WO2001006843A2 - Promotion of proliferation of adult corneal endothelial cells - Google Patents
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- WO2001006843A2 WO2001006843A2 PCT/US2000/040471 US0040471W WO0106843A2 WO 2001006843 A2 WO2001006843 A2 WO 2001006843A2 US 0040471 W US0040471 W US 0040471W WO 0106843 A2 WO0106843 A2 WO 0106843A2
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
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- C12N2500/14—Calcium; Ca chelators; Calcitonin
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- C12N2500/20—Transition metals
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- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
Definitions
- Corneal endothelial cells are different from vascular and pulmonary "endothelial cells" as they have a different embryonic tissue origin. Human corneal endothelial cells do not normally proliferate in vivo to replace those lost due to cell injury or death. Growth of these cells in culture is also extremely limited.
- corneal endothelial cells proliferate in vivo and in culture appears to be a function of age; i.e., embryonic corneal endothelial cells and cells from neonates will proliferate much more readily than cells from children and adults.
- researchers have been able to culture cells from older donors, but growth has been supported by seeding the cells onto an artificial matrix, such as chondroitin sulfate/laminin, or onto extracellular matrix secreted by corneal endothelial cells from cows, one of a number of species whose corneal endothelial cells do grow readily in culture.
- an artificial matrix such as chondroitin sulfate/laminin
- extracellular matrix secreted by corneal endothelial cells from cows one of a number of species whose corneal endothelial cells do grow readily in culture.
- a reliable way of growing and/or supporting human corneal endothelial cells, whether' in vitro or in vivo, would be highly desirable. In particular,
- the invention provides a new source of corneal endothelial cells for use in research and treatment of injured or diseased corneal endothelium.
- the invention enables the growth / proliferation of human corneal endothelial cells from adult humans, preferably those aged fifty or more years.
- the invention encompasses exposing corneal endothelial cells to an agent that interrupts cell—to-cell contact, also called “cell-cell adhesion" or “cell-cell contact”.
- Such cell-cell contact interruption step preferably is sandwiched between two incubation or exposure steps, in which adult-derived corneal endothelial cells are exposed to at least one growth factor, optionally in a growth medium.
- “Growth factor” refers to any factor that induces a corneal endothelial cell to enter the cell cycle and thus to proliferate.
- Exemplary growth factors include, but are not limited to, e.g., one or more mitogen(s) such as epidermal growth factor (EGF) , fibroblast growth factor (FGF), and/or nerve growth factor (NGF) .
- mitogen(s) such as epidermal growth factor (EGF) , fibroblast growth factor (FGF), and/or nerve growth factor (NGF) .
- Other growth factors effective for corneal endothelial proliferation are known in the art and may be used in the invention.
- the agent used to interrupt cell-cell contacts may be a calcium chelator such as ethylenediaminetetraacetic acid (EDTA) , a known chelator of calcium and magnesium ions previously used in many laboratories to release cultured cells from contact inhibition.
- EDTA ethylenediaminetetraacetic acid
- corneal endothelial cells will divide in response to wounding (11,12). In tissue culture and in organ cultured corneas, only endothelial cells adjacent to the wound edge or cells that have migrated into the wound bed will proliferate, demonstrating the importance of releasing cell-cell contacts in order to promote proliferation.
- junctional complexes formation of cell-cell contacts or adhesions is mediated by a number of proteins that are associated with different types of junctional complexes, including cadherins (13) (adhering junctions), ZO-1 (14) (tight junctions), and connexin-43 (15) (gap junctions) . These proteins all require calcium for maintenance of their adhesion function. In low- calcium environments, junctional complexes mediated by these proteins disassemble and cell-cell contact is broken. Exposure of the corneal endothelium to calcium- free medium causes disruption of apical junctional complexes, increased transendothelial perfusion, and corneal edema (16-18) . These changes can be reversed by replacing calcium in the medium (17,18), or by exposing cells to ionophores that release intracellular calcium stores (18) .
- Figures 1A-E shows representative micrographs demonsatrating the dose- and time-dependent effect of a non-lethal amount of EDTA on cell-cell contacts in a corneal endothelium culture
- FIGS 2A-D depict the Ki67-staining patterns of corneal endothelial cells in corneal pieces subjected to different treatment regimes and/or culture conditions.
- Figures 3A-C illustrate the effects of EDTA treatment on cell cycle kinetics (A) , and a determination of the optimal pre-incubation time (B) and the optimal post-incubation time (C) , by determining the percent (%) of Ki67-positive cells exhibiting staining patterns for Gl phase, S/G2 phase, or mitosis.
- ethylenediamine tetraacetic acid EDTA
- EGTA ethylene glycol- bis [ ⁇ aminoethylether] -N,N,N' ,N' -tetraacetic acid
- EDTA releases corneal endothelial cell-cell contacts in a dose- and time- dependent manner. At the doses and incubation times tested in the experiments described herein, EDTA did not induce significant cell death. Preincubation in culture medium alone (without EDTA) , for about 6-24 hrs, preferably 12-24 hours, was needed for endothelial cells to efficiently initiate proliferation in response to subsequent EDTA treatment. The endothelium of corneas incubated in mitogen-containing medium for up to 108 hrs without EDTA treatment did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared optimal and stimulated 16 - 18% of the cells to proliferate.
- the invention encompasses a method of stimulating proliferation of adult corneal endothelial cells, comprising exposing the corneal endothelial cells to an effective amount of at least one growth factor that promotes proliferation of corneal endothelial cells; and subsequently exposing the corneal endothelial cells to an effective amount of at least one agent that promotes interruption of cell-cell contacts between adjacent corneal endothelial cells.
- the method further comprises, as needed, and subsequent to the cell-cell contact interrupting step, exposing the corneal endothelial cells again to the at least one growth factor.
- the interruption step should result in cell- cell contact interruption in at least 15%, preferably at least 50%, most preferably at least 80%, of the corneal endothelial cells exposed to the interruption agent.
- the invention also relates to a new formulation of culture medium which is being successfully used to stimulate the proliferation of adult (>50 years of age) donor human corneal endothelial cells in culture directly on tissue culture plastic.
- the new formulation includes: a serum-free cell culture medium comprising insulin, transferrin, and selenium; fibroblast growth factor (pituitary) ; epidermal growth factor; nerve growth factor; an antibiotic antimycotic solution; and a calcium chelator.
- the chelator is EDTA or EGTA, preferably in an amount of about 0.02-3.0 mg/ml, advantageously 0.2-2.0 mg/ml. Further details of the formulation, with respect to the non-chelator components, are described in International Application No.
- Donor human corneas were obtained from National Disease Research Interchange (Philadelphia, PA) and from the Central Florida Lions Eye Bank. All corneas were maintained at 4°C in preservation medium for 1 week or less prior to study. Endothelial cell counts were >2000 cells/mm 2 . Criteria for exclusion of corneas from these studies included history of endothelial dystrophy, presence of central gutatta, low endothelial cell density, and ocular inflammation or disease.
- Pieces were incubated for about 24 hrs in Medium-199 containing 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor (EGF) (Upstate Biotechnologies, Lake Placid, NY) , 20 ng/ml fibroblast growth factor (FGF) (pituitary-derived, Bio edical Research Technologies, Stoughton, MA) , and 50 mg/ml gentamicin to stabilize the endothelium prior to study.
- FBS fetal bovine serum
- EGF epidermal growth factor
- FGF fibroblast growth factor
- HBSS calcium chloride, magnesium chloride, or magnesium sulfate: Life Technologies, Grand Island, NY
- pH7.4 adjusted to pH7.4
- Corneas were treated with EDTA for 10, 30, or 60 min and then returned to culture medium for up to 96 hrs.
- Negative controls for EDTA treatment included exposing corneal pieces to all manipulations and incubation conditions, including 1 hour in HBSS, but without EDTA. All corneas were maintained at 37°C in a 5% carbon dioxide, humidified atmosphere until removal for analysis of cell cycle progression.
- Laser power and gain controls were adjusted to achieve an optimal range of output signal intensity for each channel.
- Confocal images were collected and micrographs were printed using Photoshop software v4.0 (Adobe Systems, Inc., San Jose, CA) .
- the printing contrast was adjusted to provide a clearer image .
- Ki67-Positive Cells Fluorescence confocal immunocytochemistry for Ki67 was used to evaluate corneal endothelial cells for their ability to enter and complete the cell cycle (i.e., cell proliferation or mitosis) . All nuclei were stained with PI. Positive Ki67 staining patterns indicated the presence of actively cycling cells and also acted as markers for specific phases of the cell cycle. Completion of the cell cycle was determined by observation of mitotic figures stained with Ki67. Three representative confocal micrographs were taken per corneal quarter using a 40x-objective lens.
- Figure 1 shows that EDTA decreases the integrity of cell-cell contacts in a dose- and time-dependent manner.
- Corneal pieces from a 68 year old donor were incubated in the absence of EDTA (A), or in 0.02 (B) , 0.2 (C) , or 2.0 mg/ml EDTA (D,E) for 30 (B,C,D) or 60 min (E) .
- Normal cell-cell contacts visualized as an apparent single line of staining between cells, were observed in the absence of EDTA, but a gradual increase in lateral separation occurred with increasing EDTA concentration or incubation time.
- Arrows in B, C, and D indicate areas of cell-cell separation.
- FIG. 1 depicts the stimulation of corneal endothelial cell proliferation requires both preincubation in mitogen-containing medium and treatment with EDTA. Corneal quarters from a 52-year old donor were incubated under the following conditions: A) Incubation for 108 hours in medium containing 10% serum, EGF, and FGF, but without exposure to EDTA. B) No preincubation, 0.2 mg/ml EDTA for 30 min, post-incubation in medium for 48 hrs.
- Figure 3 shows the effects of EDTA on cell cycle kinetics (A) and determination of optimal pre-incubation (B) and post-incubation times (C) .
- A cell cycle kinetics
- B corneal pieces from a 64 year old donor were pre-incubated for 24 hrs in medium containing 10% serum, EGF, and FGF, then treated with 0.2 mg/ml EDTA for 60 min, followed by post-incubation in the same medium for various periods of time.
- Results in (A) are expressed as the percent of Ki67-positive cells exhibiting staining patterns for Gl phase, S/G2 phase, or mitosis.
- corneal quarters from a 54 year old donor were pre- incubated in the same medium for different periods of time, followed by 30 minutes of treatment with 2.0 mg/ml EDTA and then an approximately 48-hours post-incubation period.
- corneal quarters from a 73 year old donor were pre- incubated in the same medium for 24 hours, followed by approximately 30 minutes of treatment with 2.0 mg/ml EDTA. Quarters were then post-incubated in the same culture medium for different periods of time. Results in (B and C) are expressed as mean +/- SEM.
- FIG. 1 Representative micrographs in Figure 1 show the dose- and time-dependent effect of EDTA on cell-cell contacts.
- Fig. 1A In the absence of EDTA, cells maintained normal lateral associations (Fig. 1A) . With increasing EDTA concentration or incubation time, there was an increase in the lateral separation of cells, visualized as a double line of positive ZO-1 staining demarcating the two cell membranes (Fig. 1B-E) . Incubation in the presence of 2.0 mg/ml for 60 minutes caused cells to become rounded with only thin cytoplasmic strands connecting adjacent cells. This treatment, however, did not cause endothelial cells to lose contact with the Descemet's membrane on which the cells were cultured.
- EDTA concentrations over 3.0 mg/ml are not recommended, as they can cause cells to fall off the culture membrane.
- a most preferred concentration is around 0.2-2.0 mg/ml EDTA.
- exposing corneal endothelial cells to EDTA results in interrupted cell- contacts in at least 15% of treated cells.
- this interruption step can produce cell-cell breakages in at least 50%, even at least 80% or more, of corneal endothelial cells thus treated. (Near-complete breakages of cell-cell adhesions, i.e., in nearly all treated cells, have been observed. ) It is expected that other calcium chelators can achieve similar results.
- Fig. 2D Ki67 stained nuclei and mitotic figures
- Negative control corneas were manipulated in a manner similar to EDTA-treated corneas. These corneas were incubated for 1 hour in HBSS alone to control for the EDTA incubation and then maintained for 48 hours in the same culture medium indicated as for the EDAT-treated corneas. Fluorescence microscopy showed only a very rare Ki67-positive cell in corneas cultured in HBSS alone (data not shown) .
- Ki67 is expressed in cells from mid-to-late Gl-phase through mitosis, making it an excellent marker of actively cycling cells (20,21). Immunostaining with antibody to Ki67 also permits semi-quantitative analysis of the relative number of cells in late Gl-phase through mitosis (19,22).
- the graph in Figure 3A shows typical endothelial cell-cycle kinetics in corneal pieces from a single donor pre-incubated for 24 hours in mitogen- containing medium, treated with 0.2 mg/ml EDTA for 60 min, and post-incubated in medium for 0, 24, 48, or 72 hrs. Cells initiated proliferation by 24 hrs after EDTA treatment and completed the cycle in approximately 48 hrs, as indicated by the presence of mitotic figures at that time point.
- FIG. 3C provides a representative example, indicating that a maximum number of Ki67-positive cells was obtained 48 hrs post-incubation. Depending on the specific donor cornea, some Ki67-positive cells could be detected 72 hrs after EDTA treatment; however, no samples showed actively cycling cells by 96 hr post-incubation.
- EDTA at either 0.2 or 2.0 mg/ml was capable of promoting cell cycle progression.
- the relative percent of Ki67-positive cells observed under the four EDTA incubation conditions was significantly higher than negative controls in which corneas were incubated in HBSS alone.
- Repeated EDTA treatment cycles promoted proliferation over a longer time period, but the treatment became gradually less effective (data not shown) .
- n 2-3 donor corneas per treatment condition
- ** p-value comparison between Avg. % Ki67 (+) cells from condition #1 and other three Ki67 values
- pre-incubation of the endothelium in mitogen-containing medium was required. Possibly, pretreatment with mitogens initiates cellular responses that prepare for cell cycle entry once cell- cell contacts have been released. Pre-incubation times shorter than the 24 hrs used in this study may be even more effective, since receptor down-regulation can occur with prolonged growth factor incubation. Optimal preincubation times are expected to be in a range of about ??12-18 hours?. The possibility that pre-incubation with mitogens prepares cells to enter the cycle is supported by the fact that cells appeared to initiate proliferation as an almost synchronous population. This response differed from cell cycle entry in wounded endothelium exposed to the same culture medium (19) . Under those conditions, cells continued to enter the cycle at different start times until the wound bed was completely repopulated.
- This treatment could be applied directly to the endothelium in order to increase endothelial cell density in corneas to be used for transplantation.
- a calcium chelator e.g., EDTA or EGTA
- Other agents useful for disrupting cell-cell adhesions are antibodies directed at an antibody that specifically binds to a cell surface protein on the corneal endothelial cell that is involved in cell-cell adhesion.
- the agent could be an antibody that specifically binds to either a cadherin, ZO-1 protein, or connexin-43. the art.
- a formulation of the invention including a calcium chelator as well as serum -free culture medium comprising insulin, transferrin, and selenium; FGF (especially pituitary) ; EGF; NGF; an anti-biotic antimycotic solution, may be used for the following: Tissue Culture Medium for Research Purposes-With the use of this medium, researchers can now grow adult human corneal endothelial cells in culture without the need to seed cells onto artificial or cell-derived matrices. Previously, the greatest success in culturing corneal endothelial cells was obtained using corneas from neonates . Clearly, this does not provide a ready supply of tissue for research purposes.
- Ophthalmic Irrigating Solution- This formulation may be useful as an irrigating solution (most likely minus serum) during anterior chamber surgical procedures to maintain the overall health and stability of the endothelium (which can be compromised during these procedures) and to promote proliferation to replace cells damaged during the procedure.
- Treatment to Increase Cell Density in Vivo- This formulation may be able to be applied as topical drops or be injected into the anterior chamber to induce transient proliferation of corneal endothelial cells in older individuals whose visual acuity is impaired due to low endothelial cell counts.
- Transplantation of Corneal Endothelial Cells to Increase Cell Density One unique possibility for use of our medium formulation is to culture endothelial cells to increase cell numbers and then transplant the endothelial cell sheet back onto Descemet ' s membrane to increase cell density. This might be done by transplanting endothelial cells to donor corneas prior to transplantation or by directly applying the cultured endothelial cell sheet to Descemet ' s membrane of a recipient in vivo.
- corneal equivalents for transplantation or for in vitro testing of various cosmetic or pharmacologic agents.
- Currently, most labs are experimenting with animal corneal endothelial cells or human cells transformed by viral oncogenes, such as SV40.
- Our medium formulation may make it possible to grow normal, untransformed cells for these corneal equivalents.
- the tissue embryonic origin of the corneal endothelium is neural crest.
- neural crest cells migrate and then differentiate to form many types of tissue. Many of the cell types formed from neural crest do not readily proliferate in vivo.
- This formulation may be useful to induce proliferation in other ocular cells of similar origin, including, but not limited to, lacrimal gland acinar cells, ciliary body epithelium, corneal stromal keratocytes, skeletal muscle of the dorsal iris, and trabecular meshwork epithelium.
- Other non-ocular cells of similar embryonic origin may also proliferate upon exposure to this medium.
- non- ocular cell types of neural crest origin are sensory neurons, Schwann cells associated with the peripheral nervous system, and smooth muscle cells associated with the branchial arch arteries. If this formulation is capable of stimulating any of these cells to divide, it may prove useful as a treatment in repair of damaged tissue.
- Wilson SE Lloyd SA, He YG, McCash CS . Extended life of human corneal endothelial cells transfected with the SV40 large T antigen. Invest Ophthalmol Vis Sci. 1993;34:2112-2123.
- Treffers WF Human corneal endothelial wound repair: In vitro and in vivo. Ophthalmol. 1982; 1989:605-613.
- Ki-67 detects a nuclear matrix-associated proliferation-related antigen: Localization in mitotic cells and association with chromosomes. J Cell Sci. 1989;92:531-540.
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU73878/00A AU7387800A (en) | 1999-07-22 | 2000-07-24 | Promotion of proliferation of adult corneal endothelial cells |
US10/030,580 US6548059B1 (en) | 1999-07-22 | 2000-07-24 | Promotion of proliferation of adult corneal endothelial cells |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US14517199P | 1999-07-22 | 1999-07-22 | |
US60/145,171 | 1999-07-22 | ||
USPCT/US00/03531 | 2000-02-11 | ||
PCT/US2000/003531 WO2000047040A1 (en) | 1999-02-11 | 2000-02-11 | Growth medium for human corneal endothelial cells |
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WO2001006843A2 true WO2001006843A2 (en) | 2001-02-01 |
WO2001006843A3 WO2001006843A3 (en) | 2001-03-22 |
WO2001006843A8 WO2001006843A8 (en) | 2001-11-08 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2066334A1 (en) * | 2006-09-28 | 2009-06-10 | TissueTech, Inc. | Rnai methods and compositions for stimulating proliferation of cells with adherent junctions |
Citations (1)
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US5104787A (en) * | 1990-03-05 | 1992-04-14 | Lindstrom Richard L | Method for apparatus for a defined serumfree medical solution useful for corneal preservation |
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2000
- 2000-07-24 WO PCT/US2000/040471 patent/WO2001006843A2/en active Application Filing
- 2000-07-24 AU AU73878/00A patent/AU7387800A/en not_active Abandoned
Patent Citations (1)
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US5104787A (en) * | 1990-03-05 | 1992-04-14 | Lindstrom Richard L | Method for apparatus for a defined serumfree medical solution useful for corneal preservation |
Non-Patent Citations (3)
Title |
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CHEN ET AL.: 'TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells' INVEST. OPHTHALMOL. VIS. SCI. vol. 40, no. 11, October 1999, pages 2513 - 2519, XP002936260 * |
JOYCE ET AL.: 'Mitotic inhibition of corneal endothelium in neonatal rats' INVEST. OPHTHALMOL. VIS. SCI. vol. 39, no. 13, December 1998, pages 2572 - 2583, XP002936262 * |
SENOO T.: 'Stimulation of corneal endothelial cell proliferation by interleukin and complete mitogens' DOKKYO J. MEDICAL SCIENCES vol. 22, 1995, pages 159 - 170, XP002936261 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2066334A1 (en) * | 2006-09-28 | 2009-06-10 | TissueTech, Inc. | Rnai methods and compositions for stimulating proliferation of cells with adherent junctions |
EP2066334A4 (en) * | 2006-09-28 | 2010-12-15 | Tissuetech Inc | Rnai methods and compositions for stimulating proliferation of cells with adherent junctions |
US9006192B2 (en) | 2006-09-28 | 2015-04-14 | Tissuetech, Inc. | RNAi methods and compositions for stimulating proliferation of cells with adherent junctions |
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AU7387800A (en) | 2001-02-13 |
WO2001006843A3 (en) | 2001-03-22 |
WO2001006843A8 (en) | 2001-11-08 |
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