheets were immobilized to a re-adhesive sheet in the form of 4% PFA while adhered to the membrane of the insert.

(39) (Tissue Evaluation and Immunostaining Evaluation)

(40) The cell sheets were embedded in paraffin, cut into thin sections and subjected to HE staining. Immunostaining was performed by activating using a pressure cooker using a pH 6 activator. Subsequently, blocking was performed for 2 hours with 10% goat serum. Following blocking, primary antibody such as GFP, CK18 or vimentin was allowed to react overnight at a ratio of 1:100 and 4 C. Secondary antibody was subsequently allowed to react for 1 hour at room temperature to perform nuclear staining followed by mounting.

(41) As shown in FIG. 2, the upper portion of the cell sheet of (1) was observed to uniformly consist of CK18-positive cells expressed in epithelial cells, while vimentin-positive cells were confirmed on the opposite side on the side of the surface of the culture dish. The cell sheet of (2) consisted of cells negative for CK18 and positive for vimentin. The layered cell sheet was confirmed to have a single layer of CK18-positive cells in the upper portion thereof immediately after layering, and vimentin-positive cells present under CK-18 positive layer were confirmed. However, gaps were confirmed between the sheets. A single layer of CK18-positive cells was also confirmed in the upper portion of the re-adhesive sheet, and although a vimentin-positive layer was confirmed there below, gaps were not confirmed.

Example 2

(42) (Cell Sheet Transplantation)

(43) Ten-week-old nude rats were laparotomized to expose the uterus. The uterus was then opened by making a longitudinal incision. The myometrium and endometrium were physically separated using tweezers under a microscope. The range of separation was targeted at a distance of 1 cm in the center of the uterus. After separating the endometrium and leaving only the myometrium, the uterus was stitched 5 points each on the left and right from both sides using 7-0 Nylon sutures and positioned at the transplant site so as to spread out on both sides. Subsequently, hemostasis was performed using a heated scalpel. The layered cell sheet in the manner described above was transplanted directly to the transplant site from the plastic sheet used during layering. Subsequently, wet gauze was placed around the transplant site and a 100 mm tissue cover was placed thereon to prevent drying. The cell sheet was allowed to take by remaining undisturbed for 1.5 hours while in that state. After taking, the extended Nylon sutures were removed and the transplant site was closed by continuously suturing with 7-0 absorbable sutures. A group was simultaneously prepared as a control group in which the myometrium and endometrium were physically separated followed by closing without transplanting a cell sheet.

(44) (Evaluation of Regenerated Uterus)

(45) The regenerated uterus was evaluated 4 weeks after transplanting the cell sheet. The uterus was observed using small animal ultrasonography. The transplanted uterus was observed moving from the cervical canal towards the Fallopian tube to confirm the presence or absence of constricted sites. In addition, confirmation was also made as to the presence or absence of edema-like changes in the direction of the Fallopian tube. Constriction and obstruction were suspected in the case the presence of cystic changes was confirmed. Moreover, the uterus was injected with physiological saline to confirm the presence or absence of obstruction. The rat vagina was extended using an otolaryngological auditory canal observation instrument to expose the cervical canal. The mantle of a 22 G intravenous indwelling needle was inserted uterine cavity through the cervical canal while guided by ultrasonography. Following insertion, physiological saline was injected while observing by ultrasonography. Following injection, the uterus was judged to be free of obstruction if the entire uterus extended uniformly. Obstruction was judged to be present in the case the physiological saline did not flow in the direction of the Fallopian tubule. In the case of obstruction, the length of the obstructed site was measured by ultrasonography. Moreover, an observation was made as to whether or not GFP-positive sites can be confirmed by laparotomizing under anesthesia. In addition, an evaluation was made as to the presence or absence of cystic changes. The control group was also evaluated for the presence or absence of edema-like changes, and the endometrium separation site was confirmed to have thinned. Following confirmation, the 7-0 absorbable sutures used to suture the uterus were removed. The uterus was extracted together with the control group in order to evaluate the regenerated uterus 4 weeks after transplantation. The wounds of individuals used as pregnant uteri were performed synechiotomy against postoperative adhesion from around the uterus.

(46) The uterus at four weeks after transplantation was observed a luminal structure that was equivalent to a normal structure on the inner side of the myometrium, and endometrial gland structures were observed in the stromal layer (FIG. 3). In addition, GFP-positive tissue was observed, and CK18 and GFP positive cells were lined for shape the luminal structure. In the control group, there was hardly any tissue observed within the tissue of the myometrium, while regeneration of uterine tissue was prominently confirmed in the transplant group.

Example 3

(47) (Mating and Evaluation of Pregnancy)

(48) FIG. 4 indicates the flow of an experiment for mating and evaluation of pregnancy. More specifically, female nude rats were housed with male nude rats in the same cages at 2 weeks after evaluation of the regenerated uterus by laparotomizing. The abdomens of the animals were observed using small animal ultrasonography under anesthesia 2 weeks after the start of cohabitation. Judgement of establishment of pregnancy was confirming gestational sac and fetus by ultrasonography. Individuals in which at least one fetus was confirmed in the normal right side uterus or either the transplanted or damaged left uterus were judged to be pregnant individuals. Individuals in which a gestational sac and fetus were unable to be clearly confirmed but were suspected of being pregnant (due to changes in uterine structure) were reevaluated several days later and were judged to be pregnant individuals based on the aforementioned criteria. Those animals that did not become pregnant were housed with a different individual and then reevaluated. This procedure was repeated until pregnancy was successful. Pregnant individuals were laparotomized at the stage they were diagnosed as being pregnant followed by macroscopic and fluorescence microscope observations of the uterus. The uterus was subsequently extracted and fixed with 4% PFA. Three days after fixation, the gestational sac was cut into horizontal cross-sections and the cross-sections were observed with a light microscope and fluorescence microscope. The cross-sections were again fixed with 4% PFA following observation.

(49) (Evaluation of Gravid Uterus)

(50) In the pregnant uteri, 70% to 90% of the gestational sacs were confirmed GFP-positive sites in the cell sheet transplantation group (FIG. 5). In horizontal cross-sections, GFP-positive sites were confirmed over the entire outer circumference of the gestational sac, and GFP-positive sites were confirmed over a wide range directly beneath the placenta (FIG. 6). GFP-positive sites were similarly confirmed over the entire outer circumference of the gestational sac and GFP-positive sites were confirmed over a wide range directly beneath the placenta in tissue specimens as well (FIG. 7). In addition, GFP-positive endometrial epithelium was confirmed at non-pregnant sites as well, and an Arias-Stella reaction (reaction in which the endometrium changes to a papillary shape due to the effects of pregnancy) was observed based on the shape thereof (FIG. 8). On the basis thereof, the endometrial cell sheet was confirmed to change accompanying pregnancy following transplantation.

Example 4

(51) (Evaluation of Endometrial Cell Sheet Derived from Allogenic Origin)

(52) The same experiment as Example 3 was performed with the exception of using rats having a normal immune response in the form of SD rats for the experimental animals followed by evaluating whether or not they are able to become pregnant by transplant animals. As a result, the endometrial cell sheets were able to take and pregnancy was confirmed to be possible even in the case of using rats having a normal immune response in the form of SD rats for the transplanted animals (FIG. 9).

Comparative Example 1

(53) (Evaluation of Uterus Regenerative Effects of Uterine Stromal Cell Sheet)

(54) Since the uterine stromal cell sheet also contains epithelial cells, a study was conducted as to whether or not uterus regenerative effects are demonstrated with only a uterine stromal cell sheet. The experiment was conducted using the same methods as Example 2 and Example 3 with the exception of using cell sheets derived from adherent cells for all cell sheets layered in the cell sorting step of the method used to fabricate the cell sheets. As a result, although the transplanted cell sheets were confirmed engraftment, adhesion at the wound site was unable to be prevented, and the uterine cavity was not confirmed (FIG. 10). In addition, pregnancy was also not confirmed.

Comparative Example 2

(55) (Evaluation of Uterus Regenerative Effects of Transplantation of Uterine Stromal Cell Sheet and Silicone Tube Placement)

(56) A study was conducted as to whether or not luminal structure is maintained and the epithelial layer is reconstructed by placing a silicone tube simultaneous to transplanting a uterine stromal cell sheet. A uterine stromal cell sheet was transplanted into the uterus of nude rats from which the endometrium had been removed in the same manner as Comparative Example 1, followed by the placement of a silicone tube (AS ONE Corp.) and suturing (FIG. 11). One week after transplantation, uterine tissue sections were prepared followed by HE staining and immunostaining in accordance with established methods. As a result, the structure of the uterine cavity was maintained, cells derived from the cell sheet were confirmed, and cells positive for progesterone receptors specifically expressed in the endometrium were confirmed (FIG. 12(C)). However, a large number of inflammatory cells were observed at sites contacted by the silicone tube, while epithelial cells were not found.

(57) All publications and patent documents cited in the present description are incorporated in the present description in their entirety by reference. Furthermore, although specific embodiments of the present invention have been explained in the present description for the purpose of exemplification, it is easily understood by a person with ordinary skill in the art that the present invention may be modified in various ways without deviating from the spirit and scope thereof.