WO2006103685A2 - A method for cultivating cells derived from corneal limbal tissue and cells deived from corneal limbal tissue - Google Patents

A method for cultivating cells derived from corneal limbal tissue and cells deived from corneal limbal tissue Download PDF

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Publication number
WO2006103685A2
WO2006103685A2 PCT/IN2005/000092 IN2005000092W WO2006103685A2 WO 2006103685 A2 WO2006103685 A2 WO 2006103685A2 IN 2005000092 W IN2005000092 W IN 2005000092W WO 2006103685 A2 WO2006103685 A2 WO 2006103685A2
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gel
corneal limbal
tissue
hydrogel
cells
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PCT/IN2005/000092
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French (fr)
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WO2006103685A3 (en
Inventor
Hajib Naraharirao Madhavan
Abraham Samuel Jebakumar
Yuichi Mori
Hiroshi Yoshioka
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Vision Research Foundation
Nichi-In-Drugs & Devices (P) Ltd.
Mebiol Inc.
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Application filed by Vision Research Foundation, Nichi-In-Drugs & Devices (P) Ltd., Mebiol Inc. filed Critical Vision Research Foundation
Priority to PCT/IN2005/000092 priority Critical patent/WO2006103685A2/en
Publication of WO2006103685A2 publication Critical patent/WO2006103685A2/en
Publication of WO2006103685A3 publication Critical patent/WO2006103685A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the present invention relates to a method for cultivating cells derived from corneal limbal tissue and cells derived from corneal limbal tissue.
  • Cornea is located at the front of the eyeball and constitutes the external wall of the eyeball along with the sclera.
  • the cornea is also a transparent tissue without any blood vessels, which not only functions as a lens to permeate and refract surrounding light into the eye, but also contributes to obtaining better visual acuity by forming a smooth surface together with the lacrimal fluid.
  • One to two layers of cortical cells of corneal epithelium present at the topmost layer of the cornea are in a particular adherence structure called tight junction where intercellular space is tightly closed, and has barrier function to prevent substances from penetrating inside from the lacrimal fluid side. This barrier function makes it possible to limit the permeation of water-soluble substance into the cornea and also to prevent bacterial invasion.
  • corneal epithelial cells play an extremely important role in maintaining the transparency of the cornea and the homeostasis of the eyeball.
  • the cornea may become clouded and lose its transparency due to disorders, for example keratitis, corneal ulcer, pathological conditions such as puncture, accidents, etc.
  • disorders for example keratitis, corneal ulcer, pathological conditions such as puncture, accidents, etc.
  • therapy by corneal transplant is performed.
  • patient's cornea which had lost its transparency is removed, and a transparent cornea is
  • corneal transplantations some diseases cannot be dealt with simply by such corneal transplantations. These include, for example, Stevens-Johnson syndrome, pemphigus of the eye, chemical injury, and burns.
  • keratoconjunctival epithelial cells repeatedly divide everyday, old cells shed off, and new cells are regenerated from stem cells.
  • stem cell tissue which regenerates the corneal epithelium is called "corneal limbal tissue", and is confined in the borderline of the iris and the white of the eye and is under a particular environment of being exposed to the surroundings.
  • the stem cell tissue per se is damaged in some way and becomes eradicated. Due to the eradication of the stem cell tissue, the deficient region becomes covered by the conjunctival epithelium which is present around it, leading to lack of transparency and radical decline in visual acuity.
  • transplantated cornea cannot be retained for a long period by simply transplanting a cornea, since corneal limbus has been depleted. It is therefore necessary to transplant the corneal limbus as well to provide permanent reconstruction of the eye surface.
  • One method developed to transplant the corneal limbus is a transplantation method using amniotic membrane (Medical Asahi, September, 1999, p62-65; N Engl J Med, 340:1697-1703, 1999). Also in Japanese Patent Laid-Open No.
  • corneal limbal tissue which is stem cell tissue of corneal epithelial cells, or conjunctival perimeter, which is stem cell tissue of conjunctival epithelial cells, onto amniotic membrane of which the sponge layer and the epithelial layer are removed, and then growing the epithelial cells so that they cover the surface of amniotic membrane, are disclosed.
  • amniotic membrane Since the amniotic membrane has thick basal membrane, it acts as a matrix which keratoconjunctival epithelial cells grow and differentiate on.
  • the amniotic membrane used for the transplantation method needs to be obtained from the placenta of, for example, a pregnant woman who has undergone Caeserian section, and therefore has been difficult to obtain. Even if it is obtained, there may exist risks such as immune rejection to the donated amniotic membrane and infection from the amniotic membrane.
  • the present invention is intended to overcome the above problems.
  • the objects of the present invention are to provide corneal limbal stem cells or differentiated cells from corneal limbal stem cells which can be used for treating diseases associated with cornea without using amniotic membrane tissue, and to provide a method of cultivating corneal limbal stem cells without using amniotic membrane tissue.
  • corneal limbal stem cells which can be used to treat diseases associated with cornea
  • a method for cultivating corneal limbal stem cells characterized in that the method comprises the steps of: embedding the corneal limbal tissue within an aqueous solution in a low temperature sol state wherein the aqueous solution contains at least a hydrogel-forming polymer showing thermo-reversible sol-gel transition and wherein the aqueous solution is in a sol state at a low temperature and is in a gel state at a high temperature; heating the said aqueous solution to cultivate the said corneal limbal tissue within the said hydrogel in a high temperature gel state so that the corneal limbal stem cells spread and grow outside of the said corneal limbal tissue; cooling the said hydrogel to return from the gel state to a low temperature sol state; and collecting the spread and grown cells derived from corneal limbal tissue.
  • Figure 1 shows phase contrast microscopic observation image (10 times) of corneal limbal tissue from donor A on day 6 when cultivated in the hydrogel according to the present invention.
  • Figure 2 shows phase contrast microscopic observation image (10 times) of corneal limbal tissue from donor A on day 8 when cultivated in the hydrogel according to the present invention.
  • Figure 3 shows phase contrast microscopic observation image (20 times) of corneal limbal tissue from donor A on day 10 when cultivated in the hydrogel according to the present invention.
  • Figure 4 shows tissue image (10 times) of corneal limbal tissue from donor A
  • Figure 5 shows tissue image (10 times) of corneal limbal tissue from donor B HE stained after 5 days of cultivation in the hydrogel according to the present invention.
  • Figure 6 shows comparison of cell growth activity when corneal limbal tissue from donor J was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates
  • Figure 7 shows comparison of cell growth activity when corneal limbal tissue from donor K was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates.
  • Figure 8 shows comparison of cell growth activity when corneal limbal tissue from donor L was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates.
  • Figure 9 shows immunoperoxidase staining of donor B / 04 cultivated embedded in Mebiol Gel -P63 & Connexin 43 -POSITIVE; ABCG2 & INTEGRIN ⁇ - Negative
  • Figure 10 shows immunoperoxidase staining of Donor C / 04 cultivated embedded in Mebiol Gel P63- Negative; ABCG2 , Connexin 43 & Integrin ⁇ - Cells are POSITIVE,
  • Figure -11 shows immunoperoxidase staining of Donor D / 04 cultivated inside the Mebiol Gel P63, Connexin 43 & Integrin ⁇ - cells are POSTIVE; ABCG2 — Negative:
  • Figure 12 shows immunoperoxidase staining of Donor E / 04 cultivated inside the Mebiol Gel P 63, ABCG2 & Connexin 43 - POSTIVE; Integrin ⁇ - Negative
  • Figure 13 shows immunofluorescence staining of Donor I/ 04 cultivated inside the Mebiol Gel Immunomarker staining was done on the cells harvested day -11 Apple green staining cells - Positive for the specific markers
  • Figure 14 shows donor J / 04 - Immunomarker studies Immunofluorescence staining was done on the cells harvested day -10th day Apple green staining cells - Positive for ABCG2 & Connexin - 43 DETAILED DESCRIPTION OF THE INVENTION :
  • cells derived from corneal limbal tissue comprise corneal limbal stem cells as well as differentiated cells from corneal limbal stem cells.
  • corneal limbal stem cells are cells derived from corneal limbal tissue and are positive in p63 protein immunostaining.
  • p63 belongs to tumor suppressor gene p53 family and has a structure similar to p53.
  • p63 is a transcription factor which plays an important role in morphogenesis. A plurality of isoforms for p63 exist, and are classified roughly into TAp63 having a transcription activation domain at the N-terminus and DNp63 which do not have the domain.
  • DNp63 gene is expressed within the nucleus of keratinocytes having growing activity.
  • corneal limbal stem cells having pluripotency are cells derived from corneal limbal tissue and are positive in ABCG2 immunostaining.
  • ABCG2 ATP -binding Cassette superfamily G member 2
  • transient growing cells are cells derived from corneal limbal tissue and are positive in connexin 43 immunostaining.
  • Connexin is a gap junction protein, which forms a gap junction that transports low molecular weight proteins between cells.
  • Connexin 43 is a protein having a molecular weight of 43 kDa and is known to be expressed upon differentiation of transient growing cells.
  • mature corneal epithelial cells are cells derived from corneal limbal tissue and are positive in integrin ⁇ immunostaining.
  • Integrin is a receptor for cell adhesion factor and is a heterodimeric protein composed of ⁇ - and ⁇ -chain subunits. Integrin ⁇ is known as a marker for epithelial cells showing transient growing activity.
  • mouse monoclonal antibody IgG 2a against integrin ⁇ 4 (A9) can be used.
  • any of the above cells derived from corneal limbal tissue can be obtained, although it is particularly preferred to obtain among diseases associated with cornea corneal limbal stem cells effective for dysfunctions of corneal limbal stem cells and corneal limbal stem cells having pluripotency.
  • the hydrogel-forming polymer constituting the hydrogel according to the present invention refers to a polymer has a property such that it can form a hydrogel which has a crosslinking or network structure by retaining water (in the inside thereof) on the basis of such a structure.
  • the "hydrogel” refers to a gel, which comprises, at least a crosslinked or network structure comprising a polymer, and water (as a dispersion liquid) supported or retained by such a structure.
  • the "dispersion liquid” retained in the crosslinked or network structure is not particularly limited, as long as it is a liquid comprising water as a main or major component. More specifically, the dispersion liquid may for example be either of water per se, an aqueous solution and/or water-containing liquid.
  • the water-containing liquid may preferably contain 80 parts or more, more preferably 90 parts or more of water, based on the total 100 parts of the water-containing liquid. (Sol-gel transition temperature)
  • sol state In the present invention, the terms "sol state”, “gel state” and “sol-gel transition temperature” are defined in the following manner. With respect to these definitions, a paper (Polymer Journal, 18(5), 411-416 (1986)) may be referred to. 1 ml of a hydrogel in a sol state is poured into a test tube having an inside diameter of 1 cm, and is left standing for 12 hours in a water bath which is controlled at a predetermined temperature (constant temperature).
  • the above polymer solution is defined as a "sol state" at the above-mentioned predetermined temperature.
  • the above polymer solution is defined as a "gel state" at the above-mentioned predetermined temperature.
  • the thus determined transition temperature is defined as a "sol-gel transition temperature”.
  • the above sol-gel transition temperature at which the "gel state” is converted into the "sol state” while gradually decreasing the "predetermined temperature” (e.g., in 1 0 C decrement).
  • the definition and measurement of the "sol state,” “gel state,” and “sol-gel transition temperature” may also be carried out as mentioned below according to the definition and method described in a publication (H. Yoshioka et al, Journal of Macromolecular Science, A31(l), 113 (1994)).
  • the dynamic elastic modulus of a sample at an observed frequency of 1 Hz is determined by gradually shifting the temperature from a low temperature side to a high temperature side (1°C/1 min).
  • the sol-gel transition temperature is defined as a temperature at which the storage elastic modulus (G', elastic term) of the sample exceeds the loss elastic modulus (G", viscous term).
  • the sol state is defined as a state in which G" > G' is satisfied
  • the gel state is defined as a state in which G" ⁇ G' is satisfied.
  • the following measuring conditions can preferably be used.
  • Measuring apparatus (trade name): Stress controlled-type rheometer (model: CSL-500, mfd. by Carri-Med Co.)
  • sample solution or dispersed liquid (as a concentration of a "polymer compound having a sol-gel transition temperature"): 10% (by weight)
  • Amount of sample solution about 0.8 g
  • Shape and size of cell for measurement acrylic parallel disk (diameter: 4.0 cm), gap: 600 ⁇ m
  • the above sol-gel transition temperature may preferably be higher than 0 0 C and not higher than 45 °C, more preferably, higher than 0 0 C and not higher than 42 0 C ( particularly not lower than 4 °C and not higher than 40 0 C) in view of the prevention of a thermal damage to cells or a tissue of a living organism.
  • the hydrogel material having such a preferred sol-gel transition temperature may easily be selected from specific compounds as described below, according to the above-mentioned screening method (method of measuring the sol-gel transition temperature).
  • the above-mentioned sol-gel transition temperature (a 0 C) between the temperature at the time of the culturing of the cell or tissue (b 0 C), and the temperature at the time of the cooling for the inoculation, mixing or recovery of the cell or tissue (c 0 C).
  • the above-mentioned three kinds of temperatures of a 0 C, b 0 C and c 0 C may preferably have a relationship of b > a > c.
  • the value of (b - a) may preferably be 1 - 40 0 C, more preferably 2 - 30 0 C.
  • the value of (a - c) may preferably be 1 - 40 0 C, more preferably 2 - 30 0 C.
  • the hydrogel based on the carrier according to the present invention shows a behavior in a solid-like manner toward a higher frequency
  • the carrier shows a behavior in a liquid-like manner toward a lower frequency.
  • the property of the carrier for following the movements may preferably be measured according to the following method. (Method of measuring movement-following property)
  • the carrier according to the present invention comprising a hydrogel-forming polymer in a sol state (i.e., at a temperature lower than the sol-gel transition temperature) is poured into a test tube having an inside diameter of 1 cm, in an amount of the carrier corresponding to a volume of 1 rnL as the resultant hydrogel. Then, the above test tube is left standing for 12 hours in a water bath which is controlled at a temperature which is sufficiently higher than the sol-gel transition temperature of the carrier (e.g., a temperature which is 10 0 C higher than the sol-gel transition temperature), whereby the hydrogel material is converted into a gel state.
  • a temperature which is sufficiently higher than the sol-gel transition temperature of the carrier e.g., a temperature which is 10 0 C higher than the sol-gel transition temperature
  • T may preferably be 1 minute to 24 hours, more preferably 5 minutes to 10 hours;
  • the gel property of the hydrogel based on the carrier according to the present invention may preferably be determined by measuring the steady-state flow kinematic viscosity thereof.
  • the steady-state flow kinematic viscosity ⁇ (eta) may be measured by using a creep experiment.
  • the Steady-state flow kinematic viscosity ⁇ is defined as the ratio of the shear stress and the shear rate at this time.
  • This Steady-state flow kinematic viscosity can also be called Newtonian viscosity. However, it is required that the Steady-state flow kinematic viscosity is determined in the linear region wherein the viscosity little depends on the shear stress.
  • a stress-controlled type viscoelasticity-measuring apparatus (model: CSL-500, mfd. by Carri-Med Co., USA) is used as the measuring apparatus, and an acrylic disk (having a diameter of 4 cm) is used as the measuring device, and the resultant creep behavior (delay curve) is measured for at least five minutes with respect to a sample having a thickness of 600 ⁇ m.
  • the sampling time is once per one second for the initial 100 seconds, and once per ten seconds for subsequent period.
  • the hydrogel based on the carrier according to the present invention may preferably have an ⁇ of 5xl0 3 -5xl0 6 Pa sec, more preferably 8xl0 3 -2xl0 6 Pa sec, particularly, not less than IxIO 4 Pa sec and not more than 1x10 Pa sec, at a temperature which is about
  • Shoten, 1985 may be referred to).
  • the ratio ⁇ (tan ⁇ ) s / (tan ⁇ ) L ⁇ between the above (tan ⁇ ) s and (tan ⁇ ) L may preferably be less than 1 (mire preferably 0.8 or less, particularly, 0.5 or less).
  • Temperature a temperature which is about 10 0 C higher than the sol-gel transition temperature of the carrier
  • Measuring apparatus Stress controlled-type rheometer (model: CSL-500, mfd. by Carri-Med Co., USA) (Control of residual property in living body)
  • the hydrogel according to the present invention is mainly intended to be used in vitro. However, depending on its usage (for example, when a tissue which has been grown by using the hydrogel according to the present invention are returned into a living body), there are cases where the control of the in vivo remaining property of the hydrogel is preferred.
  • the sol-gel transition temperature of the hydrogel according to the present invention is decreased, the hydrogel tends to remain in a living body for a long period of time. In contrast, if the sol-gel transition temperature of the hydrogel according to the present invention is increased, the hydrogel tends to rapidly disappear in a living body.
  • the hydrogel tends to remain in a living body for a long period of time. If the concentration of the hydrogel-forming polymer in the hydrogel is decreased, the hydrogel tends to rapidly disappear in a living body.
  • the storage elastic modulus (G') of the hydrogel at a living body temperature (37 0 C) is increased. Further, if the concentration of the hydrogel-forming polymer in the hydrogel is increased, the storage elastic modulus (G') of the hydrogel at a living body temperature (37 0 C) is increased. That is, in order to control the residual property of the hydrogel in a living body, G' at 37°C may be controlled.
  • the following measuring conditions can preferably be used.
  • Measuring apparatus (trade name): Controlled stress rheometer CSL 500 mfd. by Carri-Med Co.
  • Amount of sample solution about 0.8 g
  • Shape and size of cell for measurement acryl parallel disk (diameter: 4.0 cm), gap: 600 ⁇ m
  • the relationship between the residual period for the hydrogel according to the present invention in a living body, and G' is also dependent on the site or portion therefor in a living body.
  • the relationship between the residual period for the hydrogel in the abdominal cavity and G' at an observation frequency of 1 Hz is as follows. That is, the desired range of G' for providing the hydrogel disappearance of 3 days or less is 10 to 500 Pa.
  • the desired range of G' for providing the hydrogel disappearance of the hydrogel remaining for a period of not less than 3 days and not more than 14 days is 200 to 1,500 Pa.
  • the desired range of G 1 for providing the hydrogel disappearance of the hydrogel remaining for a period of more than 14 days is 400 to 10,000 Pa.
  • fibroblasts exhibit substantially no growth in the hydrogel based on the hydrogel-forming polymer constituting the carrier.
  • the fibroblasts are significantly grown so as to provide a change thereof into an arboroid form peculiar to the fibroblasts (e.g., Jyunpei Enami, Baiyosaibo o Mochiiru Hoho (Method of using cultured cells); edited by Meiji Saito, Saibogai Matrix (Extracellular Matrix), published by Medical Review Co,, Ltd. (Tokyo), 1996, pp. 108-115, may be referred to)
  • fibroblasts maintain a spherical form thereof and they exhibit substantially no growth. (Presumed mechanism for inhibition of growth of fibroblasts)
  • a fibroblast has a property that it recognizes a monolayer culture, i.e., the surface of a supporting medium, and adheres thereto, whereby it actively grows two-dimensionally.
  • a collagen gel has a structure such that a large number of collage molecules (molecular weight: 300,000) with a length of 300 nm and a diameter of 1.5 nm are aggregated and are regularly arranged, and that they become collagen fibril and form a network structure in water.
  • the collagen gel Since this network structure is greater than the wavelength of visible radiation (400 nm or more), the collagen gel generally looks clouded or turbid.
  • the collagen gel is used as a carrier for a three-dimensional culture. It is presumed that since a fibroblast recognizes the surface of a thick collagen fibril as a supporting medium and adheres thereto, this cell significantly grows two-dimensionally in the collagen gel.
  • the hydrogel is constituted such that a hydrogel-forming polymer in a molecular state forms a three-dimensional network structure, the heterogeneity of the structure is smaller than that of the wavelength of visible radiation, and it has a relatively high transparency. Accordingly, it is presumed that fibroblasts do not clearly recognize the surface of a two-dimensional supporting medium in the material according to the present invention, and that as a result, an excessive growth of fibroblasts is inhibited in the material according to the present invention. (Evaluation of growing property of fibroblasts)
  • fibroblasts can be evaluated by the following method (with respect to the details thereof of this method, e.g., Tsuyoshi Yoshikawa, Ken Tsukikawa, St. Marianna University, School of Medicine, Journal Vol. 28, No. 4, pp. 161-170 (2000) may be referred to).
  • a hydrogel-forming polymer constituting the cell or tissue-culturing carrier according to the present invention is dissolved in a culture solution such as RPMI- 1640 (Life Technologies, N. Y., USA) at a low temperature (for example, 4 0 C), under stirring. Thereafter, normal human lung fibroblasts (NHLF, mfd.
  • the growth of fibroblasts is observed along with the elapse of time (e.g., on the Oth, 1st, 3rd and 7th days after the day of culture), by using a phase-contrast microscope. (Growth rate of fibroblasts)
  • the growth rate of fibroblasts can be determined in the culturing period by the following method using an enzyme activity.
  • a 24-well plate as described above is used, and fibroblasts are cultured thereby for a certain period of time in the cell or tissue-culturing carrier according to the present invention, and the temperature of the carrier is decreased to a temperature lower than the sol-gel transition temperature thereof (e.g., a temperature which is 10 0 C lower than the sol-gel transition temperature), so as to dissolve the carrier.
  • 50 ⁇ l of a WST-8 reagent mfd. by Dojin Kagaku (Dojindo Laboratories)
  • a reagent for determining the activity of succinate dehydrogenase is added to each of the wells.
  • the thus prepared 24-well plate is subjected to a reaction at a temperature which is lower than the sol-gel transition temperature (e.g., a temperature which is 10 0 C lower than the sol-gel transition temperature, for example, at 10 0 C) for 10 hours, and it is then retained at about 4 0 C for 1 hour, so that a completely homogenous aqueous solution is prepared. 200 ⁇ l of each of the thus obtained aqueous solution is poured into each well of a 96-well plate. The resultant absorbance (OD (450)) is measured at 450 nm
  • the growth rate (OD L OD,-) is ⁇ more preferably within the range of not lower than 80 % and not higher than 150 %, and particularly preferably within the range not lower than 90 % and not higher than 120 %.
  • the growth of intended cells is not inhibited relatively, while the growth of fibroblasts is inhibited.
  • the ratio (P T /P F ) between the growth rate of the intended- cells P ⁇ and the growth rate of the above fibroblasts P F may preferably be 1.1 or more.
  • the ratio (P J /P F ) may more preferably be 1.5 or more, and particularly preferably 2 or more.
  • the growth rate P ⁇ of the intended cells can be determined as follows.
  • the growth rate of cells other than fibroblasts P ⁇ may be determined in the same manner as in the above determination of the growth rate of fibroblasts P F except that human colon cancer cells (SW-948, trade name: Colonic Adenoma Cell Lines, mfd. by Dainippon Pharmaceutical Co., Ltd.) are used instead of normal human lung fibroblasts (NHLF) used in the above determination of the fibroblast growth rate P F .
  • human colon cancer cells SW-948, trade name: Colonic Adenoma Cell Lines, mfd. by Dainippon Pharmaceutical Co., Ltd.
  • NHLF normal human lung fibroblasts
  • the hydrogel-forming polymer usable for the carrier according to the present invention is not particularly limited, as long as the polymer exhibits the above-mentioned thermo -reversible sol-gel transition (that is, as long as it has a sol-gel transition temperature). It is preferable to achieve a preferred sol-gel transition temperature by adjusting the cloud point of a plurality of blocks having a cloud point and the cloud point of a hydrophilic block in the hydrogel-forming polymer, the compositions, hydrophobicity or hydropb ⁇ licity of both types of blocks, and/or their ' molecular weights, in view of easy exhibition of a preferred sol-gel transition at a physiological temperature (about O 0 C to 42 0 C).
  • polyalkylene-oxide block copolymer represented by block copolymers comprising polypropylene oxide portions and polyethylene oxide portions; etherified (or ether group-containing) celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (K. R. Holme, et al. Macromolecules, 24, 3828 (1991)), etc.
  • Pluronic F- 127 trade name, mfd. by BASF Wyandotte Chemical Co.
  • the molecular weight of the Pluronic F-127 is relatively low, and it shows an extremely high osmotic pressure at a high concentration of not less than about 20 wt. %, and simultaneously the Pluronic F-127 may easily permeate the cell membranes, whereby the Pluronic F-127 can adversely affect cells and microorganisms.
  • the sol-gel transition temperature thereof is as high as about 45 0 C or higher (N. Sarkar, J. Appl. Polym. Science, 24, 1073, (1979)).
  • such an etherified cellulose is less liable to form a gel at body temperature (about 38 0 C), and therefore it is difficult to use such a material for the above-mentioned purposes according to the present invention.
  • a conventional polymer having a sol-gel transition temperature in an aqueous solution thereof, and reversibly assuming a sol state at a temperature lower than the above transition temperature is simply used, the following problems are posed: (1) If the polymer is once converted into a gel state at a temperature higher than the sol-gel transition temperature, the resultant gel is dissolved when water is further added thereto;
  • the polymer has a sol-gel transition temperature higher than the body temperature (in the neighborhood of 38 0 C), and therefore the polymer assumes a sol state in the interior of a living body;
  • a polymer having a sol-gel transition temperature of higher than 0 0 C and not higher than 42 0 C e.g., a polymer which comprises a plurality of polymer chains having a cloud point, and a hydrophilic polymer chain block which has
  • the hydrogel-forming polymer preferably usable as the carrier according to the present invention may preferably comprise a combination of plural hydrophobic blocks having a cloud point, and a hydrophilic block bonded thereto.
  • the presence of the hydrophilic block is preferred in view of the provision of the water-solubility of the hydrogel material at a temperature lower than the sol-gel transition temperature.
  • the presence of the plural hydrophobic block having a cloud point is preferred in view of the conversion of the hydrogel material into a gel state at a temperature higher than the sol-gel transition temperature. In other words, the blocks having a cloud point become .
  • the cloud point based on the hydrophobic bonds corresponds to the above-mentioned sol-gel transition temperature
  • the cloud point corresponds to the sol-gel transition temperature. This is because the cloud point of the above-mentioned "blocks having a cloud point" is generally influenced by the bonding between the hydrophilic block and the blocks having a cloud point.
  • the hydrogel to be use in the present invention utilizes a property of hydrophobic bonds such that they are not only strengthened along with an increase in temperature, but also the change in the hydrophobic bond strength is reversible with respect to the temperature.
  • the hydrogel-forming polymer may preferably have a plurality of "blocks having cloud point".
  • the hydrophilic block in the hydrogel-forming polymer has a function of causing the hydrogel-forming polymer to be changed into a water-soluble state at a temperature lower than sol-gel transition temperature.
  • the hydrophilic block also has a function of providing the state of an aqueous (or water-containing) gel, while preventing the aggregation and precipitation of the hydrogel material due to an excess increase in the hydrophobic binding force at a temperature higher than the transition temperature.
  • the plural block having a cloud point may preferably comprise a polymer block which shows a negative solubility-temperature coefficient with respect to water.
  • such a polymer may preferably be one selected from the group consisting of: polypropylene oxide, copolymers comprising propylene oxide and another alkylene oxide, poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers comprising an N-substituted acrylamide derivative and an N-substituted methacrylamide derivative, polyvinyl methyl ether. and partially-acetylated product of polyvinyl alcohol.
  • a polypeptide comprising a hydrophobic . amino acid and a hydrophilic amino acid it is effective to use a polypeptide comprising a hydrophobic . amino acid and a hydrophilic amino acid, as the block having a cloud point.
  • a polyester-type biodegradable polymer such as polylactic acid or polyglycolic acid can also be used as a block having a cloud point which is decomposed and absorbed in a living body.
  • the above polymer (block having a cloud point) has a cloud point of higher than 4 0 C and not higher than 40 0 C, in view of the provision of a polymer (compound comprising a plurality of blocks having a cloud point, and a hydrophilic block bonded thereto) to be used in the present invention having a sol-gel transition temperature of higher than 4 0 C and not higher than 40 0 C.
  • the cloud point e.g., by the following method. That is. an about 1 wt.%-aqueous solution of the above polymer (block having a cloud point) is cooled to be converted into a transparent homogeneous solution, and thereafter the temperature of the solution is gradually increased (temperature increasing rate: abou.: 1 °C/min.), and the point at which the solution first shows a cloudy appearance i • defined as the cloud point.
  • temperature increasing rate abou.: 1 °C/min.
  • N-substituted methacrylamide derivatives are described below.
  • Poly-N-acryloyl piperidine Poly-N-n-propyl methacrylamide
  • the above polymer may be either a homopolymer or a copolymer comprising a monomer constituting the above polymer and "another monomer".
  • the "another monomer” to be used for such a purpose may be either a hydrophilic monomer, or a hydrophobic monomer.
  • the resultant cloud point may be increased.
  • the resultant cloud point may be decreased.
  • a polymer having a desired cloud point may also be obtained by selecting such a monomer to be used for the copolymerization.
  • a desired cloud point e.g., a cloud point of higher than 4 0 C and not higher than 40 °C
  • hydrophilic monomer may include: N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydro xyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, methacrylic acid and acrylic acid having an acidic group, and salts of these acids, vinyl sulfonic acid, styrenesulfonic acid, etc., and derivatives having a basic group such as N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, N,N-dirnethylaminopropyl acrylamide, salts of these derivatives, etc.
  • the hydrophilic monomer to be usable in the present invention is not restricted to these specific examples. (Hydrophobic monomer)
  • hydrophobic monomer may include: acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, and glycidyl methacrylate; N-substituted alkyl methacrylamide derivatives such as N-n-butyl methacrylamide; vinyl chloride, acrylonitrile, styrene, vinyl acetate, etc.
  • the hydrophobic monomer to be usable in the present invention is not restricted to these specific examples.
  • hydr ⁇ philic block to be combined with (or bonded to) the above-mentioned block having a cloud point may include: methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly N-methyl acrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, and salts of these acids; poly N,N-dimethylaminoethyl methacrylate, poly N,N-diethylaminoethyl methacrylate, poly N,N-dimethylaminopropyl acrylamide, and salts of these, etc.
  • the process for combining the above block having a cloud point with the hydrophilic block is not particularly limited.
  • the block having a cloud point and the hydrophilic block may also be combined or bonded with each other by preliminarily introducing reactive functional groups (such as hydroxyl group, amino group, carboxyl group, and isocyanate group) into both kinds of the blocks, and combining these blocks by using a chemical reaction. At this time, it is usual to introduce a plurality of reactive functional groups into the hydrophilic block.
  • reactive functional groups such as hydroxyl group, amino group, carboxyl group, and isocyanate group
  • polypropylene oxide having a cloud point and the hydrophilic block may be combined or bonded with each other by repetitively subjecting polypropylene oxide and a monomer constituting the above "other water-soluble block” (such as ethylene oxide) to a stepwise or consecutive polymerization, to thereby obtain a block copolymer comprising polypropylene oxide and a water-soluble block (such as polyethylene oxide) combined therewith.
  • a monomer constituting the above "other water-soluble block” such as ethylene oxide
  • Such a block copolymer may also be obtained by introducing a polymerizable group (such as acryloyl group) into the terminal of polypropylene oxide, and then copolymerizing therewith a monomer constituting the hydrophilic block.
  • a polymerizable group such as acryloyl group
  • a polymer usable in the present invention may be obtained by introducing a functional group which is reactive in a bond-forming reaction with the terminal functional group of polypropylene oxide (such as hydroxyl group) into a hydrophilic block, and reacting the resultant hydrophilic block and the polypropylene oxide.
  • a hydrogel-forming polymer usable in the present invention may be obtained by connecting materials such as one comprising polypropylene glycol and polyethylene glycol bonded to both terminals thereof (such as Pluronic F-127; trade name, mfd. by Asahi Denka Kogyo K.K.).
  • the hydrogel-forming polymer comprises a block having a cloud point
  • the polymer may completely be dissolved in water so as to assume a sol state, since the above-mentioned "block having a cloud point" present -in the polymer molecule is water-soluble together with the hydrophilic block.
  • the "block having a cloud point" present in the polymer molecule becomes hydrophobic so that separate molecules of the polymer are associated or aggregated with each other due to a hydrophobic interaction.
  • the hydrophilic block is water-soluble even at this time (i.e., even when heated up to a temperature higher than the cloud point), and therefore, the polymer according to the present invention in water is formed into a hydrogel having a three-dimensional network structure wherein hydrophobic association portions between the blocks having a cloud point constitute the crosslinking points.
  • the resultant hydrogel is again cooled to a temperature lower than the cloud point of the "block having a cloud point" present in the polymer molecule, the block having a cloud point becomes water-soluble and the above crosslinking points due to the hydrophobic association are released or liberated so that the hydrogel structure disappears, whereby the polymer according to the present invention is again formed into a complete aqueous solution.
  • the sol-gel transition in the ' polymer according to the present invention is based on the reversible hydrophilic-hydrophobic conversion in the block having a cloud point present in the polymer molecule at the cloud point, and therefore the transition has a complete reversibility in accordance with a temperature change. (Solubility of gel)
  • the hydrogel-forming polymer according to the present invention comprising at least a polymer having a sol-gel transition temperature in an aqueous solution thereof, substantially shows a water insolubility at a temperature (d 0 C) higher than the sol-gel transition temperature, and reversibly shows water solubility at a temperature (e 0 C) lower than the sol-gel transition temperature.
  • the above-mentioned temperature (d 0 C) may preferably be a temperature which is at least 1 °C, more preferably at least 2 0 C (particularly preferably, at least 5 11 C) higher than the sol-gel transition temperature.
  • the above-mentioned "substantial water insolubility" may preferably be a state wherein the amount of the above polymer to be dissolved in lOO ml of water at the above temperature (d °C) is 5.0 g or less (more preferably 0.5 g or less, particularly preferably 0.1 g or less).
  • the above-mentioned temperature (e 0 C) may preferably be a temperature which is at least 1 0 C, more preferably at least 2 0 C (particularly preferably, at least 5 0 C) lower than the sol-gel transition temperature, in terms of the absolute values of these temperatures.
  • the above-mentioned "water solubility" may preferably be a state wherein the amount of the above polymer to be dissolved in 100 ml of water at the above temperature (e 0 C) is 0.5 g or more (more preferably 1.0 g or more).
  • the above "to show a reversible water solubility” refers to a state wherein an aqueous solution of the above hydrogel-forming polymer shows the above-mentioned water solubility at a temperature lower than the sol-gel transition temperature, even after the polymer is once formed into a gel state (at a temperature higher than the sol-gel transition temperature).
  • a 10%-aqueous solution of the above polymer may preferably show a viscosity of 10 - 3,000 Pa s (10 - 3,000 centipoises), more preferably, 50 - 1,000 Pa s (50 - 1,000 centipoises) at 5 0 C.
  • Such a viscosity may preferably be measured, e.g., under the following measurement conditions: Viscometer: Stress-controlled type rheometer (model: CSL-500, mfd. by
  • the resultant solution is poured into a plastic Petri dish having a diameter of 35 mm, then the dish is warmed up to a temperature of 37 0 C to form a gel having a thickness of about 1.5 mm in the dish, and the total weight of the Petri dish (f gram) containing the gel is measured. Then, the entirety of the Petri dish containing the gel is left standing in 250 ml of water at 37 0 C for 10 hours, and thereafter the total weight of the Petri dish (g gram) containing the gel is measured, to thereby determine whether the gel has been dissolved from the gel surface or not.
  • the ratio of weight decrease in the gel i.e., the value of ⁇ (f-g)/f
  • the value of ⁇ (f-g)/f may preferably be 5.0 % or less, more preferably 1.0 % or less (particularly preferably 0.1 % or less).
  • Pluronic F- 127 comprising polypropylene oxide and polyethylene oxide bonded to both terminals thereof, the resultant gel was completely dissolved when the gel is left standing in water for several hours.
  • a hydrogel-forming polymer which can be converted into a gel state at a concentration of 20% or less (more preferably 15% or less, particularly 10% or less) in terms of the concentration of the polymer based on water, i.e., ⁇ (polymer)/(polymer + water) ⁇ x 100 (%).
  • the carrier according to the present invention comprises at least the above-mentioned polymer having a sol-gel transition temperature.
  • the carrier may also comprise another component, as desired.
  • Other components in such an embodiment may include: antibiotics, anticancer or antitumor substances, ECM such as collagen and gelatin, local chemical mediators appearing hereinafter, hormones such as insulin and growth factors, foreign genes, etc.; and other cells or tissues capable of secreting these chemical mediators and cell growth factors, etc.
  • the use amount of such "other components” is not particularly limited, as long as it exhibits an intended effect and can be retained in the gel based on the hydrogel-forming polymer for a certain period of time (e.g., for a period necessary for culturing cells or tissue).
  • the amount of the other component to be used may preferably be 2 parts or less, and more preferably 1 part or less, based on the total parts (10 parts) of the hydrogel-forming polymer.
  • (Chemical mediator) There are some cases where the regeneration of a living organism tissue requires not only cells such as precursor cells but also various chemical mediators such as cell growth factor which promotes the differentiation or the growth of the cells. The chemical mediator is generally secreted from cells. However, in order to efficiently conduct the regeneration, it is effective to previously add such a chemical mediator to the cell or tissue-culturing carrier according to the present invention, so as to supply the chemical mediator from the outside of the living organism tissue.
  • Examples of the above-mentioned chemical mediator may include: 1) local chemical mediators which can act extremely in the vicinity of the cell; T) neurotransmitters which are secreted by nerve cells and have a extremely short effective acting distance; 3) hormones which are secreted by endocrine cells and systemically act on target cells through bloodstream, etc.; and the like.
  • Examples of 1) local chemical mediator as described- above may include: proteins such as nerve cell growth factors, peptides such as chemotaxis factors, amino acid derivatives such as histamine, fatty acid derivatives such as prostaglandins, etc.
  • Examples of 2) neurotransmitter as described above may include: low-molecular weight substances including amino acids such as glycine, low-molecular peptides such as noradrenaline, acetylcholine, and enkephalin, etc.
  • cell growth factor or hormones as described above may include: cell growth factors such as fibroblast growth factor (FGF), epithelial growth factor
  • EGF vascular endothelial growth factor
  • HGF hapatocyte growth factor
  • proteins such as insulin, somatotropin, somatomedin, adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), and thyroid-stimulating hormone (TSH); glycoproteins, amino acid derivatives such as somatostatin, vasopressin, TSH releasing factor; steroids such as Cortisol, estradiol, testosteron; etc.
  • the hydrogel according to the present invention it is possible to arbitrarily control the diffusion rate of a chemical mediator in the hydrogel.
  • a hydrophilic substance and a hydrophobic substance can be diffused at different diffusion rates.
  • the diffusion of a water-soluble hydrophilic substance is controlled by the molecular sieving effect of the three-dimensional network structure of the hydrogel-forming polymer. Accordingly, in order to reduce the diffusion rate of the ' water-soluble hydrophilic substance, the concentration of the hydrogel-forming polymer constituting the hydrogel may be increased. Further, the diffusion of the water-soluble hydrophilic substance also is dependent on the molecular weight of the substance. When the concentration of the hydrogel-forming polymer constituting the hydrogel is constant, as the molecular weight of a substance is increased, the diffusion - rate thereof becomes lower.
  • the diffusion of a water-soluble hydrophobic substance in the hydrogel according to the present invention is influenced not only by the molecular sieving effect of the three-dimensional network structure of the hydrogel-forming polymer, but also by the distribution or partition thereof with respect to the hydrophobic portion of the hydrogel-forming polymer.
  • the diffusion of the water-soluble hydrophobic substance is also controlled by the ratio of the hydrophobic portion in the hydrogel-forming polymer, and therefore, the diffusion of the water-soluble hydrophobic substance is generally slower than that of the water-soluble hydrophilic substance.
  • the diffusion coefficient of a solute in the hydrogel can be obtained by the "early-time" approximation described in a publication , (Eric K. L. Lee et al, Journal of Membrane Science, 24, 125-143 (1985)).
  • a process in which a solute uniformly diffused on a hydrogel flat plate having a uniform thickness of L (cm) is eluted from both of the surfaces of the hydrogel flat plate is observed along with the elapse of time.
  • the diffusion coefficient D can be calculated from the gradient of a straight line obtained by plotting the elution rate to the elapsed time t versus the square root of the elapsed time t.
  • the ratio of diffusion coefficients of phenol red (PR), methyl blue (MB) and myoglobin (MG) may preferably be such that (D PR /D MB ) > 2 and
  • collagen as the conventional cell or tissue-culturing carrier, is a hydrophilic polymer. Unlike in the case of the hydrogel according to the present invention, a balance of hydrophilicity/hydrophobicity in collagen cannot be arbitrarily controlled. Accordingly, it has been difficult to control the diffusion rate of a chemical mediator in collagen.
  • the hydrogel according to the present invention can substantially arbitrarily control a balance of hydrophilicity/hydrophobicity as stated above, it is possible to control the diffusion rate of a chemical mediator in the gel according to the present invention so as to provide a considerable degree of freedom.
  • a known gel-forming polymer such as collagen
  • a cell or tissue is used in the present invention to mean tissues, apparatuses or organs which are present in the living bodies of animals (especially humans).
  • the in vivo tissue or organ which can be regenerated by using the carrier according to the present invention is not particularly limited. Examples of such tissue or organ may include: esophagus, stomach, small intestine, large intestine, pancreas, liver, heart, blood vessel, bone, cartilage, nerve, cornea, corium, etc. (Cell or tissue)
  • the cells or tissue which can be cultured by using culturing carrier according to the present invention is not particularly limited.
  • the culturing carrier according to the present invention can be used particularly effectively for differential cells or tissues.
  • differential cells may include stem cells and precursor cells.
  • the differential cells include any of differential unipotent cells, differential pluripotent cells, and differential totipotent cells. (Method of repairing or regenerating living organism tissue or organ)
  • the method of repairing or regenerating a cell or tissue using the carrier according to the present invention is not particularly limited. From the viewpoint of easy inoculation and recovery of cells or the like, it is preferable to utilize the sol-gel transition of a hydrogel-forming polymer. (Embodiment of using sol-gel transition)
  • a cell e.g., a stem cell or precursor cell
  • tissue containing the cell, or the like is first inoculated or mixed into the earner according to the present invention.
  • a hydrogel-forming polymer constituting the carrier used for culturing a cell or tissue of the present invention is dissolved in a culture medium such as RPMI- 1640 (Life Technologies, N.
  • a culture medium used herein is not particularly limited.
  • a culture medium in which a cell of interest (a stem cell, a precursor cell, etc.) easily grows or differentiates may be appropriately selected and used.
  • the above described chemical mediator promoting the growth or differentiation of a stem cell or precursor cell of interest may also be added to such a culture medium, as desired.
  • ECM such as collagen or gelatin may also be added thereto.
  • tissue or organ in the carrier according to the present invention for example, the above suspension is heated to a temperature (usually 37 0 C) higher than the sol-gel transition temperature of the carrier according " to the present invention, so that it is gelatinized. Thereafter, " a cell of interest or tissue containing the cell may be cultured at the temperature (usually 37°C).
  • the carrier according to the present invention When the carrier according to the present invention is gelatinized, it is also possible to gelatinize it in a desired form, using a mold having the desired form.
  • a cartilage tissue when used in repair of the ear or nose, the carrier according to the present invention is converted into a form compatible with a portion of the ear or nose to which the cartilage tissue is to be applied. Thereafter, cartilage cells are cultured in the carrier according to the present invention, so as to regenerate a cartilage tissue.
  • the cartilage tissue to be applied can be easily molded into a desired form and used.
  • the carrier according to the present invention containing the tissue or organ of interest is cooled to a temperature (for example, 4 0 C) lower than the sol-gel transition temperature, so that the carrier according to the present invention is returned to a sol state.
  • the tissue or organ of interest may be separated from the carrier according to the present invention by a common separation method such as centrifugal separation.
  • the carrier according to the present invention substantially not to inhibit (or substantially to promote) the growth or differentiation of a cell of interest (a stem cell, a precursor cell, etc.), while controlling the growth of fibroblasts. Accordingly, a cell or organ of interest can be efficiently regenerated in the carrier according to the present invention.
  • the solvent was distilled off under reduced pressure, the resultant residue was dissolved in distilled water, and subjected to ultrafiltration by using an ultrafiltration membrane having a molecular cutoff of 3 x 10 4 (Amicon PM-30) so as to fractionate the product into a low-molecular weight polymer fraction and a high-molecular weight polymer fraction.
  • the resultant aqueous solution was frozen, to thereby obtain a high-polymerization degree product of F-127 and a low-polymerization degree product of F-127.
  • This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %.
  • sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 16 0 C.
  • N-aclyloxy succinimide (mfd. by Kokusan Kagaku K.K.), and 7 g of n-butyl methacrylate (mfd.. by Kanto Kagaku K.K.) were dissolved in 4000 ml of chloroform. After the purging with nitrogen gas, 1.5 g of N,N'-azobisisobutyronitrile was added thereto, and the resultant mixture was subjected to polymerization at 60 0 C for 6 hours. The reaction mixture was concentrated, and then was reprecipitated in diethyl ether.
  • poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) was added to the thus obtained poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) to thereby obtain poly(N-isopropyl acrylamide-co-n-butyl methacrylate).
  • the thus obtained poly(N-isopropyl acrylamide-co-n-butyl methacrylate) had a sol-gel transition o temperature of about 19 °C in its aqueous solution.
  • This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %.
  • sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 21 0 C.
  • TGP-I ethylene oxide gas
  • EOG ethylene oxide gas
  • Hybrid Sterilization bag mfd. by Hogi Medical Co.
  • EOG sterilizing device trade name: Easy Pack, mfd. Inouchi Seieido Co.
  • the bag was placed in a vacuum drying chamber (40 0 C) and was left standing for half ' a day, and was sterilized while the bag was sometimes subjected to aeration.
  • the concentrated solution was diluted with 4 L of distilled water, and then, the dilution operation was carried out again.
  • the above dilution and concentration by ultrafiltration were further repeated 5 times, so as to eliminate products having a molecular weight of 10 x 10 4 or lower.
  • the product which had not been filtrated by this ultrafiltration i.e., the product remaining in the inside of the ultrafiltration membrane
  • TGP -4 (TGP -4) according to the present invention having a molecular weight of 10 x 10 4 or higher.
  • the hydrogel-forming polymer (TGP -3) according to the present invention which had been obtained in Production Example 3 was dissolved so as to provide a concentration of 10 mass % in distilled water.
  • the steady flow viscosity ⁇ thereof at 37°C was measured, it was found to be 5.8 x 10 5 Pa sec.
  • a stress rheometer (CSL 500), and an acryl disk (diameter: 4 cm) as a measuring device were used.
  • the thickness of a sample was set to 600 ⁇ m, and applying a shearing stress of 10 N/m 2 , the resultant creep was measured for 5 minutes after 5 minutes had passed.
  • Production Example 8 42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 65.1 g of water was added. The resultant solution was heated to 70 0 C under a nitrogen stream.
  • PDE 6000 polyethylene glycol dimethacrylate
  • the solution was concentrated to 2 L at 5 0 C, by using an ultrafiltration membrane with a molecular weight cutoff of 10x 10 4 .
  • 4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate The product with a molecular weight of 10x 10 or lower.
  • the product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 40 g of the hydrogel-forming polymer (TGP-6) according to the present invention with a molecular weight of 10x 10 4 or higher.
  • TGP-5 1 g was dissolved in 10 mL of Dulbecco's Minimum Essential Medium (DMEM) containing 20% Fetal Bovine Serum (FBS) at 4 0 C. Corneal Limbal tissue bits " of about 2 mm 3 were collected from donor A, and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • DMEM Dulbecco's Minimum Essential Medium
  • FBS Fetal Bovine Serum
  • Corneal Limbal tissue bits " of about 2 mm 3 were collected from donor A, and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37 0 C and converted into a gel state.
  • the above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel.
  • 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO 2 atmosphere at 37°C.
  • the layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium.
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C.
  • Corneal Limbal tissue bits of about 2 rah 3 were collected from donor B (male, 8 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were further added.
  • Tissue after 5 days of cultivation was fixed with formalin and stained with hematoxylin eosin (HE) ( Figure 5), thereby showing grown cellular layer around the tissue bits.
  • Paraffin embedded sections 4-5 mm thick were prepared from the above tissue bits after 5 days of cultivation, and peroxidase immunostaining by various markers was performed.
  • Anti-human p63 (4A4) mouse antibody (DAKO), anti-human ABCG2 (5D3) mouse antibody (DAKO), anti-human connexin 43 mouse antibody (DAKO), and anti-human integrin ⁇ 4 (A9) mouse antibody (DAKO) were used as primary antibodies.
  • the grown cellular population contained cells which are positive for p63 specific to corneal limbal stem cells and connexin 43 specific to transient growing cells, and cells which are positive for ABCG2 specific to corneal limbal stem cells having pluripotency and integrin ⁇ specific to mature corneal epithelial cells were not observed.
  • ABCG2 cells which are positive in immunoperoxidase staining on day 11 of cultivation were observed.
  • human amniotic membrane tissue obtained by Caeserian section was washed, and then trypsinized for 45 minutes to remove epithelial cells.
  • the human amniotic membrane tissue with epithelial cells removed were placed in a 24-well plate, and the corneal limbal tissue bits of about 1 mm 3 or less obtained from donor B (male, 8 years old) in Example 2 were placed on top of this.
  • 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under 10% CO 2 atmosphere at 37 0 C. The medium was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium.
  • Example 2 Tissue after 5 days of cultivation was fixed with formalin, paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers similar to Example 2 was performed. As a result, the grown cells were negative to all of p63, connexin 43, ABCG2, and mxegrin ⁇ . Comparative Example 2 (cultivation only on a plate)
  • Corneal limbal tissue bits of about 1 mm 3 or less obtained from donor B (male, 8 years old) in Example 2 were placed directly on the bottom of a 24-well plate.
  • 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under 10% CO 2 atmosphere at 37°C.
  • the medium was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium.
  • Tissue after 5 days of cultivation was fixed with formalin and stained with hematoxylin eosin (HE), thereby showing that tissue bits had disintegrated and no grown cells were observed around the perimeter.
  • HE hematoxylin eosin
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C.
  • Corneal limbal tissue bits of about 2 mm 3 were collected from donor C (male, 85 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP:5 solution in DMEM cooled to 4 0 C was placed in the center, of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • the grown cellular population was negative for p63 specific to corneal limbal stem cells, but contained cells which were positive for connexin 43 specific to transient growing cells, ABCG2 specific to corneal limbal stem cells having pluripotency, and inlegrin ⁇ specific to mature corneal epithelial cells.
  • Figure: 10 Comparative Example 2 (cultivation using amniotic membrane)
  • human amniotic membrane tissue obtained by Caeserian section was washed, and then trypsinized for 45 minutes to remove epithelial cells.
  • the human amniotic membrane tissue with epithelial cells removed were placed in a 24-well plate, and the corneal limbal tissue bits of about 1 mm 3 or less obtained from donor C (male, 85 years old) in Example 3 were placed on top of this.
  • 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under
  • Example 4 I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4 0 C. Corneal limbal tissue bits of about 2 mm 3 were collected from donor D (female, 65 years old), and the corneal limbal tissue bits of about 2 mm obtained were further cut into smaller pieces (1 mm 3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • DMEM containing 20% FBS
  • the grown cellular population was negative for ABCG2 specific to corneal limbal stem cells having pluripotency, but contained cells which were positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing cells, and integrin ⁇ specific to mature corneal epithelial cells. (Figure: 1 1)
  • Example 5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal limbal tissue bits of about 2 mm 3 were collected from donor E (male, 76 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37 0 C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • DMEM containing 20% FBS
  • Example 6 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal limbal tissue bits of about 2 mm 3 were collected from donor F (male, 32 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37 0 C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • DMEM containing 20% FBS
  • Tissue after 10 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers, was performed.
  • the grown cellular population were negative for ABCG2 specific to corneal limbal stem cells having pluripotency, but contained cells which were positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing cells, and integrin ⁇ specific to mature corneal epithelial cells.
  • Example 7 I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C.
  • Cadaveric Corneal limbal tissue bits of about 2 mm 3 were collected from donor G (female, 78 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • Tissue after 13 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed.
  • the grown cellular population contained -cells which were positive for p63 specific to corneal limbal stem cells.
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4 0 C.
  • Corneal limbal tissue bits of about 2 mm 3 were collected from donor H (male, 68 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were further added.
  • Tissue after 10 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed.
  • the grown cellular population contained cells, which were positive for p63 specific to corneal limbal stem cells.
  • Example 9 I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4 0 C. Corneal limbal tissue bits of about 2 mm 3 were collected from donor I (male, 62 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24* well plate warmed to 37°C and converted into a gel state. The. above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were further added. This was kept in cultivation at 37 0 C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4 0 C.
  • Corneal limbal tissue bits of about 2 mm 3 were collected from donor J (male, 79 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0 C were added.
  • Example 10 With a sample from donor J (male " , 79 years old) in Example 10, experiments similar to Example 10 (cultivation according to the present invention), Comparative Example 1 (cultivation on amniotic membrane tissue), and Comparative Example 2 (cultivation on a plate) were performed using DMEM medium containing 1 ⁇ Ci per well of tritium ⁇ -labelled thymidine. 50 ⁇ L per well of medium was sampled every day, the amount of radiation was measured with a scintillation counter, and the reduction of thymidine incorporated by the cell for DNA synthesis was compared, to obtain an indicator for cell growth activity.
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4 0 C.
  • Cadaveric Corneal limbal tissue bits of about 2 mm 3 were collected from donor K (male, 98 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37 0 C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were added.
  • Example 13 With a sample from donor K (male, 98 years old) in Example 12, experiments similar to Example 12 (cultivation according to the present invention), Comparative Example 1 (cultivation on amniotic membrane tissue), and Comparative Example 2 (cultivation on a plate) were performed using DMEM medium containing 1 ⁇ Ci per well of tritium 3 H-labelled thymidine. 50 ⁇ L per well of medium was sampled every day, the amount of radiation was measured with a scintillation counter, and the reduction of thymidine incorporated by the cell for DNA synthesis was compared, to obtain an indicator for cell growth activity.
  • TGP-5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C.
  • Cadaveric Corneal limbal tissue bits of about 2 mm 3 were collected from donor L (female, 56 years old), and the corneal limbal tissue bits of about 2 mm 3 obtained were further cut into smaller pieces (1 mm 3 or less).
  • 0.2 mL of TGP-5 solution in DMEM cooled to 4 0 C was placed in the center of the wells of a 24-well plate warmed to 37 0 C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm 3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were added.
  • PBS PBS
  • the cellular population collected contained p63 positive cells specific to corneal limbal stem cells, ABCG2 positive cells specific to corneal limbal stem cells having pluripotency, connexin 43 positive cells specific to transient growing cells, and integrin ⁇ positive cells specific to mature corneal epithelial cells positive.
  • cellular population from corneal limbus can be obtained according to the present invention, even when corneal limbal stem cells and corneal epithelial cells, which they differentiate into, cannot be obtained by cultivating corneal limbal tissue on amniotic membrane tissue or on typical cell cultivation plates.
  • collecting of only, the cells from corneal limbal tissue are done with ease, and the cells can be provided for various treatments for diseases associated with cornea.
  • the cellular population from corneal limbus obtained from the present invention can be stored frozen according to usual methods, to be thawed and used when a patient is in need thereof.
  • the cells derived from corneal limbal tissue obtainable from the present invention it is possible to fix only the relevant cells onto the cornea of a patient using for example therapeutic contact lens. There is no need to transplant the cells together with human amniotic membrane tissue as with conventional methods.

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Abstract

The present inventors have found that corneal limbal stem cells, which can be used to treat diseases associated with cornea can be obtained by a method for cultivating corneal limbal stem cells characterized in that the method comprises the steps of: embedding the corneal limbal tissue within an aqueous solution in a low temperature sol state wherein the aqueous solution contains at least a hydrogel-forming polymer showing thereto-reversible sol-gel transition and wherein the aqueous solution is in a sol state at a low temperature and is in a gel state at a high temperature; heating the said aqueous solution to cultivate the said corneal limbal tissue within the said hydrogel in a high temperature gel state so that the corneal limbal stem cells spread and grow outside of the said corneal limbal tissue; cooling the said hydrogel to return from the gel state to a low temperature sol state; and collecting the spread and grown cells derived from corneal limbal tissue.

Description

TITLE OF THE INVENTION
A METHOD FOR CULTIVATING CELLS DERIVED FROM CORNEAL LIMBAL TISSUE AND CELLS DERIVED FROM CORNEAL LIMBAL TISSUE
TECHNICAL FIELD OF THE INVENTION : The present invention relates to a method for cultivating cells derived from corneal limbal tissue and cells derived from corneal limbal tissue.
BACKGROUND OF THE INVNETION
Cornea is located at the front of the eyeball and constitutes the external wall of the eyeball along with the sclera. The cornea is also a transparent tissue without any blood vessels, which not only functions as a lens to permeate and refract surrounding light into the eye, but also contributes to obtaining better visual acuity by forming a smooth surface together with the lacrimal fluid. One to two layers of cortical cells of corneal epithelium present at the topmost layer of the cornea are in a particular adherence structure called tight junction where intercellular space is tightly closed, and has barrier function to prevent substances from penetrating inside from the lacrimal fluid side. This barrier function makes it possible to limit the permeation of water-soluble substance into the cornea and also to prevent bacterial invasion. In other words, corneal epithelial cells play an extremely important role in maintaining the transparency of the cornea and the homeostasis of the eyeball.
However, the cornea may become clouded and lose its transparency due to disorders, for example keratitis, corneal ulcer, pathological conditions such as puncture, accidents, etc. For such persistent decline in visual acuity due to clouding of the cornea, therapy by corneal transplant is performed. In such corneal transplantation, patient's cornea which had lost its transparency is removed, and a transparent cornea is
- i - transplanted in its place. Transparency is recovered by this transplantation, and visual acuity can be restored.
In addition, some diseases cannot be dealt with simply by such corneal transplantations. These include, for example, Stevens-Johnson syndrome, pemphigus of the eye, chemical injury, and burns. In generally, keratoconjunctival epithelial cells repeatedly divide everyday, old cells shed off, and new cells are regenerated from stem cells. In these pathological conditions, however, it has been discovered that there is a dysfunction of the stem cell tissue which regenerates the cornea. The stem cell tissue which regenerates the corneal epithelium is called "corneal limbal tissue", and is confined in the borderline of the iris and the white of the eye and is under a particular environment of being exposed to the surroundings. It is thought that because of this, under the pathological conditions described above, the stem cell tissue per se is damaged in some way and becomes eradicated. Due to the eradication of the stem cell tissue, the deficient region becomes covered by the conjunctival epithelium which is present around it, leading to lack of transparency and radical decline in visual acuity.
In such pathological conditions, transplantated cornea cannot be retained for a long period by simply transplanting a cornea, since corneal limbus has been depleted. It is therefore necessary to transplant the corneal limbus as well to provide permanent reconstruction of the eye surface. One method developed to transplant the corneal limbus is a transplantation method using amniotic membrane (Medical Asahi, September, 1999, p62-65; N Engl J Med, 340:1697-1703, 1999). Also in Japanese Patent Laid-Open No. 2001-161353, cellular bits for transplantation obtainable by attaching corneal limbal tissue, which is stem cell tissue of corneal epithelial cells, or conjunctival perimeter, which is stem cell tissue of conjunctival epithelial cells, onto amniotic membrane of which the sponge layer and the epithelial layer are removed, and then growing the epithelial cells so that they cover the surface of amniotic membrane, are disclosed.
Since the amniotic membrane has thick basal membrane, it acts as a matrix which keratoconjunctival epithelial cells grow and differentiate on. However, the amniotic membrane used for the transplantation method needs to be obtained from the placenta of, for example, a pregnant woman who has undergone Caeserian section, and therefore has been difficult to obtain. Even if it is obtained, there may exist risks such as immune rejection to the donated amniotic membrane and infection from the amniotic membrane. There has been another problem that such cells must be transplanted together with the whole amniotic membrane, since separation of the amniotic membrane from cells of corneal limbus grown using amniotic membrane as the matrix was difficult.
SUMMARY OF THE INVENTION The present invention is intended to overcome the above problems. The objects of the present invention are to provide corneal limbal stem cells or differentiated cells from corneal limbal stem cells which can be used for treating diseases associated with cornea without using amniotic membrane tissue, and to provide a method of cultivating corneal limbal stem cells without using amniotic membrane tissue. As a result of extensive investigation to achieve the above objects, the present inventors have found that corneal limbal stem cells, which can be used to treat diseases associated with cornea can be obtained by a method for cultivating corneal limbal stem cells characterized in that the method comprises the steps of: embedding the corneal limbal tissue within an aqueous solution in a low temperature sol state wherein the aqueous solution contains at least a hydrogel-forming polymer showing thermo-reversible sol-gel transition and wherein the aqueous solution is in a sol state at a low temperature and is in a gel state at a high temperature; heating the said aqueous solution to cultivate the said corneal limbal tissue within the said hydrogel in a high temperature gel state so that the corneal limbal stem cells spread and grow outside of the said corneal limbal tissue; cooling the said hydrogel to return from the gel state to a low temperature sol state; and collecting the spread and grown cells derived from corneal limbal tissue. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: shows phase contrast microscopic observation image (10 times) of corneal limbal tissue from donor A on day 6 when cultivated in the hydrogel according to the present invention.
Figure 2: shows phase contrast microscopic observation image (10 times) of corneal limbal tissue from donor A on day 8 when cultivated in the hydrogel according to the present invention.
Figure 3: shows phase contrast microscopic observation image (20 times) of corneal limbal tissue from donor A on day 10 when cultivated in the hydrogel according to the present invention.
Figure 4: shows tissue image (10 times) of corneal limbal tissue from donor A
HE stained after 10 days of cultivation in the hydrogel according to the present invention.
Figure 5: shows tissue image (10 times) of corneal limbal tissue from donor B HE stained after 5 days of cultivation in the hydrogel according to the present invention.
Figure 6 : shows comparison of cell growth activity when corneal limbal tissue from donor J was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates
Figure 7: shows comparison of cell growth activity when corneal limbal tissue from donor K was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates. Figure 8: shows comparison of cell growth activity when corneal limbal tissue from donor L was cultivated in each of in the hydrogel according to the present invention, on amniotic membrane tissues, and on cultivation plates.
Figure 9 : shows immunoperoxidase staining of donor B / 04 cultivated embedded in Mebiol Gel -P63 & Connexin 43 -POSITIVE; ABCG2 & INTEGRIN β - Negative
Figure 10 : shows immunoperoxidase staining of Donor C / 04 cultivated embedded in Mebiol Gel P63- Negative; ABCG2 , Connexin 43 & Integrin β - Cells are POSITIVE,
Figure -11 : shows immunoperoxidase staining of Donor D / 04 cultivated inside the Mebiol Gel P63, Connexin 43 & Integrin β - cells are POSTIVE; ABCG2 — Negative:
Figure 12 : shows immunoperoxidase staining of Donor E / 04 cultivated inside the Mebiol Gel P 63, ABCG2 & Connexin 43 - POSTIVE; Integrin β - Negative
Figure 13: shows immunofluorescence staining of Donor I/ 04 cultivated inside the Mebiol Gel Immunomarker staining was done on the cells harvested day -11 Apple green staining cells - Positive for the specific markers
Figure 14: shows donor J / 04 - Immunomarker studies Immunofluorescence staining was done on the cells harvested day -10th day Apple green staining cells - Positive for ABCG2 & Connexin - 43 DETAILED DESCRIPTION OF THE INVENTION :
In the present invention, cells derived from corneal limbal tissue comprise corneal limbal stem cells as well as differentiated cells from corneal limbal stem cells. In the present invention, corneal limbal stem cells are cells derived from corneal limbal tissue and are positive in p63 protein immunostaining. p63 belongs to tumor suppressor gene p53 family and has a structure similar to p53. p63 is a transcription factor which plays an important role in morphogenesis. A plurality of isoforms for p63 exist, and are classified roughly into TAp63 having a transcription activation domain at the N-terminus and DNp63 which do not have the domain. DNp63 gene is expressed within the nucleus of keratinocytes having growing activity. In p63 protein immunostaining, mouse monoclonal antibody IgG2a raised against a recombinant protein corresponding to 1-205 amino acids encoded by human DNp63, i.e. p63 (4A4) can be used. In the present invention, corneal limbal stem cells having pluripotency are cells derived from corneal limbal tissue and are positive in ABCG2 immunostaining. ABCG2 (ATP -binding Cassette superfamily G member 2) is found in various stem cells, and this is an important candidate marker protein for selecting somatic stem cells having pluripotency. . In the present invention, transient growing cells are cells derived from corneal limbal tissue and are positive in connexin 43 immunostaining. Connexin is a gap junction protein, which forms a gap junction that transports low molecular weight proteins between cells. Connexin 43 is a protein having a molecular weight of 43 kDa and is known to be expressed upon differentiation of transient growing cells. In the present invention, mature corneal epithelial cells are cells derived from corneal limbal tissue and are positive in integrin β immunostaining. Integrin is a receptor for cell adhesion factor and is a heterodimeric protein composed of α- and β-chain subunits. Integrin β is known as a marker for epithelial cells showing transient growing activity. In immunostaining, mouse monoclonal antibody IgG2a against integrin β4 (A9) can be used.
According to the present invention, any of the above cells derived from corneal limbal tissue can be obtained, although it is particularly preferred to obtain among diseases associated with cornea corneal limbal stem cells effective for dysfunctions of corneal limbal stem cells and corneal limbal stem cells having pluripotency.
(Hydrogel-forming polymer)
The hydrogel-forming polymer constituting the hydrogel according to the present invention refers to a polymer has a property such that it can form a hydrogel which has a crosslinking or network structure by retaining water (in the inside thereof) on the basis of such a structure. Further, the "hydrogel" refers to a gel, which comprises, at least a crosslinked or network structure comprising a polymer, and water (as a dispersion liquid) supported or retained by such a structure. The "dispersion liquid" retained in the crosslinked or network structure is not particularly limited, as long as it is a liquid comprising water as a main or major component. More specifically, the dispersion liquid may for example be either of water per se, an aqueous solution and/or water-containing liquid. The water-containing liquid may preferably contain 80 parts or more, more preferably 90 parts or more of water, based on the total 100 parts of the water-containing liquid. (Sol-gel transition temperature)
In the present invention, the terms "sol state", "gel state" and "sol-gel transition temperature" are defined in the following manner. With respect to these definitions, a paper (Polymer Journal, 18(5), 411-416 (1986)) may be referred to. 1 ml of a hydrogel in a sol state is poured into a test tube having an inside diameter of 1 cm, and is left standing for 12 hours in a water bath which is controlled at a predetermined temperature (constant temperature). Thereafter, when the test tube is turned upside down, in a case where the interface (meniscus) between the solution and air is deformed (inclusive a case wherein the solution flows out from the test tube) due to the weight of the solution per se, the above polymer solution is defined as a "sol state" at the above-mentioned predetermined temperature.
On the other hand, in a case where the interface (meniscus) between the solution and air is not deformed due to the weight of the solution per se, even when the test tube is turned upside down, the above polymer solution is defined as a "gel state" at the above-mentioned predetermined temperature.
In addition, in a case where a hydrogel in a sol state (solution) having a concentration of, e.g., about 8 mass % is used in the above-mentioned measurement, and the temperature at which the "sol state" is converted into the "gel state" is determined while gradually increasing the above "predetermined temperature" (e.g., in 1 0C increment), the thus determined transition temperature is defined as a "sol-gel transition temperature". At this time, alternatively, it is also possible to determine the above sol-gel transition temperature at which the "gel state" is converted into the "sol state" while gradually decreasing the "predetermined temperature" (e.g., in 1 0C decrement).
(Sol-gel transition temperature)
In the present invention, the definition and measurement of the "sol state," "gel state," and "sol-gel transition temperature" may also be carried out as mentioned below according to the definition and method described in a publication (H. Yoshioka et al, Journal of Macromolecular Science, A31(l), 113 (1994)).
That is, the dynamic elastic modulus of a sample at an observed frequency of 1 Hz is determined by gradually shifting the temperature from a low temperature side to a high temperature side (1°C/1 min). In this measurement, the sol-gel transition temperature is defined as a temperature at which the storage elastic modulus (G', elastic term) of the sample exceeds the loss elastic modulus (G", viscous term). In general, the sol state is defined as a state in which G" > G' is satisfied, and the gel state is defined as a state in which G" < G' is satisfied. For the measurement of such a solτgel transition temperature, the following measuring conditions can preferably be used.
<Measuring conditions for dynamic and loss elastic moduli> Measuring apparatus (trade name): Stress controlled-type rheometer (model: CSL-500, mfd. by Carri-Med Co.)
Concentration of sample solution (or dispersed liquid) (as a concentration of a "polymer compound having a sol-gel transition temperature"): 10% (by weight) Amount of sample solution: about 0.8 g
Shape and size of cell for measurement: acrylic parallel disk (diameter: 4.0 cm), gap: 600 μm
Measurement frequency: 1 Hz
Stress to be applied: within linear region (Preferred sol-gel transition temperature)
In the present invention, the above sol-gel transition temperature may preferably be higher than 0 0C and not higher than 45 °C, more preferably, higher than 0 0C and not higher than 42 0C ( particularly not lower than 4 °C and not higher than 40 0C) in view of the prevention of a thermal damage to cells or a tissue of a living organism. The hydrogel material having such a preferred sol-gel transition temperature may easily be selected from specific compounds as described below, according to the above-mentioned screening method (method of measuring the sol-gel transition temperature).
In a sequence of operations wherein a tissue or organ of a living organism is regenerated by using the carrier according to the present invention, it is preferred to set the above-mentioned sol-gel transition temperature (a 0C) between the temperature at the time of the culturing of the cell or tissue (b 0C), and the temperature at the time of the cooling for the inoculation, mixing or recovery of the cell or tissue (c 0C). In other words, the above-mentioned three kinds of temperatures of a 0C, b 0C and c 0C may preferably have a relationship of b > a > c. More specifically, the value of (b - a) may preferably be 1 - 40 0C, more preferably 2 - 30 0C. On the other hand, the value of (a - c) may preferably be 1 - 40 0C, more preferably 2 - 30 0C. (Movement-following property of carrier)
In view of the balance between the property of the hydrogel based on the carrier according to the present invention, and the property of the carrier for following a change in the form or shape of the tissue along with the regeneration, it is preferred that the hydrogel based on the carrier according to the present invention shows a behavior in a solid-like manner toward a higher frequency, and that the carrier shows a behavior in a liquid-like manner toward a lower frequency. More specifically, the property of the carrier for following the movements may preferably be measured according to the following method. (Method of measuring movement-following property)
The carrier according to the present invention comprising a hydrogel-forming polymer in a sol state (i.e., at a temperature lower than the sol-gel transition temperature) is poured into a test tube having an inside diameter of 1 cm, in an amount of the carrier corresponding to a volume of 1 rnL as the resultant hydrogel. Then, the above test tube is left standing for 12 hours in a water bath which is controlled at a temperature which is sufficiently higher than the sol-gel transition temperature of the carrier (e.g., a temperature which is 10 0C higher than the sol-gel transition temperature), whereby the hydrogel material is converted into a gel state.
Then, when the test tube is turned upside down, there is measured the time (T) until the interface (meniscus) between the solution and air is deformed due to the weight of the solution per se. Herein, the hydrogel will show a behavior in a liquid-like manner toward a movement having a frequency lower than 1/T (sec"1), and the hydrogel will show a behavior in a solid-like manner toward a movement having a frequency higher than 1/T (sec"1). In the case of the hydrogel according to the present invention, T may preferably be 1 minute to 24 hours, more preferably 5 minutes to 10 hours; (Steady-state flow kinematic viscosity)
Alternatively, the gel property of the hydrogel based on the carrier according to the present invention may preferably be determined by measuring the steady-state flow kinematic viscosity thereof. For example, the steady-state flow kinematic viscosity η (eta) may be measured by using a creep experiment.
In the creep experiment, a predetermined shear stress is imparted to a sample, and a time-dependent change in the resultant shear strain is observed. In general, in the creep behavior of viscoelastic material, the shear rate is changed with the elapse of time in an initial stage, but thereafter shear rate becomes constant. The Steady-state flow kinematic viscosity η is defined as the ratio of the shear stress and the shear rate at this time. This Steady-state flow kinematic viscosity can also be called Newtonian viscosity. However, it is required that the Steady-state flow kinematic viscosity is determined in the linear region wherein the viscosity little depends on the shear stress.
In a specific embodiment of the measuring method, a stress-controlled type viscoelasticity-measuring apparatus (model: CSL-500, mfd. by Carri-Med Co., USA) is used as the measuring apparatus, and an acrylic disk (having a diameter of 4 cm) is used as the measuring device, and the resultant creep behavior (delay curve) is measured for at least five minutes with respect to a sample having a thickness of 600 μm. The sampling time is once per one second for the initial 100 seconds, and once per ten seconds for subsequent period.
When the shear stress (stress) to be applied to the sample is determined, the shear stress should be set to a minimum value such that a displacement angle of 2x 10"3 rad or more is detected, when such a shear stress is loaded for ten seconds counted from the initiation of the measurement. When the resultant data is analyzed, at least 20 or more measured values are adopted with respect to the measurement after five minutes. The hydrogel based on the carrier according to the present invention may preferably have an η of 5xl03-5xl06 Pa sec, more preferably 8xl03-2xl06 Pa sec, particularly, not less than IxIO4 Pa sec and not more than 1x10 Pa sec, at a temperature which is about
10 0C higher than the sol-gel transition temperature.
When the above η is less than 5x103 Pa sec, the fluidity becomes relatively high even in a short-time observation, and the three-dimensional supporting of the cell or tissue by the gel is liable to be insufficient, and therefore, the hydrogel is less liable to function as a carrier in some cases. On the other hand, when η exceeds 5x10 Pa sec, the tendency that the gel shows little fluidity even in a long-time observation is strengthened, and difficulty in the movement-following property of a tissue of a living organism is increased. In addition, when η exceeds 5x106 Pa sec, the possibility that the gel shows a fragility is strengthened, and the tendency of brittle fracture that, after a slight pure elastic deformation, the gel is easily destroyed at a stroke is strengthened. (Dynamic elastic modulus)
Alternatively, the gel property of the hydrogel based on the carrier according to the present invention may preferably be determined by measuring the dynamic elastic modulus thereof. Provided that when a strain γ (t) = γ0 cos ωt (t is time) having an amplitude γ0, number of vibrations of ω/2π to the gel, a stressσ (t) = σ0cos (cot+ δ) having a constant stress of σ0 and a phase difference of δ is obtained. When | G = σ0/ γ0, the ratio (G1VG') between the dynamic elastic modulus G '(co) = |G| cos δ and the loss elastic modulus G "(ω) = |G| sin δ is an indicator showing the degree of gel property.
The hydrogel based on the carrier according to the present invention behaves as a solid toward a stress of ω/2π = 1 Hz (corresponding to a fast movement), and behaves as a liquid toward a stress of ω/2π =10"4 Hz (corresponding to a slow movement). More specifically, the hydrogel based on the carrier according to the present invention may preferably show the following property (with respect to the details of the method of measuring elastic modulus, e.g., literature: "Modern Industrial Chemistry" (Kindai Kyogyo Kagaku) No. 19, edited by Ryohei Oda, et al., Page 359, published by Asakura
Shoten, 1985 may be referred to).
In the case of ω/2π = 1 Hz (number ofvibrations at which the gel behaves as a solid), the ratio (G "/G ')s = (tan δ)s may preferably be less than 1 (preferably 0.8 or less, particularly, 0.5 or less). In the case of ω/2π =10" Hz (number of vibrations at which the gel behaves as a liquid), the ratio (G "/G ')L = (tan δ)L may preferably be 1 or more (preferably 1.5 or more, particularly, 2 or more).
The ratio {(tan δ)s/ (tan δ)L} between the above (tan δ)s and (tan δ)L may preferably be less than 1 (mire preferably 0.8 or less, particularly, 0.5 or less). <Measurement conditions>
Concentration of hydrogel-forming polymer (carjier): about 8 mass %
Temperature: a temperature which is about 10 0C higher than the sol-gel transition temperature of the carrier
Measuring apparatus: Stress controlled-type rheometer (model: CSL-500, mfd. by Carri-Med Co., USA) (Control of residual property in living body)
It is possible to arbitrarily control the residual (or remaining) property of the hydrogel according to the present invention in a living body (in the abdominal cavity, the subcutis, etc.), as desired. The hydrogel according to the present invention is mainly intended to be used in vitro. However, depending on its usage (for example, when a tissue which has been grown by using the hydrogel according to the present invention are returned into a living body), there are cases where the control of the in vivo remaining property of the hydrogel is preferred.
If the sol-gel transition temperature of the hydrogel according to the present invention is decreased, the hydrogel tends to remain in a living body for a long period of time. In contrast, if the sol-gel transition temperature of the hydrogel according to the present invention is increased, the hydrogel tends to rapidly disappear in a living body.
Further, if the concentration of the hydrogel-forming polymer in the hydrogel is increased, the hydrogel tends to remain in a living body for a long period of time. If the concentration of the hydrogel-forming polymer in the hydrogel is decreased, the hydrogel tends to rapidly disappear in a living body.
In the hydrogel according to the present invention, if the sol-gel transition temperature of the hydrogel according to the present invention is decreased, the storage elastic modulus (G') of the hydrogel at a living body temperature (370C) is increased. Further, if the concentration of the hydrogel-forming polymer in the hydrogel is increased, the storage elastic modulus (G') of the hydrogel at a living body temperature (370C) is increased. That is, in order to control the residual property of the hydrogel in a living body, G' at 37°C may be controlled.
To measure the value of G', the following measuring conditions can preferably be used.
<Conditions for measurement of dynamic and loss elastic moduli>
Measuring apparatus (trade name): Controlled stress rheometer CSL 500 mfd. by Carri-Med Co.
Amount of sample solution: about 0.8 g Shape and size of cell for measurement: acryl parallel disk (diameter: 4.0 cm), gap: 600 μm
Measurement frequency: 1 Hz Stress to be applied: within linear region The relationship between the residual period for the hydrogel according to the present invention in a living body, and G' is also dependent on the site or portion therefor in a living body. However, according to the findings of the present inventors, for example, the relationship between the residual period for the hydrogel in the abdominal cavity and G' at an observation frequency of 1 Hz is as follows. That is, the desired range of G' for providing the hydrogel disappearance of 3 days or less is 10 to 500 Pa. The desired range of G' for providing the hydrogel disappearance of the hydrogel remaining for a period of not less than 3 days and not more than 14 days is 200 to 1,500 Pa. The desired range of G1 for providing the hydrogel disappearance of the hydrogel remaining for a period of more than 14 days is 400 to 10,000 Pa.
(Fibroblast growing property)
In the present invention, fibroblasts exhibit substantially no growth in the hydrogel based on the hydrogel-forming polymer constituting the carrier. In general, when fibroblasts are subjected to monolayer culture on a dish (plate) for cell culture or are cultured in a collagen gel, the fibroblasts are significantly grown so as to provide a change thereof into an arboroid form peculiar to the fibroblasts (e.g., Jyunpei Enami, Baiyosaibo o Mochiiru Hoho (Method of using cultured cells); edited by Meiji Saito, Saibogai Matrix (Extracellular Matrix), published by Medical Review Co,, Ltd. (Tokyo), 1996, pp. 108-115, may be referred to) On the contrary, in the hydrogel according to the present invention, fibroblasts maintain a spherical form thereof and they exhibit substantially no growth. (Presumed mechanism for inhibition of growth of fibroblasts)
The mechanism of the fibroblast growth inhibition in the cell or tissue-culturing carrier according to the present invention is not necessarily clear, the mechanism may be presumed in the following manner according to the findings of the .present inventors. That is, a fibroblast has a property that it recognizes a monolayer culture, i.e., the surface of a supporting medium, and adheres thereto, whereby it actively grows two-dimensionally. A collagen gel has a structure such that a large number of collage molecules (molecular weight: 300,000) with a length of 300 nm and a diameter of 1.5 nm are aggregated and are regularly arranged, and that they become collagen fibril and form a network structure in water. Since this network structure is greater than the wavelength of visible radiation (400 nm or more), the collagen gel generally looks clouded or turbid. The collagen gel is used as a carrier for a three-dimensional culture. It is presumed that since a fibroblast recognizes the surface of a thick collagen fibril as a supporting medium and adheres thereto, this cell significantly grows two-dimensionally in the collagen gel.
In contrast, in the cell or tissue-culturing carrier according to the present invention, since the hydrogel is constituted such that a hydrogel-forming polymer in a molecular state forms a three-dimensional network structure, the heterogeneity of the structure is smaller than that of the wavelength of visible radiation, and it has a relatively high transparency. Accordingly, it is presumed that fibroblasts do not clearly recognize the surface of a two-dimensional supporting medium in the material according to the present invention, and that as a result, an excessive growth of fibroblasts is inhibited in the material according to the present invention. (Evaluation of growing property of fibroblasts)
The growth of fibroblasts can be evaluated by the following method (with respect to the details thereof of this method, e.g., Tsuyoshi Yoshikawa, Ken Tsukikawa, St. Marianna University, School of Medicine, Journal Vol. 28, No. 4, pp. 161-170 (2000) may be referred to). A hydrogel-forming polymer constituting the cell or tissue-culturing carrier according to the present invention is dissolved in a culture solution such as RPMI- 1640 (Life Technologies, N. Y., USA) at a low temperature (for example, 40C), under stirring. Thereafter, normal human lung fibroblasts (NHLF, mfd. by Takara Shuzo Co., Ltd.) are dispersed in the above solution, so that the cell density is set to 6 x 104 cells/mL. 0.2 mL of the resultant NHLF dispersion is.poured into each well of a 24-well plate (material: plastic; the size of a well: about 15 mm long, 15 mm wide, and 20 mm depth; e.g., a commercial item such as Multiwell (trade name) mfd. by Becton-Dickinson), and then is formed into a gel state at 37°C. Thereafter, 0.4 mL of culture solution is added thereto, and then is cultured at 370C under 5% CO2, atmospheric pressure. The growth of fibroblasts is observed along with the elapse of time (e.g., on the Oth, 1st, 3rd and 7th days after the day of culture), by using a phase-contrast microscope. (Growth rate of fibroblasts)
Further, the growth rate of fibroblasts can be determined in the culturing period by the following method using an enzyme activity. For example, a 24-well plate as described above is used, and fibroblasts are cultured thereby for a certain period of time in the cell or tissue-culturing carrier according to the present invention, and the temperature of the carrier is decreased to a temperature lower than the sol-gel transition temperature thereof (e.g., a temperature which is 100C lower than the sol-gel transition temperature), so as to dissolve the carrier. Thereafter, 50 μl of a WST-8 reagent (mfd. by Dojin Kagaku (Dojindo Laboratories)) as a reagent for determining the activity of succinate dehydrogenase is added to each of the wells.
The thus prepared 24-well plate is subjected to a reaction at a temperature which is lower than the sol-gel transition temperature (e.g., a temperature which is 100C lower than the sol-gel transition temperature, for example, at 100C) for 10 hours, and it is then retained at about 40C for 1 hour, so that a completely homogenous aqueous solution is prepared. 200 μl of each of the thus obtained aqueous solution is poured into each well of a 96-well plate. The resultant absorbance (OD (450)) is measured at 450 nm
(reference wavelength: 620 nm) by using a chromatometer for microplates. It has been confirmed that there is a proportional relationship between the thus obtained OD (450) and the number of vital cells (e.g., Furukawa T. et al., "High in vitro-in vitro correlation of drug response using sponge gel-supported three-dimensional histoculture and MTT end point," Int. J. Cancer 51: 489, 1992 may be referred to). That is, the growth rate of fibroblasts is obtained as a ratio (ODL/ODf) between the absorbance at the beginning of the culture ODf = (OD (450)) and the absorbance after the culture ODL = (OD (450)). . In the present invention, the growth rate of fibroblasts PF = (ODL/ODt) which has been obtained after the culture thereof at 370C for 3 days may preferably be within the range of not lower than 70 % and not higher than 200 %. The growth rate (ODL OD,-) is ■ more preferably within the range of not lower than 80 % and not higher than 150 %, and particularly preferably within the range not lower than 90 % and not higher than 120 %. (Relative growing property of fibroblasts)
In the present invention, it is preferred that in gel based on the hydrogel-forming polymer, the growth of intended cells (other than fibroblasts) is not inhibited relatively, while the growth of fibroblasts is inhibited. More specifically, the ratio (PT/PF) between the growth rate of the intended- cells Pτ and the growth rate of the above fibroblasts PF may preferably be 1.1 or more. The ratio (PJ/PF) may more preferably be 1.5 or more, and particularly preferably 2 or more. The growth rate Pτ of the intended cells can be determined as follows.
The growth rate of cells other than fibroblasts Pτ may be determined in the same manner as in the above determination of the growth rate of fibroblasts PF except that human colon cancer cells (SW-948, trade name: Colonic Adenoma Cell Lines, mfd. by Dainippon Pharmaceutical Co., Ltd.) are used instead of normal human lung fibroblasts (NHLF) used in the above determination of the fibroblast growth rate PF. (The growth rates PT and PF are determined under the same conditions). The ratio (Pτ/PF) is obtained from the values of the growth rates Pτ and PF as determined above. (Hydrogel-forming polymer)
The hydrogel-forming polymer usable for the carrier according to the present invention is not particularly limited, as long as the polymer exhibits the above-mentioned thermo -reversible sol-gel transition (that is, as long as it has a sol-gel transition temperature). It is preferable to achieve a preferred sol-gel transition temperature by adjusting the cloud point of a plurality of blocks having a cloud point and the cloud point of a hydrophilic block in the hydrogel-forming polymer, the compositions, hydrophobicity or hydropbϋlicity of both types of blocks, and/or their ' molecular weights, in view of easy exhibition of a preferred sol-gel transition at a physiological temperature (about O0C to 420C).
As specific examples of the polymer such that an aqueous solution thereof has a sol-gel transition temperature, and it reversibly assumes a sol state at a temperature lower than the sol-gel transition temperature., there have been known, e.g., polyalkylene-oxide block copolymer represented by block copolymers comprising polypropylene oxide portions and polyethylene oxide portions; etherified (or ether group-containing) celluloses such as methyl cellulose and hydroxypropyl cellulose; chitosan derivatives (K. R. Holme, et al. Macromolecules, 24, 3828 (1991)), etc. In addition, there has been developed a gel utilizing Pluronic F- 127 (trade name, mfd. by BASF Wyandotte Chemical Co.) comprising a polypropylene oxide portion and polyethylene oxide portions bonded to the both terminals thereof.
It is known that a high-concentration aqueous solution of the above Pluronic F-127 is converted into a hydrogel at a temperature of not lower than about 20 0C, is converted into an aqueous solution at a temperature lower than this temperature. However, this material can assume a gel state only at a high concentration of not lower than about 20 wt. %. In addition, even when -such a gel having a high concentration of not lower than about 20 wt. % is maintained at a temperature higher than the gel-forming temperature, the gel is dissolved when water is further added thereto. In addition, since the molecular weight of the Pluronic F-127 is relatively low, and it shows an extremely high osmotic pressure at a high concentration of not less than about 20 wt. %, and simultaneously the Pluronic F-127 may easily permeate the cell membranes, whereby the Pluronic F-127 can adversely affect cells and microorganisms. On the other hand, in the case of an etherified cellulose represented by methyl cellulose, hydroxypropyl cellulose, etc., the sol-gel transition temperature thereof is as high as about 45 0C or higher (N. Sarkar, J. Appl. Polym. Science, 24, 1073, (1979)). Accordingly, such an etherified cellulose is less liable to form a gel at body temperature (about 38 0C), and therefore it is difficult to use such a material for the above-mentioned purposes according to the present invention. As described above, when a conventional polymer having a sol-gel transition temperature in an aqueous solution thereof, and reversibly assuming a sol state at a temperature lower than the above transition temperature is simply used, the following problems are posed: (1) If the polymer is once converted into a gel state at a temperature higher than the sol-gel transition temperature, the resultant gel is dissolved when water is further added thereto;
(2) The polymer has a sol-gel transition temperature higher than the body temperature (in the neighborhood of 38 0C), and therefore the polymer assumes a sol state in the interior of a living body;
(3) It is necessary to increase the concentration of the polymer in an aqueous solution thereof to an extremely high value, in order to convert the polymer into a gel state; etc.
On the other hand, according to the present inventor's investigation, it has been found that the above problem can be solved by constituting-the carrier according to the present invention by use of a polymer having a sol-gel transition temperature of higher than 0 0C and not higher than 42 0C (e.g., a polymer which comprises a plurality of polymer chains having a cloud point, and a hydrophilic polymer chain block which has
' been bonded thereto; and an aqueous solution of which has a sol-gel transition temperature, and reversibly assumes a sol state at a temperature lower than the sol-gel transition temperature).
(Preferred hydrogel-forming polymers)
The hydrogel-forming polymer preferably usable as the carrier according to the present invention may preferably comprise a combination of plural hydrophobic blocks having a cloud point, and a hydrophilic block bonded thereto. The presence of the hydrophilic block is preferred in view of the provision of the water-solubility of the hydrogel material at a temperature lower than the sol-gel transition temperature. The presence of the plural hydrophobic block having a cloud point is preferred in view of the conversion of the hydrogel material into a gel state at a temperature higher than the sol-gel transition temperature. In other words, the blocks having a cloud point become . water-soluble at a temperature lower than the cloud point, and are converted into a water-insoluble state at a temperature higher than the cloud point, and therefore these blocks function as crosslinking points constituted by hydrophobic bonds for forming a gel at a temperature higher than the cloud point. That is, the cloud point based on the hydrophobic bonds corresponds to the above-mentioned sol-gel transition temperature
• ofthe hydrogel.
However, it is not always necessary that the cloud point corresponds to the sol-gel transition temperature. This is because the cloud point of the above-mentioned "blocks having a cloud point" is generally influenced by the bonding between the hydrophilic block and the blocks having a cloud point.
The hydrogel to be use in the present invention utilizes a property of hydrophobic bonds such that they are not only strengthened along with an increase in temperature, but also the change in the hydrophobic bond strength is reversible with respect to the temperature. In view of the formation of plural crosslinking points in one molecule, and the formation of a gel having a good stability, the hydrogel-forming polymer may preferably have a plurality of "blocks having cloud point".
On the other hand, as described above, the hydrophilic block in the hydrogel-forming polymer has a function of causing the hydrogel-forming polymer to be changed into a water-soluble state at a temperature lower than sol-gel transition temperature. The hydrophilic block also has a function of providing the state of an aqueous (or water-containing) gel, while preventing the aggregation and precipitation of the hydrogel material due to an excess increase in the hydrophobic binding force at a temperature higher than the transition temperature. (Plural blocks having cloud point) The plural block having a cloud point may preferably comprise a polymer block which shows a negative solubility-temperature coefficient with respect to water. More specifically, such a polymer may preferably be one selected from the group consisting of: polypropylene oxide, copolymers comprising propylene oxide and another alkylene oxide, poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers comprising an N-substituted acrylamide derivative and an N-substituted methacrylamide derivative, polyvinyl methyl ether. and partially-acetylated product of polyvinyl alcohol.
In order to prepare a block having a cloud point which is decomposed and absorbed in a living body, it is effective to use a polypeptide comprising a hydrophobic . amino acid and a hydrophilic amino acid, as the block having a cloud point. Alternatively, a polyester-type biodegradable polymer such as polylactic acid or polyglycolic acid can also be used as a block having a cloud point which is decomposed and absorbed in a living body.
It is preferred that the above polymer (block having a cloud point) has a cloud point of higher than 4 0C and not higher than 40 0C, in view of the provision of a polymer (compound comprising a plurality of blocks having a cloud point, and a hydrophilic block bonded thereto) to be used in the present invention having a sol-gel transition temperature of higher than 4 0C and not higher than 40 0C.
It is possible to measure the cloud point, e.g., by the following method. That is. an about 1 wt.%-aqueous solution of the above polymer (block having a cloud point) is cooled to be converted into a transparent homogeneous solution, and thereafter the temperature of the solution is gradually increased (temperature increasing rate: abou.: 1 °C/min.), and the point at which the solution first shows a cloudy appearance i • defined as the cloud point. Specific examples of the poly N-substituted acrylamide derivatives and po!"
N-substituted methacrylamide derivatives are described below. Poly-N-acryloyl piperidine Poly-N-n-propyl methacrylamide Poly-N-isopropyl acrylamide Poly-N,N-diethyl acrylamide
Poly-N-isopropyl methacrylamide Poly-N-cyclopropyl acrylamide Poly-N-acryloyl pyrrolidine Poly-N,N-ethyl methyl acrylamide Poly-N-cyclopropyl methacrylamide Poly-N-ethyl acrylamide
The above polymer may be either a homopolymer or a copolymer comprising a monomer constituting the above polymer and "another monomer". The "another monomer" to be used for such a purpose may be either a hydrophilic monomer, or a hydrophobic monomer. In general, when copolymerization with a hydrophilic monomer is conducted, the resultant cloud point may be increased. On the other hand, when copolymerization with a hydrophobic monomer is conducted, the resultant cloud point may be decreased. Accordingly, a polymer having a desired cloud point (e.g., a cloud point of higher than 4 0C and not higher than 40 °C ) may also be obtained by selecting such a monomer to be used for the copolymerization. (Hydrophilic monomer)
Specific examples of the above hydrophilic monomer may include: N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydro xyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, methacrylic acid and acrylic acid having an acidic group, and salts of these acids, vinyl sulfonic acid, styrenesulfonic acid, etc., and derivatives having a basic group such as N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, N,N-dirnethylaminopropyl acrylamide, salts of these derivatives, etc. However, the hydrophilic monomer to be usable in the present invention is not restricted to these specific examples. (Hydrophobic monomer)
On the other hand, specific examples of the above hydrophobic monomer may include: acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, and glycidyl methacrylate; N-substituted alkyl methacrylamide derivatives such as N-n-butyl methacrylamide; vinyl chloride, acrylonitrile, styrene, vinyl acetate, etc. However, the hydrophobic monomer to be usable in the present invention is not restricted to these specific examples. (Hydrophilic block) On the other hand, specific examples of the hydrόphilic block to be combined with (or bonded to) the above-mentioned block having a cloud point may include: methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly N-methyl acrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, and salts of these acids; poly N,N-dimethylaminoethyl methacrylate, poly N,N-diethylaminoethyl methacrylate, poly N,N-dimethylaminopropyl acrylamide, and salts of these, etc.
The process for combining the above block having a cloud point with the hydrophilic block is not particularly limited. For example, it is preferred to obtain a block copolymer, or a graft copolymer, or a dendrimer-type copolymer containing these blocks.
It is also possible to conduct such a combination by introducing a polymerizable functional group (such as acryloyl group) into either one of the above blocks, and copolymerizing with the resultant product a monomer capable of providing the other block.
Alternatively, it is also possible to obtain a combination product of the above block having a cloud point with the hydrophilic block by copolymerizing a monomer capable of providing the block having a cloud point with a monomer capable of providing the hydrophilic block.
In addition, the block having a cloud point and the hydrophilic block may also be combined or bonded with each other by preliminarily introducing reactive functional groups (such as hydroxyl group, amino group, carboxyl group, and isocyanate group) into both kinds of the blocks, and combining these blocks by using a chemical reaction. At this time, it is usual to introduce a plurality of reactive functional groups into the hydrophilic block.
Further, the polypropylene oxide having a cloud point and the hydrophilic block may be combined or bonded with each other by repetitively subjecting polypropylene oxide and a monomer constituting the above "other water-soluble block" (such as ethylene oxide) to a stepwise or consecutive polymerization, to thereby obtain a block copolymer comprising polypropylene oxide and a water-soluble block (such as polyethylene oxide) combined therewith.
Such a block copolymer may also be obtained by introducing a polymerizable group (such as acryloyl group) into the terminal of polypropylene oxide, and then copolymerizing therewith a monomer constituting the hydrophilic block.
Further, a polymer usable in the present invention may be obtained by introducing a functional group which is reactive in a bond-forming reaction with the terminal functional group of polypropylene oxide (such as hydroxyl group) into a hydrophilic block, and reacting the resultant hydrophilic block and the polypropylene oxide. In addition, a hydrogel-forming polymer usable in the present invention may be obtained by connecting materials such as one comprising polypropylene glycol and polyethylene glycol bonded to both terminals thereof (such as Pluronic F-127; trade name, mfd. by Asahi Denka Kogyo K.K.).
In an embodiment of the present invention wherein the hydrogel-forming polymer comprises a block having a cloud point, at a temperature lower than the cloud point, the polymer may completely be dissolved in water so as to assume a sol state, since the above-mentioned "block having a cloud point" present -in the polymer molecule is water-soluble together with the hydrophilic block. However, when a solution of the above polymer is heated up to a temperature higher than the cloud point, the "block having a cloud point" present in the polymer molecule becomes hydrophobic so that separate molecules of the polymer are associated or aggregated with each other due to a hydrophobic interaction.
On the other hand, the hydrophilic block is water-soluble even at this time (i.e., even when heated up to a temperature higher than the cloud point), and therefore, the polymer according to the present invention in water is formed into a hydrogel having a three-dimensional network structure wherein hydrophobic association portions between the blocks having a cloud point constitute the crosslinking points. The resultant hydrogel is again cooled to a temperature lower than the cloud point of the "block having a cloud point" present in the polymer molecule, the block having a cloud point becomes water-soluble and the above crosslinking points due to the hydrophobic association are released or liberated so that the hydrogel structure disappears, whereby the polymer according to the present invention is again formed into a complete aqueous solution. In the above-described manner, the sol-gel transition in the ' polymer according to the present invention is based on the reversible hydrophilic-hydrophobic conversion in the block having a cloud point present in the polymer molecule at the cloud point, and therefore the transition has a complete reversibility in accordance with a temperature change. (Solubility of gel)
As described above, the hydrogel-forming polymer according to the present invention comprising at least a polymer having a sol-gel transition temperature in an aqueous solution thereof, substantially shows a water insolubility at a temperature (d 0C) higher than the sol-gel transition temperature, and reversibly shows water solubility at a temperature (e 0C) lower than the sol-gel transition temperature.
The above-mentioned temperature (d 0C) may preferably be a temperature which is at least 1 °C, more preferably at least 2 0C (particularly preferably, at least 5 11C) higher than the sol-gel transition temperature. Further, the above-mentioned "substantial water insolubility" may preferably be a state wherein the amount of the above polymer to be dissolved in lOO ml of water at the above temperature (d °C) is 5.0 g or less (more preferably 0.5 g or less, particularly preferably 0.1 g or less). On the other hand, the above-mentioned temperature (e 0C) may preferably be a temperature which is at least 1 0C, more preferably at least 2 0C (particularly preferably, at least 5 0C) lower than the sol-gel transition temperature, in terms of the absolute values of these temperatures. Further, the above-mentioned "water solubility" may preferably be a state wherein the amount of the above polymer to be dissolved in 100 ml of water at the above temperature (e 0C) is 0.5 g or more (more preferably 1.0 g or more). The above "to show a reversible water solubility" refers to a state wherein an aqueous solution of the above hydrogel-forming polymer shows the above-mentioned water solubility at a temperature lower than the sol-gel transition temperature, even after the polymer is once formed into a gel state (at a temperature higher than the sol-gel transition temperature). A 10%-aqueous solution of the above polymer may preferably show a viscosity of 10 - 3,000 Pa s (10 - 3,000 centipoises), more preferably, 50 - 1,000 Pa s (50 - 1,000 centipoises) at 5 0C. Such a viscosity may preferably be measured, e.g., under the following measurement conditions: Viscometer: Stress-controlled type rheometer (model: CSL-500, mfd. by
Carri.Med Co., USA)
Rotor diameter: 60 mm
Rotor configuration: Parallel-plate type
Measurement frequency: 1 Hz (hertz) Even when the an aqueous solution of the hydrogel-forming polymer according to the present invention is formed into a gel state at a temperature higher than the sol-gel transition temperature, and thereafter the resultant gel is immersed in a large amount of water, the gel is not substantially dissolved in water. For example, such a characteristic of the above carrier may be confirmed in the following manner. More specifically, 0.15 g of the hydrogel-forming polymer according to the present invention is dissolved in 1.35 g of distilled water at a temperature lower than the above sol-gel transition temperature (e.g., under cooling with ice) to thereby prepare a 10 mass %-aqueous solution. Then, the resultant solution is poured into a plastic Petri dish having a diameter of 35 mm, then the dish is warmed up to a temperature of 37 0C to form a gel having a thickness of about 1.5 mm in the dish, and the total weight of the Petri dish (f gram) containing the gel is measured. Then, the entirety of the Petri dish containing the gel is left standing in 250 ml of water at 37 0C for 10 hours, and thereafter the total weight of the Petri dish (g gram) containing the gel is measured, to thereby determine whether the gel has been dissolved from the gel surface or not. At this time, in the hydrogel-forming polymer according to the present invention, the ratio of weight decrease in the gel, i.e., the value of {(f-g)/f) may preferably be 5.0 % or less, more preferably 1.0 % or less (particularly preferably 0.1 % or less).
Even when an aqueous solution of the hydrogel-forming polymer according to the present invention was converted into a gel state at a temperature higher than the sol-gel transition temperature, and then the resultant gel was immersed in a large amount (about 0.1 - 100 times larger than the gel, by volume ratio), the gel was not dissolved for a long period of time. Such a property of the polymer to be used in the present invention may be achieved, e.g., by the presence of at least two (a plurality of) blocks having a cloud point in the polymer molecule. On the contrary, according to the present inventors' experiments, in a case where a similar gel was formed by using the above-mentioned. Pluronic F- 127 comprising polypropylene oxide and polyethylene oxide bonded to both terminals thereof, the resultant gel was completely dissolved when the gel is left standing in water for several hours. In order to suppress the cytotoxicity of a non-gel state to a low level as completely as possible, it is preferred to use a hydrogel-forming polymer which can be converted into a gel state at a concentration of 20% or less (more preferably 15% or less, particularly 10% or less) in terms of the concentration of the polymer based on water, i.e., {(polymer)/(polymer + water)} x 100 (%). (Other components)
The carrier according to the present invention comprises at least the above-mentioned polymer having a sol-gel transition temperature. However, the carrier may also comprise another component, as desired. Specific examples of "other components" in such an embodiment may include: antibiotics, anticancer or antitumor substances, ECM such as collagen and gelatin, local chemical mediators appearing hereinafter, hormones such as insulin and growth factors, foreign genes, etc.; and other cells or tissues capable of secreting these chemical mediators and cell growth factors, etc.
The use amount of such "other components" is not particularly limited, as long as it exhibits an intended effect and can be retained in the gel based on the hydrogel-forming polymer for a certain period of time (e.g., for a period necessary for culturing cells or tissue). In general, the amount of the other component to be used may preferably be 2 parts or less, and more preferably 1 part or less, based on the total parts (10 parts) of the hydrogel-forming polymer. (Chemical mediator) There are some cases where the regeneration of a living organism tissue requires not only cells such as precursor cells but also various chemical mediators such as cell growth factor which promotes the differentiation or the growth of the cells. The chemical mediator is generally secreted from cells. However, in order to efficiently conduct the regeneration, it is effective to previously add such a chemical mediator to the cell or tissue-culturing carrier according to the present invention, so as to supply the chemical mediator from the outside of the living organism tissue.
Examples of the above-mentioned chemical mediator may include: 1) local chemical mediators which can act extremely in the vicinity of the cell; T) neurotransmitters which are secreted by nerve cells and have a extremely short effective acting distance; 3) hormones which are secreted by endocrine cells and systemically act on target cells through bloodstream, etc.; and the like.
Examples of 1) local chemical mediator as described- above may include: proteins such as nerve cell growth factors, peptides such as chemotaxis factors, amino acid derivatives such as histamine, fatty acid derivatives such as prostaglandins, etc.
Examples of 2) neurotransmitter as described above may include: low-molecular weight substances including amino acids such as glycine, low-molecular peptides such as noradrenaline, acetylcholine, and enkephalin, etc.
Examples of 3) cell growth factor or hormones as described above may include: cell growth factors such as fibroblast growth factor (FGF), epithelial growth factor
(EGF), vascular endothelial growth factor (VEGF) and hapatocyte growth factor (HGF); proteins such as insulin, somatotropin, somatomedin, adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH), and thyroid-stimulating hormone (TSH); glycoproteins, amino acid derivatives such as somatostatin, vasopressin, TSH releasing factor; steroids such as Cortisol, estradiol, testosteron; etc. (Diffusion of chemical mediator in gel)
In the hydrogel according to the present invention, it is possible to arbitrarily control the diffusion rate of a chemical mediator in the hydrogel. Especially, a hydrophilic substance and a hydrophobic substance can be diffused at different diffusion rates. The diffusion of a water-soluble hydrophilic substance is controlled by the molecular sieving effect of the three-dimensional network structure of the hydrogel-forming polymer. Accordingly, in order to reduce the diffusion rate of the ' water-soluble hydrophilic substance, the concentration of the hydrogel-forming polymer constituting the hydrogel may be increased. Further, the diffusion of the water-soluble hydrophilic substance also is dependent on the molecular weight of the substance. When the concentration of the hydrogel-forming polymer constituting the hydrogel is constant, as the molecular weight of a substance is increased, the diffusion - rate thereof becomes lower.
The diffusion of a water-soluble hydrophobic substance in the hydrogel according to the present invention is influenced not only by the molecular sieving effect of the three-dimensional network structure of the hydrogel-forming polymer, but also by the distribution or partition thereof with respect to the hydrophobic portion of the hydrogel-forming polymer. Thus, the diffusion of the water-soluble hydrophobic substance is also controlled by the ratio of the hydrophobic portion in the hydrogel-forming polymer, and therefore, the diffusion of the water-soluble hydrophobic substance is generally slower than that of the water-soluble hydrophilic substance.
The diffusion coefficient of a solute in the hydrogel can be obtained by the "early-time" approximation described in a publication, (Eric K. L. Lee et al, Journal of Membrane Science, 24, 125-143 (1985)). In this method, a process in which a solute uniformly diffused on a hydrogel flat plate having a uniform thickness of L (cm) is eluted from both of the surfaces of the hydrogel flat plate is observed along with the elapse of time. When the elution amount of the solute at a time "t" (sec) is represented as Mt, and the elution amount after an infinite time passed is represented by M00, the relationship represented by the following formula (1) is satisfied, with respect to a diffusion coefficient D (cm2/sec) of the solute in the hydrogel within the range of MtZM00 < 0.6:
M1ZM00 = (Dt/π)1/2 x 4/L (1) • Accordingly, the diffusion coefficient D can be calculated from the gradient of a straight line obtained by plotting the elution rate to the elapsed time t versus the square root of the elapsed time t.
In view of balance between the retention/diffusion (or release) performances with respect to various substances, the ratio of diffusion coefficients of phenol red (PR), methyl blue (MB) and myoglobin (MG) may preferably be such that (DPR/DMB) > 2 and
(DpR/DMG) > 1.2 in the cell or tissue-culturing carrier according to the present invention.
The more preferred ranges of the values are as follows:
(1) (DPR/DMB): more preferably 10 or more, further more preferably 20 or more and particularly preferably 50 or more, and preferably 1 x 105 or less, more preferably 1 x 104 or less and further more preferably 1 x 10J or less
(2) (DPR/D,VJG): more preferably 1.5 or more, further more preferably 3 or more and particularly preferably 5 or more, and preferably 1 x 104 or less, more preferably 1 x 103 or less and further more preferably 1 x 102 or less
(Case of collagen, etc.)
As stated above, collagen, as the conventional cell or tissue-culturing carrier, is a hydrophilic polymer. Unlike in the case of the hydrogel according to the present invention, a balance of hydrophilicity/hydrophobicity in collagen cannot be arbitrarily controlled. Accordingly, it has been difficult to control the diffusion rate of a chemical mediator in collagen.
In the case of polylactic acid or polyglycolic acid, since these polymers have a strong hydrophobic ity, it has also been difficult to control the diffusion rate of a chemical mediator in these polymers.
In contrast, since the hydrogel according to the present invention can substantially arbitrarily control a balance of hydrophilicity/hydrophobicity as stated above, it is possible to control the diffusion rate of a chemical mediator in the gel according to the present invention so as to provide a considerable degree of freedom. When the gel according to the present invention is used in combination with a known gel-forming polymer such as collagen (that is, when the polymer according to the present invention and a known gel-forming polymer such as collagen are co-present as gel-forming polymers), it is also possible to control the diffusion rate of a chemical mediator so as to provide a considerable degree of freedom, even in the gel also containing a known gel-forming polymer such as collagen. (Tissue or organ in living body)
The term "a cell or tissue" is used in the present invention to mean tissues, apparatuses or organs which are present in the living bodies of animals (especially humans). The in vivo tissue or organ which can be regenerated by using the carrier according to the present invention is not particularly limited. Examples of such tissue or organ may include: esophagus, stomach, small intestine, large intestine, pancreas, liver, heart, blood vessel, bone, cartilage, nerve, cornea, corium, etc. (Cell or tissue) The cells or tissue which can be cultured by using culturing carrier according to the present invention is not particularly limited.
The culturing carrier according to the present invention can be used particularly effectively for differential cells or tissues. Examples of such differential cells may include stem cells and precursor cells. The differential cells include any of differential unipotent cells, differential pluripotent cells, and differential totipotent cells. (Method of repairing or regenerating living organism tissue or organ)
The method of repairing or regenerating a cell or tissue using the carrier according to the present invention is not particularly limited. From the viewpoint of easy inoculation and recovery of cells or the like, it is preferable to utilize the sol-gel transition of a hydrogel-forming polymer. (Embodiment of using sol-gel transition)
In an embodiment of using such sol-gel transition, a cell (e.g., a stem cell or precursor cell), tissue containing the cell, or the like is first inoculated or mixed into the earner according to the present invention. In order to carry out such inoculation or mixing, for example, a hydrogel-forming polymer constituting the carrier used for culturing a cell or tissue of the present invention is dissolved in a culture medium such as RPMI- 1640 (Life Technologies, N. Y., USA) at a low temperature (e.g., 4°C) while stirring, so that the carrier according to the present invention is converted into a state of an aqueous solution (a sol state) with a temperature lower than its sol-gel transition temperature, and then the above cell or tissue may be added or suspended therein. A culture medium used herein is not particularly limited. A culture medium in which a cell of interest (a stem cell, a precursor cell, etc.) easily grows or differentiates may be appropriately selected and used. In addition, the above described chemical mediator promoting the growth or differentiation of a stem cell or precursor cell of interest may also be added to such a culture medium, as desired. Moreover, ECM such as collagen or gelatin may also be added thereto.
In order to regenerate a living organism, tissue or organ in the carrier according to the present invention, for example, the above suspension is heated to a temperature (usually 370C) higher than the sol-gel transition temperature of the carrier according "to the present invention, so that it is gelatinized. Thereafter, "a cell of interest or tissue containing the cell may be cultured at the temperature (usually 37°C).
When the carrier according to the present invention is gelatinized, it is also possible to gelatinize it in a desired form, using a mold having the desired form. For example, when a cartilage tissue is used in repair of the ear or nose, the carrier according to the present invention is converted into a form compatible with a portion of the ear or nose to which the cartilage tissue is to be applied. Thereafter, cartilage cells are cultured in the carrier according to the present invention, so as to regenerate a cartilage tissue. Thus, the cartilage tissue to be applied can be easily molded into a desired form and used. In order to recover a tissue or organ of interest from the carrier according to the present invention after the tissue or organ is regenerated therein, the carrier according to the present invention containing the tissue or organ of interest is cooled to a temperature (for example, 40C) lower than the sol-gel transition temperature, so that the carrier according to the present invention is returned to a sol state. Thereafter, the tissue or organ of interest may be separated from the carrier according to the present invention by a common separation method such as centrifugal separation.
As stated above, it is possible for the carrier according to the present invention substantially not to inhibit (or substantially to promote) the growth or differentiation of a cell of interest (a stem cell, a precursor cell, etc.), while controlling the growth of fibroblasts. Accordingly, a cell or organ of interest can be efficiently regenerated in the carrier according to the present invention. Examples
Hereinbelow, the present invention will be described in more detail with reference to Examples. However, it should be noted that the present invention is defined by Claims, but is not limited by the following Examples.
Production Example 1
10 g of a polypropylene oxide-polyethylene oxide copolymer (average polymerization degree of propylene oxide/ethylene oxide = about 60, Pluronic F-127. mfd. by Asahi Denka K.K.) was dissolved in 30 ml of dry chloroform, and in the co-presence of phosphorus pentaoxide, 0.13 g of hexamethylene diisocyanate was added thereto, and the resultant mixture was subjected to reaction under re fluxing at the boiling point for six hours. The solvent was distilled off under reduced pressure, the resultant residue was dissolved in distilled water, and subjected to ultrafiltration by using an ultrafiltration membrane having a molecular cutoff of 3 x 104 (Amicon PM-30) so as to fractionate the product into a low-molecular weight polymer fraction and a high-molecular weight polymer fraction. The resultant aqueous solution was frozen, to thereby obtain a high-polymerization degree product of F-127 and a low-polymerization degree product of F-127.
When the above high-polymerization degree product of F-127 (TGP-I, a hydrogel-forming polymer according to the present invention) was dissolved in distilled water under ice-cooling in an amount of 8 mass %. When the resultant aqueous solution was gradually warmed, it was found that the viscosity was gradually increased from 21 0C, and was solidified at about 270C so as to be converted into a hydrogel state. When the resultant hydrogel was cooled, it was returned to an aqueous solution at 21 0C. Such a conversion was reversibly and repetitively observed. On the other hand, a solution which had been obtained by dissolving the above low-polymerization degree product of F- 127 in distilled water under ice-cooling in an amount of 8 mass %, was not converted into a gel state at all even when it was heated to 60 0C or higher.
Production Example 2
160 mol of ethylene oxide was subjected to an addition reaction with 1 mol of trimethylol propane by cationic polymerization, to thereby obtain polyethylene oxide triol having an average molecular weight of about 7000.
100 g of the thus obtained polyethyleneoxide triol was dissolved in 1000 ml of distilled water, and then 12 g of potassium permanganate was slowly added thereto at room temperature, and the resultant mixture was subjected to an oxidization reaction at this temperature for about one hour. The resultant solid content was removed by filtration, and the product was subjected to extraction with chloroform, and the solvent (chloroform) was distilled off, to thereby obtain 90 g of a polyethylene oxide tricarboxyl derivative.
1O g of the thus obtained polyethylene oxide tricarboxyl derivative, and 10 g of polypropylene oxide diamino derivative (average propylene oxide polymerization degree: about 65, trade name: Jeffamine D-4000, mfd. by Jefferson Chemical Co.,
U:S.A., cloud point: about 9 0C) were dissolved in 1000 ml of carbon tetrachloride, and then 1.2 g of dicyclohexyl carbodiimide was added thereto, and the resultant mixture was allowed to cause a reaction for 6 hours under refluxing at boiling point. The resultant reaction mixture was cooled and the solid content was removed by filtration, and thereafter the solvent (carbon tetrachloride) therein was distilled off under reduced pressure. Then, the resultant residue was dried under vacuum, to thereby obtain a polymer for coating (TGP-2) comprising plural polypropylene oxide blocks, and polyethylene oxide block combined therewith. This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %. When the sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 16 0C.
Production Example 3 96 g of N-isopropyl acrylamide (mfd. by Eastman Kodak Co.). 17 g of
N-aclyloxy succinimide (mfd. by Kokusan Kagaku K.K.), and 7 g of n-butyl methacrylate (mfd.. by Kanto Kagaku K.K.) were dissolved in 4000 ml of chloroform. After the purging with nitrogen gas, 1.5 g of N,N'-azobisisobutyronitrile was added thereto, and the resultant mixture was subjected to polymerization at 60 0C for 6 hours. The reaction mixture was concentrated, and then was reprecipitated in diethyl ether. The resultant solid content was recovered by filtration, and then was dried under vacuum, to thereby obtain 78 g of poly (N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate).
Then, an excess of isopropylamine was added to the thus obtained poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) to thereby obtain poly(N-isopropyl acrylamide-co-n-butyl methacrylate). The thus obtained poly(N-isopropyl acrylamide-co-n-butyl methacrylate) had a sol-gel transition o temperature of about 19 °C in its aqueous solution.
Then, 10 g of the thus obtained poly(N-isopropyl acrylamide-co-N-aclyloxy succinimide-co-n-butyl methacrylate) and 5 g of both terminal-aminated polyethylene oxide (molecular weight = 6000, mfd. by Kawaken Fine Chemical K.K.) were dissolved in 1000 ml of chloroform, and the resultant mixture was allowed to cause a reaction at 50 0C for 3 hours. The reaction mixture was cooled to room temperature, and thereafter 1 g of isopropylamine was added thereto, and was left standing for 1 hour. The reaction mixture was concentrated, and then was precipitated in diethyl ether. The solid content was recovered by filtration, and thereafter was dried under vacuum, to thereby obtain a polymer for coating (TGP-3) comprising plural poly(N-isopropyl acrylamide-co-n-butyl methacrylate) blocks and polyethylene oxide block combined therewith. _ _
This polymer was dissolved in distilled water under cooling with ice so as to provide a concentration of 5 mass %. When the sol-gel transition temperature of the resultant aqueous solution was measured, it was found that the sol-gel transition temperature was about 21 0C.
Production Example 4 (Sterilization method)
2.0 g of the above-mentioned polymer (TGP-I) was placed in an EOG (ethylene oxide gas) sterilizing bag (trade name: Hybrid Sterilization bag, mfd. by Hogi Medical Co.), and was filled up with EOG by use of an EOG sterilizing device (trade name: Easy Pack, mfd. Inouchi Seieido Co.) and then the bag was left standing at room temperature for twenty-four hours. Further, the bag was left standing at 40 0C for half a day, EOG was removed from the bag and the bag was subjected to aeration. The bag was placed in a vacuum drying chamber (40 0C) and was left standing for half 'a day, and was sterilized while the bag was sometimes subjected to aeration.
Separately, it was confirmed that the sol-gel transition temperature of the polymer was not changed even after this sterilization operation.
Production Example 5 37 g of N-isopropylacrylamide, 3 g of n-butyl methacrylate, and 28 g of polyethylene oxide monoacrylate (having a molecular weight of 4,000, PME-4000 mfd. by Nihon Yushi K.K. (NOF Corporation)) were dissolved in 340 mL of benzene. Thereafter, 0.8 g of 2,2'-azobisisobutyronitrile was added to the resultant solution, and then was subjected to a reaction at 6O0C for 6 hours. 600 mL of chloroform was added to the thus obtained reaction product so as to be dissolved therein , and the resultant solution was dropped into 20 L (liter) of ether so as to be precipitated therein. The resultant precipitate was recovered by filtration, and the precipitate was then subjected to vacuum drying at about 4O0C for 24 hours. Thereafter, the resultant product was again dissolved in 6 L of distilled water. The solution was concentrated to a volume of 2 L at 10°C by using a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10 x 104 (H1P100-43 mfd. by Amicon),.
The concentrated solution was diluted with 4 L of distilled water, and then, the dilution operation was carried out again. The above dilution and concentration by ultrafiltration were further repeated 5 times, so as to eliminate products having a molecular weight of 10 x 104 or lower. The product which had not been filtrated by this ultrafiltration (i.e., the product remaining in the inside of the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 60 g of a hydrogel-forming polymer
(TGP -4) according to the present invention having a molecular weight of 10 x 104 or higher.
1 g of the thus obtained hydrogel-forming polymer (TGP-4) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of the obtained aqueous solution was measured, it was found to be 250C.
Production Example 6
The hydrogel-forming polymer (TGP -3) according to the present invention which had been obtained in Production Example 3 was dissolved so as to provide a concentration of 10 mass % in distilled water. When the steady flow viscosity η thereof at 37°C was measured, it was found to be 5.8 x 105 Pa sec. In the measurement of the steady flow viscosity η, a stress rheometer (CSL 500), and an acryl disk (diameter: 4 cm) as a measuring device were used. The thickness of a sample was set to 600 μm, and applying a shearing stress of 10 N/m2, the resultant creep was measured for 5 minutes after 5 minutes had passed. On the other hand, agar was dissolved so as to provide a concentration of 2 mass % in distilled water at 90°C, and the mixed solution was converted into a gel state at 10°C for 1 hour. Thereafter, η thereof at 37°C was measured. As a result, the obtained value exceeded the measurement limit (1 x 107 Pa- sec) of the apparatus. Production Example 7
71.0 g of N-isopropylacrylamide and 4.4 g of n-butyl methacrylate were dissolved in 1,117 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 22.6 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 773 g of water was added. The oresultant solution was heated to 7O0C under a nitrogen stream. While the solution was maintaining at 70°C under a nitrogen stream, 0.8 mL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 8 mL of 10 % ammonium persulfate (APS) aqueous solution were added to the solution, and then was subjected to a reaction for 30 minutes under stirring. Further, 0.8 mL of TEMED and 8 mL of 10 % APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 1O0C or lower, it was diluted with 5 L of cold distilled water with a temperature of 100C. Thereafter, the solution was concentrated to 2 L at 100C, by using an ultrafiltration membrane with a molecular weight cutoff of 1 Ox 104.
4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate products with a molecular weight of 10x104 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 72 g of the hydrogel-forming polymer (TGP-5) according to the present invention with a molecular weight of 1 Ox 104 or higher.
1 g of the thus obtained hydrogel-forming polymer (TGP-5) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be 2O0C.
Production Example 8 42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 65.1 g of water was added. The resultant solution was heated to 700C under a nitrogen stream. While the solution was maintained at 7O0C under a nitrogen stream, 0.4 rηL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 4 mL of 10 % ammonium persulfate (APS) aqueous solution were added to the solution, and then, the thus obtained solution was subjected to a reaction for 30 minutes under stirring. Further, 0.4 mL of TEMED and 4 mL of 10 % APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 50C or lower, it was diluted with 5 L of cold distilled water with a temperature of 5°C. Thereafter, the solution was concentrated to 2 L at 50C, by using an ultrafiltration membrane with a molecular weight cutoff of 10x 104. 4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate The product with a molecular weight of 10x 10 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 40 g of the hydrogel-forming polymer (TGP-6) according to the present invention with a molecular weight of 10x 104 or higher.
1 g of the thus obtained hydrogel-forming polymer (TGP-6) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be
70C.
Production Example 9
45.5 g of N-isopropylacrylamide and 0.56 g of n-butyl methacrylate were dissolved in 592 g of ethanol. To the resultant mixture solution, an aqueous solution which had been obtained by dissolving 11.5 g of polyethylene glycol dimethacrylate (PDE 6000, mfd. by NOF Corporation) in 65.1 g of water was added. The resultant solution was heated to 700C under a nitrogen stream. While the solution was maintained at 70°C under a nitrogen stream, 0.4 mL of ' N,N,N',N'-tetramethylethylenediamine (TEMED) and 4 mL of 10 % ammonium persulfate (APS) aqueous solution were added to the solution, and then was subjected to a reaction for 30 minutes under stirring. Further, 0.4 mL of TEMED and 4 mL of 10 % APS aqueous solution were added thereto 4 times at 30-minute intervals, and the polymerization reaction was terminated. The reaction mixture was cooled to 1O0C or lower, it was diluted with 5 L of cold distilled water with a temperature of 100C. Thereafter, the solution was concentrated to 2 L at 1O0C, by using an ultrafiltration membrane with a molecular weight cutoff of 10x104.
4 L of cold distilled water was added to the concentrated solution for dilution, and the above concentration operation using the ultrafiltration was conducted again. Thereafter, the above dilution and ultrafiltration concentration were repeated 5 times, so as to eliminate The product with a molecular weight of 10x 104 or lower. The product which had not been filtrated by the above ultrafiltration (product remaining in the ultrafiltration membrane) was recovered and freeze-dried, so as to obtain 22 g of the hydrogel-forming polymer (TGP-7) according to the present invention with a molecular weight of 1 Ox 104 or higher.
1 g of the thus obtained hydrogel-forming polymer (TGP-7) according to the present invention was dissolved in 9 g of distilled water under ice cooling. When the sol-gel transition temperature of this aqueous solution was measured, it was found to be 370C. the hydrogel TGP-5 was 7.6.
Example 1
1 g of TGP-5 was dissolved in 10 mL of Dulbecco's Minimum Essential Medium (DMEM) containing 20% Fetal Bovine Serum (FBS) at 40C. Corneal Limbal tissue bits" of about 2 mm3 were collected from donor A, and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 370C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy on days 6 (Figure 1), 8 (Figure 2), and 10 (Figure 3) of cultivation, growth of cells which spread and grew over time on the perimeter of the corneal limbal tissue bits was observed. Tissue after 10 days of cultivation was fixed with formalin and stained with hematoxylin eosin (HE) (Figure 4), thereby showing an image of multilayered cellular growth with keratinized outermost layer in' the perimeter of the tissue bits.
Example 2
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal Limbal tissue bits of about 2 rah3 were collected from donor B (male, 8 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, actively growing cells were observed by day 2 of cultivation, and by day 5 of cultivation the grown cells had formed a monolayer and almost formed full sheath around the tissue bits. Tissue after 5 days of cultivation was fixed with formalin and stained with hematoxylin eosin (HE) (Figure 5), thereby showing grown cellular layer around the tissue bits. Paraffin embedded sections 4-5 mm thick were prepared from the above tissue bits after 5 days of cultivation, and peroxidase immunostaining by various markers was performed. Anti-human p63 (4A4) mouse antibody (DAKO), anti-human ABCG2 (5D3) mouse antibody (DAKO), anti-human connexin 43 mouse antibody (DAKO), and anti-human integrin β4 (A9) mouse antibody (DAKO) were used as primary antibodies. As a result, the grown cellular population contained cells which are positive for p63 specific to corneal limbal stem cells and connexin 43 specific to transient growing cells, and cells which are positive for ABCG2 specific to corneal limbal stem cells having pluripotency and integrin β specific to mature corneal epithelial cells were not observed. With ABCG2 however, cells which are positive in immunoperoxidase staining on day 11 of cultivation were observed. (Figure: 9)
Comparative .Example 1 (cultivation using amniotic membrane)
With informed consent of the donor, human amniotic membrane tissue obtained by Caeserian section was washed, and then trypsinized for 45 minutes to remove epithelial cells. The human amniotic membrane tissue with epithelial cells removed were placed in a 24-well plate, and the corneal limbal tissue bits of about 1 mm3 or less obtained from donor B (male, 8 years old) in Example 2 were placed on top of this. 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under 10% CO2 atmosphere at 370C. The medium was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. Tissue after 5 days of cultivation was fixed with formalin, paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers similar to Example 2 was performed. As a result, the grown cells were negative to all of p63, connexin 43, ABCG2, and mxegrin β. Comparative Example 2 (cultivation only on a plate)
Corneal limbal tissue bits of about 1 mm3 or less obtained from donor B (male, 8 years old) in Example 2 were placed directly on the bottom of a 24-well plate. 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under 10% CO2 atmosphere at 37°C. The medium was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. Tissue after 5 days of cultivation was fixed with formalin and stained with hematoxylin eosin (HE), thereby showing that tissue bits had disintegrated and no grown cells were observed around the perimeter.
Example 3
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal limbal tissue bits of about 2 mm3 were collected from donor C (male, 85 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP:5 solution in DMEM cooled to 40C was placed in the center, of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. As observed by phase contrast microscopy of the tissue bits in the gel, cellular growth was not active until day 3 of cultivation, and migration of the grown cells to the outside of gel was observed. Cells which migrated to the outside of gel died. Tissue after 3 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, at the perimeter of the tissue bits, the grown cellular population was negative for p63 specific to corneal limbal stem cells, but contained cells which were positive for connexin 43 specific to transient growing cells, ABCG2 specific to corneal limbal stem cells having pluripotency, and inlegrin β specific to mature corneal epithelial cells.(Figure: 10) Comparative Example 2 (cultivation using amniotic membrane)
With informed consent of the donor, human amniotic membrane tissue obtained by Caeserian section was washed, and then trypsinized for 45 minutes to remove epithelial cells. The human amniotic membrane tissue with epithelial cells removed were placed in a 24-well plate, and the corneal limbal tissue bits of about 1 mm3 or less obtained from donor C (male, 85 years old) in Example 3 were placed on top of this. 0.5 mL of DMEM (containing 20% FBS) medium was added and this was cultivated under
10% CO2 atmosphere at 370C. Tissue after 3 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, the grown cells were negative to all of p63, connexin 43, ABCG2, and integrin β.
Example 4. I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 40C. Corneal limbal tissue bits of about 2 mm3 were collected from donor D (female, 65 years old), and the corneal limbal tissue bits of about 2 mm obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. Tissue after 3 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, at the perimeter of the tissue bits, the grown cellular population was negative for ABCG2 specific to corneal limbal stem cells having pluripotency, but contained cells which were positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing cells, and integrin β specific to mature corneal epithelial cells. (Figure: 1 1)
Example 5 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal limbal tissue bits of about 2 mm3 were collected from donor E (male, 76 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 370C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, good cellular growth was observed after day 6, and by day 10 the grown cells had almost formed full sheath around the tissue bits. Tissue after 11 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, at the perimeter of the tissue bits, the grown cellular population contained cells which were positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing cells, and ABCG2 specific to corneal limbal stem cells having pluripotency, and cells which were positive for integrin β specific to mature corneal epithelial cells were not observed. (Figure 12)
Example 6 1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Corneal limbal tissue bits of about 2 mm3 were collected from donor F (male, 32 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 370C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 370C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, good cellular growth was observed after day 2, and by day 10 the grown cells almost formed full sheath around the tissue bits.
Tissue after 10 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers, was performed. As a result, at the perimeter of the tissue bits, the grown cellular population were negative for ABCG2 specific to corneal limbal stem cells having pluripotency, but contained cells which were positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing cells, and integrin β specific to mature corneal epithelial cells.
Example 7 I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Cadaveric Corneal limbal tissue bits of about 2 mm3 were collected from donor G (female, 78 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were further added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 niL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, good cellular growth was observed after day 3, and by day 6 the grown cells had almost formed full sheath around the tissue bits.
Tissue after 13 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, at the perimeter of the tissue bits, the grown cellular population contained -cells which were positive for p63 specific to corneal limbal stem cells.
Example 8
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 40C. Corneal limbal tissue bits of about 2 mm3 were collected from donor H (male, 68 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4 0C were further added. This was kept in cultivation at 370C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, good cellular growth was observed after day 3.
Tissue after 10 days of cultivation was fixed with formalin and paraffin embedded sections 4-5 mm thick were prepared, and peroxidase immunostaining by various markers was performed. As a result, at the perimeter of the tissue bits, the grown cellular population contained cells, which were positive for p63 specific to corneal limbal stem cells.
Example 9 I g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 40C. Corneal limbal tissue bits of about 2 mm3 were collected from donor I (male, 62 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24* well plate warmed to 37°C and converted into a gel state. The. above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were further added. This was kept in cultivation at 370C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at
370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, good cellular growth was observed after day 3, and by day 10 migration of the grown cells away from the corneal limbal tissue bits to the outside of gel was observed. After 11 days of cultivation, plates were cooled for 2 hours to 80C to melt the gel, the grown cells detached from corneal limbal tissue bits were precipitated, and cultivated for 1 day at 370C to form cellular monolayer so that immunofluorescence staining by various markers could be performed. As a result, cells which are positive for p63 specific to corneal limbal stem cells, connexin 43 specific to transient growing .
cells, ABCG2 specific to corneal limbal stem cells having pluripotency. and integrin β specific to mature corneal epithelial cells were observed in the collected cellular population.(Figure 13)
Example 10
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 40C. Corneal limbal tissue bits of about 2 mm3 were collected from donor J (male, 79 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 4°C was placed in the center of the wells of a 24-well plate warmed to 37°C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 40C were added. This was kept in cultivation at 370C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 37°C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast' microscopy of the tissue bits in the gel, by day 7 migration of the grown cells away from the corneal limbal tissue bits to the outside of gel was observed. After 8 days of cultivation, plates were cooled for 2 hours to 80C to melt the gel, the grown cells detached from corneal limbal tissue bits were precipitated, and cultivated for 1 day at 37°C to form a cellular monolayer so that immunofluorescence staining by various markers could be performed. As a result, cells which are positive for connexin 43 specific to transient growing cells and ABCG2 specific to corneal limbal stem cells having pluripotency were observed in the collected cellular population.
(Figure 14)
Example 11
With a sample from donor J (male", 79 years old) in Example 10, experiments similar to Example 10 (cultivation according to the present invention), Comparative Example 1 (cultivation on amniotic membrane tissue), and Comparative Example 2 (cultivation on a plate) were performed using DMEM medium containing 1 μCi per well of tritium Η-labelled thymidine. 50 μL per well of medium was sampled every day, the amount of radiation was measured with a scintillation counter, and the reduction of thymidine incorporated by the cell for DNA synthesis was compared, to obtain an indicator for cell growth activity. As shown in Figure 6, in the three dimensional cultivation within the gel of the present invention, high thymidine incorporation was observed from day 3 to 8 of cultivation, whereas in cultivation on amniotic membrane tissue and on cultivation plates, only low level of thymidine incorporation was observed from day 3 to 8 of cultivation.
Example 12
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 40C. Cadaveric Corneal limbal tissue bits of about 2 mm3 were collected from donor K (male, 98 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 370C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were added. This was kept in cultivation at 37°C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 37°C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, by day 4 migration of the grown cells away from the corneal limbal tissue bits to the outside of gel was observed.
After 7 days of cultivation, plates were cooled for 2 hours to 80C to melt the gel, the grown cells detached from corneal limbal tissue bits were precipitated, and cultivated for 1 day at 370C to form a cellular monolayer so that immunofluorescence staining by various markers could be performed. As a result, cells, which are positive for p63 specific to corneal limbal stem cells, ABCG2 specific to corneal limbal stem cells having pluripotency were observed in the collected cellular population.
Example 13 With a sample from donor K (male, 98 years old) in Example 12, experiments similar to Example 12 (cultivation according to the present invention), Comparative Example 1 (cultivation on amniotic membrane tissue), and Comparative Example 2 (cultivation on a plate) were performed using DMEM medium containing 1 μCi per well of tritium 3H-labelled thymidine. 50 μL per well of medium was sampled every day, the amount of radiation was measured with a scintillation counter, and the reduction of thymidine incorporated by the cell for DNA synthesis was compared, to obtain an indicator for cell growth activity. As shown in Figure 7, in the three dimensional cultivation within the gel of the present invention, high thymidine- incorporation was observed from day 1 to 8 of cultivation. On the other hand, in cultivation on amniotic membrane tissue and on cultivation plates, high thymidine incorporation was observed on day 1 of cultivation, but in both cases thymidine ' incorporation rapidly decreased, and after day 4 of cultivation, only' low level of thymidine incorporation was observed.
Example 14
1 g of TGP-5 was dissolved in 10 mL of DMEM (containing 20% FBS) medium at 4°C. Cadaveric Corneal limbal tissue bits of about 2 mm3 were collected from donor L (female, 56 years old), and the corneal limbal tissue bits of about 2 mm3 obtained were further cut into smaller pieces (1 mm3 or less). 0.2 mL of TGP-5 solution in DMEM cooled to 40C was placed in the center of the wells of a 24-well plate warmed to 370C and converted into a gel state. The above corneal limbal tissue bits of about 1 mm3 or less were placed on top of this, and a few drops of TGP-5 solution in DMEM cooled to 4°C were added. This was kept in cultivation at 370C, after which corneal limbal tissue bits were embedded within the TGP-5 gel. 0.5 mL of DMEM (containing 20% FBS) medium at 370C was added on top of the gel, and incubated under 10% CO2 atmosphere at 370C. The layered medium on top of the gel (0.5 mL) was exchanged every 3 days with 0.5 mL of fresh DMEM (containing 20% FBS) medium. As observed by phase contrast microscopy of the tissue bits in the gel, by day 4 migration of the grown cells away from the corneal limbal tissue bits to the outside of gel was observed.
After 10 days of cultivation, plates were cooled for 2 hours to 8°C to melt the gel, and the grown cells detached from corneal limbal tissue bits were precipitated. The medium was removed, and the grown cells were dispersed in 1 mL of phosphate buffer
(PBS) cooled to 4°C. The cellular suspension was transferred into a 1.5 mL centrifugation tube, and spinned at 2000 rpm to precipitate cells. 1 mL of PBS cooled to
4°C was added to the centrifugation tube, the precipitated cells were dispersed, spinned again at 2000 rpm to precipitate the cells. This wash process was repeated 4 times, and the cells were finally suspended in 100 μL of PBS. A smear was prepared on a slide by Cytospin, fixed with acetone, and imrnunostaining by various markers was performed. As a result, the cellular population collected contained p63 positive cells specific to corneal limbal stem cells, ABCG2 positive cells specific to corneal limbal stem cells having pluripotency, connexin 43 positive cells specific to transient growing cells, and integrin β positive cells specific to mature corneal epithelial cells positive.
Example 15
With a sample form donor L (female, 56 years old) in Experiment 14, experiments similar to Example 12 (cultivation according to the present invention), Comparative Example 1 (cultivation on amniotic membrane tissue), and Comparative Example 2 (cultivation on a plate) were performed using DMEM medium containing 1 μCi per well of tritium 3H-labelled thymidine. 50 μL per well of medium was sampled every day, the amount of radiation was measured with a scintillation counter, and the reduction of thymidine incorporated by the cell for DNA synthesis was compared, to obtain an indicator for cell growth activity. As shown in Figure 8, in the three dimensional cultivation within the gel of the present invention, high thymidine incorporation was observed from day 1 to 10 of cultivation. On the other hand, in cultivation on amniotic membrane tissue, high thymidine incorporation was observed in the early stage, but gradually decreased. In addition, in cultivation on cultivation plates, only low level of thymidine incorporation was observed in the early stage up until day 10 of cultivation.
Advantages of the Invention As described above, cellular population from corneal limbus can be obtained according to the present invention, even when corneal limbal stem cells and corneal epithelial cells, which they differentiate into, cannot be obtained by cultivating corneal limbal tissue on amniotic membrane tissue or on typical cell cultivation plates. In the conventional method of cultivating corneal limbal tissue on .amniotic membrane tissue, it was difficult to separately collect cells from corneal limbal tissue and amniotic membrane tissue. According to the present invention, collecting of only, the cells from corneal limbal tissue are done with ease, and the cells can be provided for various treatments for diseases associated with cornea. The cellular population from corneal limbus obtained from the present invention can be stored frozen according to usual methods, to be thawed and used when a patient is in need thereof. Regarding the cells derived from corneal limbal tissue obtainable from the present invention, it is possible to fix only the relevant cells onto the cornea of a patient using for example therapeutic contact lens. There is no need to transplant the cells together with human amniotic membrane tissue as with conventional methods.

Claims

1. A method for cultivating cells derived from corneal limbal tissue, characterized in that the method comprises the steps of: embedding the corneal limbal tissue within an aqueous solution in a low temperature sol state wherein the aqueous solution contains at least a hydrogel-forming polymer showing thermo-reversible sol-gel transition and wherein the aqueous solution is in a sol state at a low temperature and is in a gel state at a high temperature; heating the said aqueous solution to cultivate the said corneal limbal tissue within the said hydrogel in a high temperature gel state so that the corneal limbal stem cells spread and grow outside of the said corneal limbal tissue; cooling the said hydrogel to return from the gel state to a low temperature sol state; and collecting the spread and grown cells derived from corneal limbal tissue.
2. The method as claimed in claim 1, wherein corneal limbal stem cells-are cells derived from corneal limbal tissue and are positive in p63 protein immunostaining.
3. The method as claimed in claim 1, wherein corneal limbal stem cells having pluripotency are cells derived from corneal limbal tissue and are positive in
ABCG2 immunostaining.
4. The method as claimed in claim 1, wherein transient growing cells are cells derived from corneal limbal tissue and are positive in connexin 43 immunostaining.
5. The method as claimed in claim 1, wherein mature corneal epithelial cells are cells derived from corneal limbal tissue and are positive in integrin β immunostaining.
6. The method as claimed in claim 1, wherein the hydrogel forming polymer forms a hydrogel which has a crosslinking or network structure by retaining water.
7. Cells derived from corneal limbal tissue by the cultivation method claimed in claim 1.
PCT/IN2005/000092 2005-03-28 2005-03-28 A method for cultivating cells derived from corneal limbal tissue and cells deived from corneal limbal tissue WO2006103685A2 (en)

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US9561264B2 (en) 2000-06-05 2017-02-07 The Brigham And Women's Hospital, Inc. Gene encoding a multidrug resistance human P-glycoprotein homologue on chromosome 7p15-21 and uses thereof
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EP3865573A1 (en) * 2013-02-19 2021-08-18 Children's Medical Center Corporation Abcb5(+) stem cells for treating ocular disease
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CN106591216A (en) * 2016-12-12 2017-04-26 深圳市眼科医院 Human normal corneal epithelium cells and application thereof
RU2809076C1 (en) * 2023-07-14 2023-12-06 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт глазных болезней им. М.М. Краснова" (ФГБНУ "НИИ глазных болезней им. М.М. Краснова") Method of obtaining limbal stem cells in collagen hydrogel

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