WO2009110647A1 - Tissue sheet of cornea or conjunctiva using nasal mucosa epithelium - Google Patents
Tissue sheet of cornea or conjunctiva using nasal mucosa epithelium Download PDFInfo
- Publication number
- WO2009110647A1 WO2009110647A1 PCT/KR2008/001219 KR2008001219W WO2009110647A1 WO 2009110647 A1 WO2009110647 A1 WO 2009110647A1 KR 2008001219 W KR2008001219 W KR 2008001219W WO 2009110647 A1 WO2009110647 A1 WO 2009110647A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nasal mucosa
- cells
- amnion
- epithelial cells
- corneal
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
Definitions
- the present invention relates to a corneal or conjunctival tissue sheet comprising nasal mucosa epithelial cells grown on the amnion, more particularly to a corneal or conjunctival tissue sheet derived from nasal mucosa epithelial cells prepared by seeding nasal mucosa epithelial cells on the amnion and growing them in the presence of feeder cells to form multiple layers.
- Ocular surface diseases such as Stevens-Johnson syndrome and ocular pemphigoid or chemical burns may be accompanied by the damage of the cornea and the conjunctiva as well as the corneal epithelium and the corneal stem cells (the limbus), which results in blood vessel formation on the damaged ocular surface, vascularization and opacity of the cornea and, ultimately, blindness.
- corneal allografting transplantation of other person's cornea
- transplantation of autologous corneal limbus the border of the cornea where corneal stem cells are located
- amnion transplantation transplantation of cultured corneal epithelial cells, or the like have been developed during the last decade, there are many problems.
- the percentage of those who can get help from transplantation of donated cornea among the patients who need corneal transplantation is merely about 1% (WHO), and the long-term use of immunosuppressants after the transplantation may result in various adverse reactions.
- the autologous limbal transplantation refers to the transplantation of intact limbus of the patient.
- the autologous transplantation is restricted because both eyes are harmed in most cases. Even when it is possible, the donor tissue may be insufficient and the stem cells of the intact eye may be damaged.
- amnion is without antigenicity, is similar to collagen, has various cytokines and growth factors, and contains angiogenesis Inhibitors, it is useful in cornea transplantation and tissue engineering.
- Oral mucosal epithelial cells have been used because they are easily accessible, can be obtained in large quantity, leave no scar at the donor site, can be grown in short time, and can be maintained for a long time without being comified under the laboratory condition.
- the oral mucosa may be effective in reconstructing corneal epithelial cells, it does not have mucin-secreting goblet cells.
- Fig. 1 shows the result of mucin staining of human normal conjunctiva (Fig. 1 (a)), human normal oral mucosa (Fig. 1 (b)) and human normal nasal mucosa (Fig. 1 (c)). Mucin-secreting goblet cells are stained with purple color in the conjunctiva (Fig.
- the inventors of the present invention have researched for a method of reconstructing the cornea or conjunctiva using nasal mucosa epithelial cells including the goblet cells. Further, the inventors of the present invention recognized that corneal or limbal implantation for corneal reconstruction needs to be preceded by the improvement of ocular microenvironment because, in most cases, ocular injuries involve both the cornea and the conjunctiva not just the cornea. Especially, the maintenance of normal tear film is important in differentiation and proliferation of ocular surface epithelial cells. A severe eye dryness may result in the loss of corneal epithelium and damage to the corneal stem cells, thereby severely affecting the vision.
- the ocular microenvironment can be restored by culturing and transplanting normal conjunctival cells including healthy goblet cells (mucin-secreting cells), it may provide a fundamental cure for xerophthalmia, provide a most biocompatible therapy for intractable keratoconjunctivitis with few side effects, extend availability of existing artificial cornea, and enable permanent vision preservation.
- nasal mucosal epithelium cultured according to the present invention is structurally identical with the corneal or conjunctival epithelium, it may be useful for the development of techniques replacing animal tests, e.g. drug absorption, respiratory inflammation therapy, etc.
- the inventors of the present invention carried out researches in order to solve the aforesaid problems of the related art. They identified that mucin-secreting corneal or conjunctival tissue sheet can be reconstructed by using nasal mucosa epithelial cells including goblet cells and completed the present invention.
- an object of the present invention is to provide a corneal or conjunctival tissue sheet comprising mucin-secreting cells.
- Another object of the present invention is to provide a method for preparing the corneal or conjunctival tissue sheet.
- the present invention provides a corneal or conjunctival tissue sheet comprising nasal mucosa epithelial cells grown on the amnion.
- the term "corneal or conjunctival tissue sheet” refers to multiple layers of cells derived from nasal mucosa epithelial cells.
- the cell layers are formed by culturing and proliferating the cells of the nasal mucosal epithelium on a carrier, so that at least part of the cells are differentiated and form multiple layers.
- the carrier may be the amnion as well as actual tissue from various organisms, e.g. fibrin and collagen, or biocompatible tissue engineering supports. In an embodiment of the present invention, the amnion is used as the carrier.
- the corneal or conjunctival tissue sheet may be used as an implant material (replacement for corneal or conjunctival epithelium) for patients whose cornea or conjunctiva is damaged or lost.
- a graft may be fixed to nearby tissues using suture in order to promote its survival or may be attached using fibrin glue.
- the surface of the implant site may be protected by temporarily covering with a contact lens.
- the nasal mucosa epithelial cells include mucin-secreting cells. Since the existing sheets for reconstruction of the cornea derived from oral mucosa do not include mucin-secreting goblet cells, they cannot produce mucus which stabilizes tears. However, because the corneal or conjunctival tissue sheet derived from nasal mucosa according to the present invention include healthy goblet cells, it can secrete mucin after being implanted into the conjunctival tissue (see Fig. 1 and Fig. 6).
- the original ocular microenvironment may be recovered through implantation of artificial cornea or artificial conjunctiva using the sheet of the present invention may, it may provide a fundamental cure for xerophthalmia and provide a therapy for intractable keratoconjunctivitis with few side effects.
- tissue sheet of the present invention in order to verify whether the original ocular microenvironment may be recovered using the tissue sheet of the present invention, expression of marker proteins - epithelial stem cell marker p63 (Chemicon), cornified epithelial markers cytokeratin 1 (Novocastra) and cytokeratin 10 (Dako), corneal epithelial markers cytokeratin 3/12 (Chemicon), epithelial basal layer marker cytokeratin 5 (Abeam), conjunctival epithelial marker cytokeratin 13 (Biogenex), epithelial basal layer marker CD44 (Bender Medsytem), marker for mucin 5 expressed in comeal/conjunctival/nasal mucosa MUC5AC (Lab Vision), marker for mucin 1 expressed in comeal/conjunctival/nasal mucosa MUC1 (Novocastra), and corneal antiangiogenic factor thrombospondin-1 (TSP-1 , Santa Cruz)
- nasal mucosal epithelium employed in the present invention includes mucin-secreting goblet cells and expresses keratin 3, which is expressed specifically in the differentiated corneal epithelium only, but not the cornified keratins keratin 1 and keratin 10, it will provide structure and function similar to those of actual corneal or conjunctival epithelium.
- the amnion may be used as a substrate for culturing nasal mucosa epithelial cells without any treatment.
- the amnion may be one with the epithelium removed, for example, by curettage.
- the "amnion” is a membranous sac that surrounds the outermost layer of the uterus and the placenta. It consists of a basement membrane and an amnion epithelial layer on top of the parenchyma abundant in collagen. All human epithelial cells are strongly attached to the basement membrane, or the basal layer, to form a sheet.
- the basement membrane consists of collagen type IV, laminin 5, or the like.
- amnion is employed in order to strongly attach the cultured nasal mucosa epithelial cells to the basement membrane.
- the amniotic epithelium is selectively removed by treating with enzyme.
- the amnion contributes to the attachment and proliferation of cells, with the amniotic epithelial cells being removed and the basement membrane conserved well.
- This characteristic feature of the amnion employed in the present invention can be confirmed in Fig. 4. It can be seen that the amniotic epithelial cells has been removed and the collagen type IV of the basement membrane is conserved well (Fig. 4 (c)), and that the laminin 5 of the basement membrane is expressed in all the layers of the basement membrane (Fig. 4 (d)).
- the amnion used in the present invention may be acquired, for example, from human fetal membrane or placenta obtained after childbirth.
- human fetal membrane, placenta and umbilical cord obtained after childbirth may be processed and purified to obtain human amnion.
- the processing and purification may be carried out according to a method known in the art. That is, the human amnion may be prepared by peeling off the amnion from the fetal membrane obtained after childbirth and removing remaining tissues through physical processing e.g. ultrasonic cleaning and enzyme treatment followed by adequate washing.
- the medium used for culturing nasal mucosa epithelial cells on the amnion are is particularly limited as long as it is possible to grow the cells and form multiple layers.
- a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium at specific proportions to which fetal bovine serum (FBS), growth factors, antibiotics, or the like are added may be used as culture medium.
- the nasal mucosa epithelial cells form multiple layers on the amnion.
- multiple layers on the amnion means that after the nasal mucosa epithelial cells are attached on the amnion, they grow uniformly to form a confluent monolayer sheet, and then the nasal mucosa epithelial cells are differentiated as they are completely exposed to the air to form multiple (3 to 5) layers of cells (Fig. 6 (b)).
- the cell growth on the amnion may be carried out in the presence of feeder cells.
- the feeder cells excrete various growth factors and provide intercellular substances, thereby helping the growth and adhesion of the epithelial cells.
- the feeder cells are in mitotically inactive states.
- the cells may be mitotically inactivated by irradiating with ⁇ -ray or by treating with mitomycin C. Such inactivation treatment is made to prevent the proliferation of the feeder cells themselves, which may interfere with the proliferation of nasal mucosa epithelial cells, thereby enhancing the proliferation of the nasal mucosa epithelial cells.
- the amnion may serve both as a carrier of the nasal mucosa epithelial cells and as a barrier membrane that prevents the cocultured feeder cells from infiltrating into the nasal mucosa epithelial cells. This ensures that the feeder cells are not present in the finally obtained corneal or conjunctival epithelial sheet. Accordingly, preparation of a corneal or conjunctival epithelial sheet with no concern of immune rejection by the feeder cells is possible, which is clinically of great significance.
- stainless steel mesh 150 mm squares, Ted PeIIa
- Ted PeIIa was used as a support for supporting the amnion.
- the support was designed to have legs with predetermined heights, so that it is separated from the feeder cells seeded at the bottom. The support helps the transport of various growth factors secreted from the feeder cells.
- Fig. 3 (a) shows the lateral view of a culture dish in which the stainless steel mesh is put.
- Fig. 3 (b) shows the amnion 2 cm in diameter fixed on the stainless steel mesh.
- the density of the feeder cells may be, for example, about 1 x10 2 cells/cm 2 or more, preferably about 1 *10 2 cells/cm 2 to about 1 ⁇ 10 7 cells/cm 2 , more preferably about 1 *10 2 cells/cm 2 to about 1 *10 5 cells/cm 2 .
- the number of the feeder cells may be, for example, 0.001 to 100 times, preferably 0.01 to 1 time that of the nasal mucosa epithelial cells. If the number of the feeder cells is too small, the nasal mucosa epithelial cells may not proliferate well and desired multiple layers of the nasal mucosa epithelial cells may not be obtained.
- the feeder cells may be any cells known in the art. Preferred examples include those selected from a group consisting of mouse 3T3 cells, human fibroblasts, human corneal keratocytes, amniotic epithelial cells, bone marrow-derived stem cells and adipose-derived stem cells.
- the nasal mucosa epithelial cells may be originated from the nasal septum or turbinate tissue.
- the nasal mucosa epithelial cells may be obtained by finely cutting the nasal septum or turbinate tissue using e.g. a scalpel or by scraping the epithelial surface using e.g. a surgical knife.
- the present invention provides a method for preparing a corneal or conjunctival tissue sheet comprising: a) preparing nasal mucosa epithelial cells; b) seeding the nasal mucosa epithelial cells on the amnion; and c) culturing the seeded nasal mucosa epithelial cells along with feeder cells to form multiple layers.
- the preparation of the nasal mucosa epithelial cells may be carried out by finely cutting the nasal mucosa tissue, treating with dispase, scraping the tissue surface using a surgical tool, and culturing in an adequate medium (e.g. epithelial culture medium such as bronchial epithelial growth medium (BEGM)).
- an adequate medium e.g. epithelial culture medium such as bronchial epithelial growth medium (BEGM)
- the amnion in the stage b) may be one from which the amniotic epithelial cells have been removed using trypsin- thylenediaminetetraacetic acid (EDTA).
- EDTA trypsin- thylenediaminetetraacetic acid
- the trypsin-EDTA may be prepared by diluting trypsin-EDTA solution (Sigma, USA) commonly used in the art to an adequate concentration.
- the trypsin-EDTA may be removed by washing with physiologically compatible buffer, e.g. phosphate buffered saline (PBS), or culture medium.
- physiologically compatible buffer e.g. phosphate buffered saline (PBS), or culture medium.
- the culturing in the stage c) may be carried out in a mixed culture medium of DMEM and Ham's F12 for 2-10 days, so that the nasal mucosa epithelial cells grow uniformly on the amnion.
- the preparation method of the present invention may further comprise, after the stage c), culturing the multiple layers of nasal mucosa epithelial cells for 2-14 days under exposure to the air.
- the culturing of the nasal mucosa epithelial cells seeded on the amnion is carried out in two stages.
- the first stage of culturing (until the nasal mucosa epithelial cells on the amnion form a confluent monolayer) is carried out under a submerged condition, which is a typical culturing condition.
- a preferred culturing period is 2-10 days, most preferably 7 days.
- the second stage of culturing is carried out under an air-liquid condition.
- a preferred culturing period is 2-10 days, most preferably 7 days.
- the submerged condition means a state where the nasal mucosa epithelial cells seeded on the amnion are submerged in the culture medium. Under this condition, the nasal mucosa epithelial cells are attached to the amnion and proliferate uniformly to form a confluent monolayer sheet.
- the air-liquid condition means a state where the nasal mucosa epithelial cells seeded on the amnion are completely exposed to the air. Under this condition, the confluent epithelial cell layer is exposed to the air as in the human body, and the supply of nutrients is carried out through the amnion which is in contact with the culture medium. During the air-liquid culturing, the cells are differentiated to form a multiple layered epithelial sheet.
- the present invention provides an artificial corneal or conjunctival tissue sheet for replacing animal test, which comprises amnion and nasal mucosa epithelial cells grown on the amnion.
- the artificial corneal or conjunctival tissue sheet according to the present invention has a histological structure identical to human corneal or conjunctival epithelial tissue, it may be employed to replace animal test for testing drug absorption, respiratory inflammation, etc. or to develop alternative test methods in the field of cosmetics, medicine, pharmaceutics, or the like.
- Fig. 1 shows a result of staining mucin by periodic acid-Schiff (PAS) staining [(a): human normal conjunctiva; (b): human normal oral mucosa; (c): human normal nasal mucosa];
- PAS periodic acid-Schiff
- Fig. 2 shows a result of culturing nasal mucosa epithelial cells in early stage [(a): human normal nasal mucosa tissue; (b): tissue remaining after isolation of epithelium; (c): isolated and cultured nasal mucosa epithelial cells];
- Fig. 3 shows culturing of nasal mucosa epithelial cells seeded on the amnion, which is placed on stainless steel mesh, along with feeder cells at the bottom;
- Fig. 4 shows nasal mucosal epithelium three-dimensionally cultured on the amnion [(a): hematoxylin and eosin (H&E) staining of amnion; (b): H&E staining after removing amniotic epithelium using trypsin-ethylenediaminetetraacetic acid (EDTA); (c): immunohistologic staining of collagen type IV remaining in amnion after removal of amniotic epithelium; (d): immunohistologic staining of laminin-5 remaining in amnion after removal of amniotic epithelium; (e): nasal mucosa epithelial cells seeded on amnion after removal of amniotic epithelium and submerge-cultured for 48 hours; (f) nasal mucosa epithelial sheet after 7 days of submerge culturing];
- Fig. 5 shows the three-dimensionally cultured nasal mucosa sheet attached on ring-shaped nitrocellulose film
- Fig. 6 shows immunostaining of three-dimensionally cultured nasal mucosal epithelium [(a), (b): H&E staining of human normal conjunctival tissue and nasal mucosal epithelium three-dimensionally cultured on amnion, respectively; (c), (d): immunohistologic staining of cytokeratin 3 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (e), (f): immunohistologic staining of cytokeratin 5 in human normal corneal tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (g), (h): immunohistologic staining of cytokeratin 10 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (i), (j): immunohistologic staining of cytokeratin 13 in human normal conjunctival tissue and three-dimensionally cultured nasal
- Nasal mucosa tissue was isolated from nasal septum or turbinate tissue.
- the isolated nasal mucosa tissue was washed with phosphate buffered saline (PBS) containing 50 ⁇ g/mL gentamicin (Gibco) to remove blood clots and contaminants.
- PBS phosphate buffered saline
- Gibco gentamicin
- the washed nasal mucosa tissue was finely cut to a size of 5 mm, and treated with 1 img/mL dispase (Roche, Germany) at 37 0 C for 2 hours.
- nasal mucosa epithelial cells acquire by scraping the epithelial surface using a surgical knife.
- the dispase acts at the interface between the basal surface of the epithelial cells and the basement membrane to weaken the binding between the epithelium and the substrate, thereby aiding in the detachment of the epithelial cells under physical stimulation.
- the isolated nasal mucosa epithelial cells may be subjected to three-dimensional culturing immediately or after culturing for increasing cell numbers.
- the nasal mucosa epithelial cells isolated in Example 1 were uniformly suspended bronchial epithelial growth medium (BEGM 1 Lonza, USA) and cultured after being seeded at a concentration of 5x10 3 cells/cm 2 .
- the BEGM included epidermal growth factor (EGF), a growth factor commonly used in cell culturing, as well as insulin, transferrin, hydrocortisone, retinoic acid, epinephrine, pituitary extract, antibiotic, etc.
- EGF epidermal growth factor
- Culturing following the seeding was carried out as follows.
- the seeded nasal mucosa epithelial cells were cultured in a 37 0 C CO 2 incubator. The medium was replaced once in 2 days.
- the culture medium was removed from the culture dish and washed once with PBS. After treating with 2 mL trypsin-ethylenediaminetetraacetic acid (EDTA) for 10 minutes by adding trypsin-EDTA solution (1.5 mL) to the incubator, nasal mucosa epithelial cells were recovered from the culture dish. The number of the nasal mucosa epithelial cells was counted, and centrifuge was carried out at 300*g to leave only the nasal mucosa epithelial cells. After removing the supernatant, culture medium was added to uniformly suspend the nasal mucosa epithelial cells. The cell suspension was added to a 100 mm culture dish with a cell number of 3x10 5 , and was subjected to subculturing.
- EDTA trypsin-ethylenediaminetetraacetic acid
- Fig. 2 shows a result of culturing nasal mucosa epithelial cells in early stage.
- Fig. 2 shows human normal nasal mucosa tissue
- b shows the tissue remaining after isolation of the epithelium
- c shows the isolated and cultured nasal mucosa epithelial cells.
- nasal mucosa epithelial cells were effectively isolated from human normal nasal mucosa tissue using dispase and physical stimulation only and were effectively cultured in accordance with the present invention.
- the cultured nasal mucosa epithelial cells exhibited a pebble-like shape characteristic of epithelial cells.
- the nasal mucosa epithelial cells are cultured in the presence of feeder cells.
- the feeder cells are in mitotically inactive states.
- the cells may be mitotically inactivated by irradiating with ⁇ -ray or by treating with mitomycin C.
- fibroblasts 3T3 cells were used as feeder cells.
- 3T3 cells were seeded on a 100 mm culture dish at a concentration of 10 4 cells/cm 2 . After incubation in a 37 0 C, 5% CO 2 incubator for 18 hours, the attached 3T3 cells were immersed in 4 ⁇ g/mL mitomycin C solution (10 ml_) for 2 hours to suppress the proliferation activity of the 3T3 cells.
- the culture medium used to prepare the mitomycin C solution was F-medium, which is commonly used to culture fibroblasts.
- the F-medium is a 3:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 containing 10% fetal bovine serum (FBS). Subsequently, after washing several times with PBS to remove mitomycin C, the culture medium was replaced with fresh F-medium.
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- Example 4 Construction of nasal mucosa epithelial sheet (artificial cornea or conjunctiva) using nasal mucosa epithelial cells
- Nasal mucosa epithelial cells were cultured along with the fibroblasts prepared in Example 3 using epithelium-removed amnion as substrate.
- a 100 mm culture dish and stainless steel mesh (150 mm squares, Ted PeIIa) the same as that of Fig. 3 were used.
- the amnion was isolated from the placenta obtained after childbirth.
- Amnion epithelial cells were removed from the amnion using trypsin-EDTA.
- Figs. 4 (b) through (d) show the result of immunohistologic staining of the amniotic basement membrane after selectively removing the amniotic epithelial cells from the amnion.
- the amnion was used as a medium providing a basal surface helping the attachment of the nasal mucosa epithelial cells and transporting the cultured nasal mucosa cells in sheet form.
- Example 3 stainless steel mesh was placed on the fibroblasts (feeder cells) prepared in Example 3, and the amnion was settled and adhered on the mesh, so that the basement membrane side of the epithelium-removed amnion faced upward.
- Culture medium was added in an amount such that the level of the medium did not rise over the height of the amnion, so that the cell suspension might not be diluted by the culture medium and remain on the amnion surface during the seeding of the nasal mucosa cell suspension.
- Example 1 The suspension of nasal mucosa epithelial cells acquired in Example 1 or Example 2 was diluted with culture medium to a concentration of about 1 x10 4 cells/cm 2 to about 5 ⁇ 10 5 cells/cm 2 , to a volume of 50-100 ⁇ l_, and uniformly seeded on the amnion settled on the mesh.
- the seeded nasal mucosa epithelial cells were cultured in the presence of the fibroblasts. Culturing was carried out in at 37 0 C in a CO 2 incubator. Culture medium was replaced once in 2 days. The medium was a 3:1 mixture of DMEM and Ham's F12 used for culturing of animal cells. The medium contained 10% FBS, 5 ⁇ g/mL insulin, 5 ⁇ g/mL transferrin, 400 ng/mL hydrocortisone, 10 "9 M cholera toxin, 10 ⁇ g/mL EGF, 10 unit/mL penicillin and 10 ⁇ g/mL streptomycin.
- the nasal mucosa epithelial cells were uniformly grown for 7 days on the amnion in the submerged state (Fig. 2 (a)). They were further cultured by so-called air lifting for 7 days under the air-liquid condition, where the nasal mucosa epithelial cells are exposed to the air, in order to induce differentiation into multiple layered epithelial sheet (Fig. 2 (b)).
- the air lifting technique is a method of limiting the level of culture medium below the nasal mucosa epithelial cells grown on the amnion, thereby exposing the cells to the air.
- Fig. 4 shows hematoxylin and eosin (H&E) staining of the amnion, (b) shows H&E staining after removing the amniotic epithelium using trypsin-EDTA. (c) shows immunohistologic staining of collagen type IV remaining in the amnion after removal of the amniotic epithelium, and (d) shows immunohistologic staining of laminin-5 remaining in the amnion after removal of the amniotic epithelium.
- Fig. 5 shows the three-dimensionally cultured nasal mucosa sheet attached on ring-shaped nitrocellulose film. It can be seen that the nasal mucosa cells differentiated through air-liquid culturing form a thin, transparent sheet. The nasal mucosa cells are attached on the nitrocellulose film so that the adhesion surface faces upward. For implantation, the sheet may be cut to an adequate size, for example, using a biopsy punch.
- Example 5 Histological characteristics of cultured nasal mucosa epithelial sheet
- the three-dimensionally cultured tissue embedded in paraffin was sliced with a thickness of 4 ⁇ m and attached on a slide.
- the slide was immersed in xylene for 10 minutes in order to remove paraffin around the tissue. Then, after hydrating the tissue by treating sequentially with 100%, 90%, 80% and 70% ethanol for 2 minutes each, the slide was washed with flowing water for 10 minutes. After staining the nucleus by treating with hematoxylin (Auto Hematoxylin, ResGen) for 5 minutes, the slide was washed with flowing water for 10 minutes. Subsequently, after staining the cytoplasm by treating with eosin (Sigma) for 1 minute, the slide was washed with flowing water for 10 minutes.
- Figs. 6 (a) and (b) show H&E staining of human normal conjunctival tissue and the nasal mucosal epithelium three-dimensionally cultured on the amnion, respectively. As seen in (b), the nasal mucosa epithelial cells are uniformly attached on the amnion and form cell layers of 3-5 layers.
- the three-dimensionally cultured tissue embedded in paraffin was sliced with a thickness of 4 ⁇ m and attached on a slide.
- the slide was immersed in xylene in order to remove paraffin around the tissue.
- the tissue was hydrated by treating sequentially with 100%, 90%, 80% and 70% ethanol.
- marker proteins - epithelial stem cell marker p63 (Chemicon), cornified epithelial markers cytokeratin 1 (Novocastra) and cytokeratin 10 (Dako), corneal epithelial markers cytokeratin 3/12 (Chemicon), epithelial basal layer marker cytokeratin 5 (Abeam), conjunctival epithelial marker cytokeratin 13 (Biogenex), epithelial basal layer marker CD44 (Bender Medsytem), marker for mucin 5 expressed in comeal/conjunctival/nasal mucosa MUC5AC (Lab Vision), marker for mucin 1 expressed in corneal/conjunctival/nasal mucosa MUC1 (Novocastra), and corneal antiangiogenic factor thrombospondin-1 (TSP-1 , Santa Cruz) - was investigated using antibodies against the marker proteins, with the titer value recommended by the manufacturers (1 :50 or 1 :100).
- FIG. 6 (c) through (r) compare the immunohistologic staining result of the markers for human normal conjunctival tissue and the three-dimensionally cultured nasal mucosal epithelium.
- Expression of the corneal/conjunctival epithelium-specific markers cytokeratin 3 and cytokeratin 13 are identified in (d) and (h).
- expression of the epithelial stem cell marker p63 in the basal layer is identified in (I). Therefore, it can be seen that engraftment and proliferation were successful.
- Fig. 6 (m) through (r) it is revealed that the sheet using nasal mucosa according to the present invention secretes the same mucin secreted by normal conjunctiva in a same manner, whereas the recently proposed sheet using oral mucosa is without mucin-secreting cells, (m) shows expression of mucin 1 , which exists between cell membranes, in normal cornea and conjunctiva. A similar result is observed in the nasal mucosa sheet of the present invention, in (n). Although various mucins contribute to the stabilization of ocular surface, the most important among them is mucin 5AC.
- Mucin 5AC a gel-forming mucin, absorbs water and forms a gel-like water layer on the ocular surface, thereby protecting the eyes, (o) shows expression of mucin 5AC, in the form of sac, in normal conjunctiva. A similar result is observed in the nasal mucosa sheet of the present invention, in (p).
- Fig. 6 (s) and (t) show immunohistologic staining of the antiangiogenic factor TSP-1 in human normal corneal tissue and the three-dimensionally cultured nasal mucosal epithelial sheet, respectively.
- TSP-1 is known as the most important factor that inhibits angiogenesis on the cornea. It can be seen that a similar result is observed in the nasal mucosa sheet of the present invention as that of the normal cornea. Therefore, it is considered that when the nasal mucosal epithelial sheet according to the present invention is employed for corneal implantation, it may inhibit angiogenesis on the cornea and maintain the cornea transparent.
- the nasal mucosa epithelial sheet prepared in accordance with the present invention exhibits expression patterns similar to that of human normal cornea or conjunctiva, contributes to the stabilization of ocular surface with abundant mucin-secreting cells, and may be employed for permanent corneal or conjunctival implantation with superior engraftment and consistency.
- the corneal or conjunctival tissue sheet of the present invention since the corneal or conjunctival tissue sheet of the present invention includes mucin-secreting cells, it may restore ocular microenvironment and contribute to stabilization of ocular surface, whereas the existing corneal epithelial sheet using oral mucosa does not include mucin-secreting cells and has difficulty in restoring ocular microenvironment. Accordingly, the corneal or conjunctival tissue sheet of the present invention will be employed for permanent corneal or conjunctival implantation with superior engraftment and consistency.
Abstract
Disclosed is a corneal or conjunctival tissue sheet including amnion and nasal mucosa epithelial cells grown on the amnion, more particularly a corneal or conjunctival tissue sheet derived from nasal mucosa epithelial cells prepared by seeding nasal mucosa epithelial cells on the amnion and growing them in the presence of feeder cells to form multiple layers. The disclosed nasal mucosa epithelial sheet exhibits expression patterns similar to that of human normal cornea or conjunctiva, contributes to the stabilization of ocular surface with abundant mucin-secreting cells, and may be employed for permanent corneal or conjunctival implantation with superior engraftment and consistency.
Description
[DESCRIPTION] [Invention Title]
Tissue Sheet of Cornea or Conjunctiva Using Nasal Mucosa Epithelium
[Technical Field]
The present invention relates to a corneal or conjunctival tissue sheet comprising nasal mucosa epithelial cells grown on the amnion, more particularly to a corneal or conjunctival tissue sheet derived from nasal mucosa epithelial cells prepared by seeding nasal mucosa epithelial cells on the amnion and growing them in the presence of feeder cells to form multiple layers.
[Background Art]
Ocular surface diseases such as Stevens-Johnson syndrome and ocular pemphigoid or chemical burns may be accompanied by the damage of the cornea and the conjunctiva as well as the corneal epithelium and the corneal stem cells (the limbus), which results in blood vessel formation on the damaged ocular surface, vascularization and opacity of the cornea and, ultimately, blindness. For treatment of these diseases, although corneal allografting (transplantation of other person's cornea), transplantation of autologous corneal limbus (the border of the cornea where corneal stem cells are located), amnion transplantation, transplantation of cultured corneal epithelial cells, or the like have been developed during the last decade, there are many problems.
As for allografting, the percentage of those who can get help from transplantation of donated cornea among the patients who need corneal transplantation is merely about
1% (WHO), and the long-term use of immunosuppressants after the transplantation may result in various adverse reactions.
The autologous limbal transplantation refers to the transplantation of intact limbus of the patient. However, the autologous transplantation is restricted because both eyes are harmed in most cases. Even when it is possible, the donor tissue may be insufficient and the stem cells of the intact eye may be damaged.
Therefore, a need for the development of artificial cornea has been proposed. Although soft and smooth synthetic artificial cornea (AlphaCor, Australia) made of polymer is commercialized, adverse reactions such as cell infiltration into the artificial cornea, deterioration of transparency due to protein deposition, loss of the basal layer of the cornea are reported. Therefore, the artificial cornea is not permanent but has limited life span. Thus, a need for tissue-engineered artificial cornea is increasing. Recently, studies are underway about the implantation of cornea limbal cells cultured on the amnion and the implantation of cultured oral epithelium to the cornea (Takahiro Nakamura, et al., IOVS, January 2003, Vol. 44, No.1 ; Kohji Nishida, M. D., et al., The New England Journal of Medicine, 2004; 351 : 1187-96. 2004; 351 : 1187-96). Since the amnion is without antigenicity, is similar to collagen, has various cytokines and growth factors, and contains angiogenesis Inhibitors, it is useful in cornea transplantation and tissue engineering.
Oral mucosal epithelial cells have been used because they are easily accessible, can be obtained in large quantity, leave no scar at the donor site, can be grown in short time, and can be maintained for a long time without being comified under the laboratory condition. Although the oral mucosa may be effective in reconstructing corneal epithelial cells, it does not have mucin-secreting goblet cells. Fig. 1 shows the result of
mucin staining of human normal conjunctiva (Fig. 1 (a)), human normal oral mucosa (Fig. 1 (b)) and human normal nasal mucosa (Fig. 1 (c)). Mucin-secreting goblet cells are stained with purple color in the conjunctiva (Fig. 1 (a)), but they are not observed in the oral mucosa (Fig. 1 (b)). They are expressed abundantly in the nasal mucosa (Fig. 1 (c)). In consideration of the fact that the mucus which is required to stabilize tears cannot be produced without goblet cells, the inventors of the present invention have researched for a method of reconstructing the cornea or conjunctiva using nasal mucosa epithelial cells including the goblet cells. Further, the inventors of the present invention recognized that corneal or limbal implantation for corneal reconstruction needs to be preceded by the improvement of ocular microenvironment because, in most cases, ocular injuries involve both the cornea and the conjunctiva not just the cornea. Especially, the maintenance of normal tear film is important in differentiation and proliferation of ocular surface epithelial cells. A severe eye dryness may result in the loss of corneal epithelium and damage to the corneal stem cells, thereby severely affecting the vision.
If the ocular microenvironment can be restored by culturing and transplanting normal conjunctival cells including healthy goblet cells (mucin-secreting cells), it may provide a fundamental cure for xerophthalmia, provide a most biocompatible therapy for intractable keratoconjunctivitis with few side effects, extend availability of existing artificial cornea, and enable permanent vision preservation.
Worldwide, experiments on animals are criticized in view of protection and welfare of animals. In this regard, studies on in vitro experiments capable of replacing animal tests are actively carried out. For example, test methods using various tissue models cultured from human-derived cells have been established. Especially, with the
rise of ethical concerns as to whether it is justifiable to sacrifice animals for the development of cosmetics not for medicines, as well as the development of alternative safety test methods using human tissue models, Cosmetics Directive 76/768/EEC of the European Union bans the production and marketing of cosmetic products based on animal testing since 2009. And, the OECD Test Guideline (OECD TG 431) adopts the skin corrosion test using artificial skin model as the standard test method. Since the nasal mucosal epithelium cultured according to the present invention is structurally identical with the corneal or conjunctival epithelium, it may be useful for the development of techniques replacing animal tests, e.g. drug absorption, respiratory inflammation therapy, etc.
[Disclosure] [Technical Problem]
The inventors of the present invention carried out researches in order to solve the aforesaid problems of the related art. They identified that mucin-secreting corneal or conjunctival tissue sheet can be reconstructed by using nasal mucosa epithelial cells including goblet cells and completed the present invention.
Accordingly, an object of the present invention is to provide a corneal or conjunctival tissue sheet comprising mucin-secreting cells.
Another object of the present invention is to provide a method for preparing the corneal or conjunctival tissue sheet.
[Technical Solution]
In an aspect, the present invention provides a corneal or conjunctival tissue sheet comprising nasal mucosa epithelial cells grown on the amnion.
In the present invention, the term "corneal or conjunctival tissue sheet" refers to multiple layers of cells derived from nasal mucosa epithelial cells. The cell layers are formed by culturing and proliferating the cells of the nasal mucosal epithelium on a carrier, so that at least part of the cells are differentiated and form multiple layers. The carrier may be the amnion as well as actual tissue from various organisms, e.g. fibrin and collagen, or biocompatible tissue engineering supports. In an embodiment of the present invention, the amnion is used as the carrier. The corneal or conjunctival tissue sheet may be used as an implant material (replacement for corneal or conjunctival epithelium) for patients whose cornea or conjunctiva is damaged or lost. During implantation, a graft may be fixed to nearby tissues using suture in order to promote its survival or may be attached using fibrin glue. After the implantation, the surface of the implant site may be protected by temporarily covering with a contact lens.
In the corneal or conjunctival tissue sheet of the present invention, the nasal mucosa epithelial cells include mucin-secreting cells. Since the existing sheets for reconstruction of the cornea derived from oral mucosa do not include mucin-secreting goblet cells, they cannot produce mucus which stabilizes tears. However, because the corneal or conjunctival tissue sheet derived from nasal mucosa according to the present invention include healthy goblet cells, it can secrete mucin after being implanted into the conjunctival tissue (see Fig. 1 and Fig. 6). Further, because the original ocular microenvironment may be recovered through implantation of artificial cornea or artificial conjunctiva using the sheet of the present invention may, it may provide a fundamental cure for xerophthalmia and provide a therapy for intractable keratoconjunctivitis with few
side effects.
In the examples of the present invention, in order to verify whether the original ocular microenvironment may be recovered using the tissue sheet of the present invention, expression of marker proteins - epithelial stem cell marker p63 (Chemicon), cornified epithelial markers cytokeratin 1 (Novocastra) and cytokeratin 10 (Dako), corneal epithelial markers cytokeratin 3/12 (Chemicon), epithelial basal layer marker cytokeratin 5 (Abeam), conjunctival epithelial marker cytokeratin 13 (Biogenex), epithelial basal layer marker CD44 (Bender Medsytem), marker for mucin 5 expressed in comeal/conjunctival/nasal mucosa MUC5AC (Lab Vision), marker for mucin 1 expressed in comeal/conjunctival/nasal mucosa MUC1 (Novocastra), and corneal antiangiogenic factor thrombospondin-1 (TSP-1 , Santa Cruz) - was investigated using antibodies against the marker proteins. As a result, all the histological features desired by the present invention were confirmed.
Recently, transplantation of the nasal mucosa as a supplementary therapy of corneal transplantation for contributing to stabilization of tears and solving eye dryness was reported (Hartmut Wenkel, et al., Br. J. Ophthalmol. 2000; 84; 279-284). In contrast, in the present invention, not the nasal mucosa tissue itself, but a sheet of epithelial cells prepared by culturing nasal mucosa epithelial cells is used. It avoids the need of technical and economical difficulties to acquire tissues from human body and can be prepared from relatively small amount of cells. Therefore, it may be more effective for the reconstruction of the cornea or conjunctiva. Further, since the nasal mucosal epithelium employed in the present invention includes mucin-secreting goblet cells and expresses keratin 3, which is expressed specifically in the differentiated corneal epithelium only, but not the cornified keratins keratin 1 and keratin 10, it will
provide structure and function similar to those of actual corneal or conjunctival epithelium.
In the corneal or conjunctival tissue sheet of the present invention, the amnion may be used as a substrate for culturing nasal mucosa epithelial cells without any treatment. Alternatively, the amnion may be one with the epithelium removed, for example, by curettage. As used herein, the "amnion" is a membranous sac that surrounds the outermost layer of the uterus and the placenta. It consists of a basement membrane and an amnion epithelial layer on top of the parenchyma abundant in collagen. All human epithelial cells are strongly attached to the basement membrane, or the basal layer, to form a sheet. The basement membrane consists of collagen type IV, laminin 5, or the like. One of the reasons that the amnion is employed is to strongly attach the cultured nasal mucosa epithelial cells to the basement membrane. In order to expose the basal layer for the attachment of the nasal mucosal epithelium, the amniotic epithelium is selectively removed by treating with enzyme. In the present invention, the amnion contributes to the attachment and proliferation of cells, with the amniotic epithelial cells being removed and the basement membrane conserved well. This characteristic feature of the amnion employed in the present invention can be confirmed in Fig. 4. It can be seen that the amniotic epithelial cells has been removed and the collagen type IV of the basement membrane is conserved well (Fig. 4 (c)), and that the laminin 5 of the basement membrane is expressed in all the layers of the basement membrane (Fig. 4 (d)).
The amnion used in the present invention may be acquired, for example, from human fetal membrane or placenta obtained after childbirth. Specifically, human fetal membrane, placenta and umbilical cord obtained after childbirth may be processed and
purified to obtain human amnion. The processing and purification may be carried out according to a method known in the art. That is, the human amnion may be prepared by peeling off the amnion from the fetal membrane obtained after childbirth and removing remaining tissues through physical processing e.g. ultrasonic cleaning and enzyme treatment followed by adequate washing.
In the present invention, the medium used for culturing nasal mucosa epithelial cells on the amnion are is particularly limited as long as it is possible to grow the cells and form multiple layers. For example, as commonly used in for culturing epithelial cells, a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium at specific proportions to which fetal bovine serum (FBS), growth factors, antibiotics, or the like are added may be used as culture medium.
In the corneal or conjunctival tissue sheet of the present invention, the nasal mucosa epithelial cells form multiple layers on the amnion. As used herein, "multiple layers on the amnion" means that after the nasal mucosa epithelial cells are attached on the amnion, they grow uniformly to form a confluent monolayer sheet, and then the nasal mucosa epithelial cells are differentiated as they are completely exposed to the air to form multiple (3 to 5) layers of cells (Fig. 6 (b)).
In the corneal or conjunctival tissue sheet of the present invention, the cell growth on the amnion may be carried out in the presence of feeder cells. The feeder cells excrete various growth factors and provide intercellular substances, thereby helping the growth and adhesion of the epithelial cells. The feeder cells are in mitotically inactive states. The cells may be mitotically inactivated by irradiating with γ-ray or by treating with mitomycin C. Such inactivation treatment is made to prevent the proliferation of the feeder cells themselves, which may interfere with the proliferation
of nasal mucosa epithelial cells, thereby enhancing the proliferation of the nasal mucosa epithelial cells.
In the present invention, the amnion may serve both as a carrier of the nasal mucosa epithelial cells and as a barrier membrane that prevents the cocultured feeder cells from infiltrating into the nasal mucosa epithelial cells. This ensures that the feeder cells are not present in the finally obtained corneal or conjunctival epithelial sheet. Accordingly, preparation of a corneal or conjunctival epithelial sheet with no concern of immune rejection by the feeder cells is possible, which is clinically of great significance.
In the examples of the present invention, stainless steel mesh (150 mm squares, Ted PeIIa) was used as a support for supporting the amnion. The support was designed to have legs with predetermined heights, so that it is separated from the feeder cells seeded at the bottom. The support helps the transport of various growth factors secreted from the feeder cells. Fig. 3 (a) shows the lateral view of a culture dish in which the stainless steel mesh is put. Fig. 3 (b) shows the amnion 2 cm in diameter fixed on the stainless steel mesh.
In the present invention, the density of the feeder cells may be, for example, about 1 x102 cells/cm2 or more, preferably about 1 *102 cells/cm2 to about 1 χ107 cells/cm2, more preferably about 1 *102 cells/cm2 to about 1 *105 cells/cm2. To state as a proportion to the nasal mucosa epithelial cells, the number of the feeder cells may be, for example, 0.001 to 100 times, preferably 0.01 to 1 time that of the nasal mucosa epithelial cells. If the number of the feeder cells is too small, the nasal mucosa epithelial cells may not proliferate well and desired multiple layers of the nasal mucosa epithelial cells may not be obtained. And, if the number of the feeder cells is too large, the proliferation of the nasal mucosa epithelial cells may be interrupted.
In the present invention, the feeder cells may be any cells known in the art. Preferred examples include those selected from a group consisting of mouse 3T3 cells, human fibroblasts, human corneal keratocytes, amniotic epithelial cells, bone marrow-derived stem cells and adipose-derived stem cells.
In the corneal or conjunctival tissue sheet of the present invention, the nasal mucosa epithelial cells may be originated from the nasal septum or turbinate tissue. The nasal mucosa epithelial cells may be obtained by finely cutting the nasal septum or turbinate tissue using e.g. a scalpel or by scraping the epithelial surface using e.g. a surgical knife.
In another embodiment, the present invention provides a method for preparing a corneal or conjunctival tissue sheet comprising: a) preparing nasal mucosa epithelial cells; b) seeding the nasal mucosa epithelial cells on the amnion; and c) culturing the seeded nasal mucosa epithelial cells along with feeder cells to form multiple layers.
In the stage a), the preparation of the nasal mucosa epithelial cells may be carried out by finely cutting the nasal mucosa tissue, treating with dispase, scraping the tissue surface using a surgical tool, and culturing in an adequate medium (e.g. epithelial culture medium such as bronchial epithelial growth medium (BEGM)).
In the preparation method of the present invention, the amnion in the stage b) may be one from which the amniotic epithelial cells have been removed using trypsin- thylenediaminetetraacetic acid (EDTA). The trypsin-EDTA may be prepared by diluting trypsin-EDTA solution (Sigma, USA) commonly used in the art to an adequate concentration. The trypsin-EDTA may be removed by washing with physiologically
compatible buffer, e.g. phosphate buffered saline (PBS), or culture medium.
In the preparation method of the present invention, the culturing in the stage c) may be carried out in a mixed culture medium of DMEM and Ham's F12 for 2-10 days, so that the nasal mucosa epithelial cells grow uniformly on the amnion.
The preparation method of the present invention may further comprise, after the stage c), culturing the multiple layers of nasal mucosa epithelial cells for 2-14 days under exposure to the air.
In the present invention, the culturing of the nasal mucosa epithelial cells seeded on the amnion is carried out in two stages. The first stage of culturing (until the nasal mucosa epithelial cells on the amnion form a confluent monolayer) is carried out under a submerged condition, which is a typical culturing condition. A preferred culturing period is 2-10 days, most preferably 7 days. The second stage of culturing is carried out under an air-liquid condition. A preferred culturing period is 2-10 days, most preferably 7 days. In the present invention, the submerged condition means a state where the nasal mucosa epithelial cells seeded on the amnion are submerged in the culture medium. Under this condition, the nasal mucosa epithelial cells are attached to the amnion and proliferate uniformly to form a confluent monolayer sheet. And, the air-liquid condition means a state where the nasal mucosa epithelial cells seeded on the amnion are completely exposed to the air. Under this condition, the confluent epithelial cell layer is exposed to the air as in the human body, and the supply of nutrients is carried out through the amnion which is in contact with the culture medium. During the air-liquid culturing, the cells are differentiated to form a multiple layered epithelial sheet.
In another embodiment, the present invention provides an artificial corneal or conjunctival tissue sheet for replacing animal test, which comprises amnion and nasal
mucosa epithelial cells grown on the amnion.
Since the artificial corneal or conjunctival tissue sheet according to the present invention has a histological structure identical to human corneal or conjunctival epithelial tissue, it may be employed to replace animal test for testing drug absorption, respiratory inflammation, etc. or to develop alternative test methods in the field of cosmetics, medicine, pharmaceutics, or the like.
[Description of Drawings]
The above and other aspects, features and advantages of the disclosed exemplary embodiments will be more apparent from the following detailed description taken in conjunction with the accompanying drawings in which:
Fig. 1 shows a result of staining mucin by periodic acid-Schiff (PAS) staining [(a): human normal conjunctiva; (b): human normal oral mucosa; (c): human normal nasal mucosa];
Fig. 2 shows a result of culturing nasal mucosa epithelial cells in early stage [(a): human normal nasal mucosa tissue; (b): tissue remaining after isolation of epithelium; (c): isolated and cultured nasal mucosa epithelial cells];
Fig. 3 shows culturing of nasal mucosa epithelial cells seeded on the amnion, which is placed on stainless steel mesh, along with feeder cells at the bottom;
Fig. 4 shows nasal mucosal epithelium three-dimensionally cultured on the amnion [(a): hematoxylin and eosin (H&E) staining of amnion; (b): H&E staining after removing amniotic epithelium using trypsin-ethylenediaminetetraacetic acid (EDTA); (c): immunohistologic staining of collagen type IV remaining in amnion after removal of amniotic epithelium; (d): immunohistologic staining of laminin-5 remaining in amnion
after removal of amniotic epithelium; (e): nasal mucosa epithelial cells seeded on amnion after removal of amniotic epithelium and submerge-cultured for 48 hours; (f) nasal mucosa epithelial sheet after 7 days of submerge culturing];
Fig. 5 shows the three-dimensionally cultured nasal mucosa sheet attached on ring-shaped nitrocellulose film; and
Fig. 6 shows immunostaining of three-dimensionally cultured nasal mucosal epithelium [(a), (b): H&E staining of human normal conjunctival tissue and nasal mucosal epithelium three-dimensionally cultured on amnion, respectively; (c), (d): immunohistologic staining of cytokeratin 3 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (e), (f): immunohistologic staining of cytokeratin 5 in human normal corneal tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (g), (h): immunohistologic staining of cytokeratin 10 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (i), (j): immunohistologic staining of cytokeratin 13 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (k), (I): immunohistologic staining of p63 in human normal corneal tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (m), (n): immunohistologic staining of MUC 1 in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (o), (p): immunohistologic staining of MUC 5AC in human normal conjunctival tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (q), (r): immunohistologic staining of CD44v6 in human normal corneal tissue and three-dimensionally cultured nasal mucosal epithelium, respectively; (s), (t):
immunohistologic staining of thrombospondin-1 in human normal corneal tissue and three-dimensionally cultured nasal mucosal epithelium, respectively.
[Best Mode]
The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of this disclosure.
Example 1. Isolation of nasal mucosa epithelial cells
Nasal mucosa tissue was isolated from nasal septum or turbinate tissue. The isolated nasal mucosa tissue was washed with phosphate buffered saline (PBS) containing 50 μg/mL gentamicin (Gibco) to remove blood clots and contaminants. The washed nasal mucosa tissue was finely cut to a size of 5 mm, and treated with 1 img/mL dispase (Roche, Germany) at 37 0C for 2 hours. Then, nasal mucosa epithelial cells acquire by scraping the epithelial surface using a surgical knife. The dispase acts at the interface between the basal surface of the epithelial cells and the basement membrane to weaken the binding between the epithelium and the substrate, thereby aiding in the detachment of the epithelial cells under physical stimulation. The isolated nasal mucosa epithelial cells may be subjected to three-dimensional culturing immediately or after culturing for increasing cell numbers.
Example 2. Culturing of nasal mucosa epithelial cells
The nasal mucosa epithelial cells isolated in Example 1 were uniformly suspended bronchial epithelial growth medium (BEGM1 Lonza, USA) and cultured after being seeded at a concentration of 5x103 cells/cm2. The BEGM included epidermal growth factor (EGF), a growth factor commonly used in cell culturing, as well as insulin,
transferrin, hydrocortisone, retinoic acid, epinephrine, pituitary extract, antibiotic, etc. Culturing following the seeding was carried out as follows. The seeded nasal mucosa epithelial cells were cultured in a 37 0C CO2 incubator. The medium was replaced once in 2 days. When the culture dish was densely populated with cells, the culture medium was removed from the culture dish and washed once with PBS. After treating with 2 mL trypsin-ethylenediaminetetraacetic acid (EDTA) for 10 minutes by adding trypsin-EDTA solution (1.5 mL) to the incubator, nasal mucosa epithelial cells were recovered from the culture dish. The number of the nasal mucosa epithelial cells was counted, and centrifuge was carried out at 300*g to leave only the nasal mucosa epithelial cells. After removing the supernatant, culture medium was added to uniformly suspend the nasal mucosa epithelial cells. The cell suspension was added to a 100 mm culture dish with a cell number of 3x105, and was subjected to subculturing.
Fig. 2 shows a result of culturing nasal mucosa epithelial cells in early stage. In Fig. 2, (a) shows human normal nasal mucosa tissue, (b) shows the tissue remaining after isolation of the epithelium, and (c) shows the isolated and cultured nasal mucosa epithelial cells. As seen in Fig. 2, nasal mucosa epithelial cells were effectively isolated from human normal nasal mucosa tissue using dispase and physical stimulation only and were effectively cultured in accordance with the present invention. The cultured nasal mucosa epithelial cells exhibited a pebble-like shape characteristic of epithelial cells.
Example 3. Preparation of feeder cells
The nasal mucosa epithelial cells are cultured in the presence of feeder cells.
Preferably, the feeder cells are in mitotically inactive states. The cells may be
mitotically inactivated by irradiating with γ-ray or by treating with mitomycin C. In this example, fibroblasts (3T3 cells) were used as feeder cells.
Specifically, 3T3 cells were seeded on a 100 mm culture dish at a concentration of 104 cells/cm2. After incubation in a 37 0C, 5% CO2 incubator for 18 hours, the attached 3T3 cells were immersed in 4 μg/mL mitomycin C solution (10 ml_) for 2 hours to suppress the proliferation activity of the 3T3 cells. The culture medium used to prepare the mitomycin C solution was F-medium, which is commonly used to culture fibroblasts. The F-medium is a 3:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 containing 10% fetal bovine serum (FBS). Subsequently, after washing several times with PBS to remove mitomycin C, the culture medium was replaced with fresh F-medium.
Example 4. Construction of nasal mucosa epithelial sheet (artificial cornea or conjunctiva) using nasal mucosa epithelial cells
In order to verify whether the nasal mucosa epithelial cells acquired in Examples 1 and 2 can be constructed into cornea or conjunctiva, three-dimensional culturing was carried out on the amnion and the expression of corneal or conjunctival marker was identified by immunohistologic staining.
Nasal mucosa epithelial cells were cultured along with the fibroblasts prepared in Example 3 using epithelium-removed amnion as substrate. A 100 mm culture dish and stainless steel mesh (150 mm squares, Ted PeIIa) the same as that of Fig. 3 were used. The amnion was isolated from the placenta obtained after childbirth. Amnion epithelial cells were removed from the amnion using trypsin-EDTA. Figs. 4 (b) through (d) show the result of immunohistologic staining of the amniotic basement membrane after selectively removing the amniotic epithelial cells from the amnion. The amnion was
used as a medium providing a basal surface helping the attachment of the nasal mucosa epithelial cells and transporting the cultured nasal mucosa cells in sheet form.
Specifically, stainless steel mesh was placed on the fibroblasts (feeder cells) prepared in Example 3, and the amnion was settled and adhered on the mesh, so that the basement membrane side of the epithelium-removed amnion faced upward. Culture medium was added in an amount such that the level of the medium did not rise over the height of the amnion, so that the cell suspension might not be diluted by the culture medium and remain on the amnion surface during the seeding of the nasal mucosa cell suspension. The suspension of nasal mucosa epithelial cells acquired in Example 1 or Example 2 was diluted with culture medium to a concentration of about 1 x104 cells/cm2 to about 5χ105 cells/cm2, to a volume of 50-100 μl_, and uniformly seeded on the amnion settled on the mesh.
The seeded nasal mucosa epithelial cells were cultured in the presence of the fibroblasts. Culturing was carried out in at 37 0C in a CO2 incubator. Culture medium was replaced once in 2 days. The medium was a 3:1 mixture of DMEM and Ham's F12 used for culturing of animal cells. The medium contained 10% FBS, 5 μg/mL insulin, 5 μg/mL transferrin, 400 ng/mL hydrocortisone, 10"9 M cholera toxin, 10 μg/mL EGF, 10 unit/mL penicillin and 10 μg/mL streptomycin.
The nasal mucosa epithelial cells were uniformly grown for 7 days on the amnion in the submerged state (Fig. 2 (a)). They were further cultured by so-called air lifting for 7 days under the air-liquid condition, where the nasal mucosa epithelial cells are exposed to the air, in order to induce differentiation into multiple layered epithelial sheet (Fig. 2 (b)). The air lifting technique is a method of limiting the level of culture medium below the nasal mucosa epithelial cells grown on the amnion, thereby exposing the cells
to the air.
In Fig. 4, (a) shows hematoxylin and eosin (H&E) staining of the amnion, (b) shows H&E staining after removing the amniotic epithelium using trypsin-EDTA. (c) shows immunohistologic staining of collagen type IV remaining in the amnion after removal of the amniotic epithelium, and (d) shows immunohistologic staining of laminin-5 remaining in the amnion after removal of the amniotic epithelium. It can be seen that the epithelium was completely removed and the main basement membrane components collagen type IV and laminin-5 are conserved well, (e) shows the nasal mucosa epithelial cells seeded on the amnion after removal of the amniotic epithelium and submerge-cultured for 48 hours, and (f) shows the nasal mucosa epithelial sheet after 7 days of submerge culturing. It can be seen that the nasal mucosa epithelial cells are uniformly adhered on the amnion surface and form a sheet.
Fig. 5 shows the three-dimensionally cultured nasal mucosa sheet attached on ring-shaped nitrocellulose film. It can be seen that the nasal mucosa cells differentiated through air-liquid culturing form a thin, transparent sheet. The nasal mucosa cells are attached on the nitrocellulose film so that the adhesion surface faces upward. For implantation, the sheet may be cut to an adequate size, for example, using a biopsy punch.
Example 5. Histological characteristics of cultured nasal mucosa epithelial sheet
1) H&E staining
The three-dimensionally cultured tissue embedded in paraffin was sliced with a thickness of 4 μm and attached on a slide. The slide was immersed in xylene for 10 minutes in order to remove paraffin around the tissue. Then, after hydrating the tissue by treating sequentially with 100%, 90%, 80% and 70% ethanol for 2 minutes each, the
slide was washed with flowing water for 10 minutes. After staining the nucleus by treating with hematoxylin (Auto Hematoxylin, ResGen) for 5 minutes, the slide was washed with flowing water for 10 minutes. Subsequently, after staining the cytoplasm by treating with eosin (Sigma) for 1 minute, the slide was washed with flowing water for 10 minutes. Then, after hydrating the tissue by treating sequentially with 70%, 80%, 90% and 100% ethanol for 2 minutes each, the slide was treated with xylene for 10 minutes. After dropping mounting solution (Shandon Synthetic Mountant, Thermo) on the tissue portion of the slide, a slide cover was attached for mounting.
Figs. 6 (a) and (b) show H&E staining of human normal conjunctival tissue and the nasal mucosal epithelium three-dimensionally cultured on the amnion, respectively. As seen in (b), the nasal mucosa epithelial cells are uniformly attached on the amnion and form cell layers of 3-5 layers.
2) lmmunohistologic staining
The three-dimensionally cultured tissue embedded in paraffin was sliced with a thickness of 4 μm and attached on a slide. The slide was immersed in xylene in order to remove paraffin around the tissue. Then, the tissue was hydrated by treating sequentially with 100%, 90%, 80% and 70% ethanol. Expression of marker proteins - epithelial stem cell marker p63 (Chemicon), cornified epithelial markers cytokeratin 1 (Novocastra) and cytokeratin 10 (Dako), corneal epithelial markers cytokeratin 3/12 (Chemicon), epithelial basal layer marker cytokeratin 5 (Abeam), conjunctival epithelial marker cytokeratin 13 (Biogenex), epithelial basal layer marker CD44 (Bender Medsytem), marker for mucin 5 expressed in comeal/conjunctival/nasal mucosa MUC5AC (Lab Vision), marker for mucin 1 expressed in corneal/conjunctival/nasal mucosa MUC1 (Novocastra), and corneal antiangiogenic factor thrombospondin-1
(TSP-1 , Santa Cruz) - was investigated using antibodies against the marker proteins, with the titer value recommended by the manufacturers (1 :50 or 1 :100). Then, the nucleus was counter stained with hematoxylin (ResGen).
In Fig, 6, (c) through (r) compare the immunohistologic staining result of the markers for human normal conjunctival tissue and the three-dimensionally cultured nasal mucosal epithelium. Expression of the corneal/conjunctival epithelium-specific markers cytokeratin 3 and cytokeratin 13 are identified in (d) and (h). Further, expression of the epithelial stem cell marker p63 in the basal layer is identified in (I). Therefore, it can be seen that engraftment and proliferation were successful.
In Fig. 6 (m) through (r), it is revealed that the sheet using nasal mucosa according to the present invention secretes the same mucin secreted by normal conjunctiva in a same manner, whereas the recently proposed sheet using oral mucosa is without mucin-secreting cells, (m) shows expression of mucin 1 , which exists between cell membranes, in normal cornea and conjunctiva. A similar result is observed in the nasal mucosa sheet of the present invention, in (n). Although various mucins contribute to the stabilization of ocular surface, the most important among them is mucin 5AC. Mucin 5AC, a gel-forming mucin, absorbs water and forms a gel-like water layer on the ocular surface, thereby protecting the eyes, (o) shows expression of mucin 5AC, in the form of sac, in normal conjunctiva. A similar result is observed in the nasal mucosa sheet of the present invention, in (p).
Fig. 6 (s) and (t) show immunohistologic staining of the antiangiogenic factor TSP-1 in human normal corneal tissue and the three-dimensionally cultured nasal mucosal epithelial sheet, respectively. TSP-1 is known as the most important factor that inhibits angiogenesis on the cornea. It can be seen that a similar result is
observed in the nasal mucosa sheet of the present invention as that of the normal cornea. Therefore, it is considered that when the nasal mucosal epithelial sheet according to the present invention is employed for corneal implantation, it may inhibit angiogenesis on the cornea and maintain the cornea transparent.
As described, the nasal mucosa epithelial sheet prepared in accordance with the present invention exhibits expression patterns similar to that of human normal cornea or conjunctiva, contributes to the stabilization of ocular surface with abundant mucin-secreting cells, and may be employed for permanent corneal or conjunctival implantation with superior engraftment and consistency.
While the exemplary embodiments have been shown and described, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of this disclosure as defined by the appended claims.
In addition, many modifications can be made to adapt a particular situation or material to the teachings of this disclosure without departing from the essential scope thereof. Therefore, it is intended that this disclosure not be limited to the particular exemplary embodiments disclosed as the best mode contemplated for carrying out this disclosure, but that this disclosure will include all embodiments falling within the scope of the appended claims.
[Industrial Applicability]
As described above, since the corneal or conjunctival tissue sheet of the present invention includes mucin-secreting cells, it may restore ocular microenvironment and contribute to stabilization of ocular surface, whereas the existing corneal epithelial sheet
using oral mucosa does not include mucin-secreting cells and has difficulty in restoring ocular microenvironment. Accordingly, the corneal or conjunctival tissue sheet of the present invention will be employed for permanent corneal or conjunctival implantation with superior engraftment and consistency.
Claims
[CLAIMS] [Claim 1 ]
A corneal or conjunctival tissue sheet comprising amnion and nasal mucosa epithelial cells grown on the amnion.
[Claim 2]
The corneal or conjunctival tissue sheet according to claim 1 , wherein the nasal mucosa epithelial cells include mucin-secreting cells.
[Claim 3]
The corneal or conjunctival tissue sheet according to claim 1 , wherein the amnion is amnion from which the epithelium is removed.
[Claim 4]
The corneal or conjunctival tissue sheet according to claim 1 , wherein the nasal mucosa epithelial cells form multiple layers on the amnion.
[Claim 5]
The corneal or conjunctival tissue sheet according to claim 1 , wherein the nasal mucosa epithelial cells are grown on the amnion in the presence of feeder cells.
[Claim 6]
The corneal or conjunctival tissue sheet according to claim 5, wherein the feeder
cells are selected from a group consisting of mouse 3T3 cells, human fibroblasts, human corneal keratocytes, amniotic epithelial cells, bone marrow-derived stem cells and adipose-derived stem cells.
[Claim 7]
The corneal or conjunctival tissue sheet according to claim 1 , wherein the nasal mucosa epithelial cells are originated from nasal septum or turbinate tissue.
[Claim 8]
A method for preparing a corneal or conjunctival tissue sheet comprising: preparing nasal mucosa epithelial cells; seeding the nasal mucosa epithelial cells on amnion; and culturing the seeded nasal mucosa epithelial cells in the presence of feeder cells to form multiple layers.
[Claim 9]
The method according to claim 8, wherein, in said seeding, the amnion is amnion from which amniotic epithelial cells are removed using trypsin-EDTA.
[Claim 10]
The method according to claim 8, wherein, in said culturing, the nasal mucosa epithelial cells are cultured as submerged in a mixed culture medium of DMEM and Ham's F12 medium for 2-10 days, so that the cells are grown uniformly on the amnion.
[Claim 11 ]
The method according to claim 8, further comprising, after said culturing, culturing the multiple layered nasal mucosa epithelial cells for 2-14 days under exposure to the air.
[Claim 12]
An artificial corneal or conjunctival tissue sheet for replacing animal test comprising amnion and nasal mucosa epithelial cells grown on the amnion.
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/KR2008/001219 WO2009110647A1 (en) | 2008-03-03 | 2008-03-03 | Tissue sheet of cornea or conjunctiva using nasal mucosa epithelium |
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/KR2008/001219 WO2009110647A1 (en) | 2008-03-03 | 2008-03-03 | Tissue sheet of cornea or conjunctiva using nasal mucosa epithelium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2417933A3 (en) * | 2010-08-11 | 2012-08-22 | Body Organ Biomedical Corp. | Bio-sheet for eye tissue repair |
| CN112553142A (en) * | 2020-12-11 | 2021-03-26 | 山东大学 | 3D organ of nasal mucosa epithelial cells and culture method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003532A1 (en) * | 2001-11-19 | 2005-01-06 | Takahiro Nakamura | Ectocornea-like sheet and method of constructing the same |
| US20060153928A1 (en) * | 2003-02-26 | 2006-07-13 | Shigeru Kinoshita | Aminion-origin medical material and method of preparing the same |
| US20070280993A1 (en) * | 2004-03-11 | 2007-12-06 | Arblast Co., Ltd., | Corneal Epithelial Sheet, Method Of constructing The Same, And Transplantation Method Using The Sheet |
| US20080026030A1 (en) * | 2004-06-30 | 2008-01-31 | Arblast Co., Ltd. | Corneal Epithelial Sheet and Process for Producing the Same |
-
2008
- 2008-03-03 WO PCT/KR2008/001219 patent/WO2009110647A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003532A1 (en) * | 2001-11-19 | 2005-01-06 | Takahiro Nakamura | Ectocornea-like sheet and method of constructing the same |
| US20060153928A1 (en) * | 2003-02-26 | 2006-07-13 | Shigeru Kinoshita | Aminion-origin medical material and method of preparing the same |
| US20070280993A1 (en) * | 2004-03-11 | 2007-12-06 | Arblast Co., Ltd., | Corneal Epithelial Sheet, Method Of constructing The Same, And Transplantation Method Using The Sheet |
| US20080026030A1 (en) * | 2004-06-30 | 2008-01-31 | Arblast Co., Ltd. | Corneal Epithelial Sheet and Process for Producing the Same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2417933A3 (en) * | 2010-08-11 | 2012-08-22 | Body Organ Biomedical Corp. | Bio-sheet for eye tissue repair |
| CN112553142A (en) * | 2020-12-11 | 2021-03-26 | 山东大学 | 3D organ of nasal mucosa epithelial cells and culture method and application thereof |
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