WO2003002154A1 - Artificial autologous skin secreting leptin and method for obtaining the same - Google Patents

Artificial autologous skin secreting leptin and method for obtaining the same Download PDF

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Publication number
WO2003002154A1
WO2003002154A1 PCT/ES2002/000320 ES0200320W WO03002154A1 WO 2003002154 A1 WO2003002154 A1 WO 2003002154A1 ES 0200320 W ES0200320 W ES 0200320W WO 03002154 A1 WO03002154 A1 WO 03002154A1
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Prior art keywords
keratinocytes
leptin
skin
artificial
incubation
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PCT/ES2002/000320
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Spanish (es)
French (fr)
Inventor
Jose Luis Jorcano Noval
Fernando Larcher Laguzzi
Alvaro Meana Infiesta
Marcela Del Rio Nechaevsky
Original Assignee
Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.)
Centro Comunitario De Transfusion De Asturias-Cruz Roja Española
Fundacion Marcelino Botin
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0625Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629Keratinocytes; Whole skin
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0697Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698Skin equivalents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, E.G. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02Prostheses implantable into the body
    • A61F2/10Hair or skin implants
    • A61F2/105Skin implants, e.g. artificial skin
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00Coculture with; Conditioned medium produced by
    • C12N2502/09Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00Coculture with; Conditioned medium produced by
    • C12N2502/13Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323Adult fibroblasts
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00Genetically modified cells

Abstract

The invention relates to an artificial autologous skin that includes genetically modified keratinocytes by infection with a retroviral vector carrying the gene coding for human leptin on an artificial autologous skin basically obtained by gelification with Ca++ from plasma with platelets. The method to obtain said skin involves the following steps: obtaining the plasma with platelets by means of light centrifugation; cultivating dermal cells from a biopsy; gelifying the plasma with platelets using calcium salts and seeding the cultivated dermal cells inside the fibrin matrix; cultivating keratinocytes from a biopsy; genetically modifying the keratinocytes by infection with a retroviral vector carrying the gene coding for human leptin; cultivating the genetically modified keratinocytes until their confluence; selecting the genetically modified keratinocytes using a marker gene; seeding the selected keratinocytes on the artificial dermis obtained after the first three steps.

Description

ARTIFICIAL SKIN secreting EPTINA AUTOLOGOUS AND METHOD

OBTAINING

The present invention relates to a usable artificial skin for treatment of diseases caused by a permanent impairment of protein hormone leptin.

BACKGROUND OF THE INVENTION

Leptin is a protein hormone essential mainly secreted by adipocytes responsible for regulating both food intake and neuroendocrine function through hypothalamic receptors. They have been collected so far also experimental data show a clear effect of leptin on acting directly on peripheral tissues including the endocrine pancreas, pituitary gland, ovary, skeletal muscle and liver include cellular function. Leptin could also play a key role in processes related to angiogenesis and bone remodeling. Congenital leptin deficiency has been identified as the etiologic agent of a disease, low incidence in the population, that causes early onset severe obesity (Montague CT, Farooqi IS, Whitehead JP, Soos, MA, Rau, H., Wareham, NJ, Sewter, CP, Digby, JE, Mohammed, SN., Hurst, JA, Cheetham, O, Earley, A., Barnett, A., Prins, J. And O'Rahilly S. (1997) Congenital leptin deficiency is associated With early-onset severe obesity n humans. Nature 387, 903-908). Low levels of circulating leptin have also been observed in patients affected by different forms of generalized lipoatrophic diabetes. Patients suffering from this disease have a significant involution of adipose tissue (source hormone) and hipergiucemia and severe insulin resistance. Other clinical manifestations of this disease include the appearance of an anabolic syndrome characterized by organomegaly and hypertrophic cardiomyopathy (Pardini, VC, Victoria, IM, Rocha, SM, Andrade, DG, Rocha, AM, Pieroni FB, Milagres G., Purisch, S . and Velho, G. (1998) Leptin levéis, beta-cell function, and insulin sensitivity in congenital and acquired families With generalized lipoatropic diabetes. J Clin Endocrine! Metah. 83, 503-508).

The latter is the leading cause of death among patients suffering from generalized lipodystrophic diabetes. The administration of leptin has been shown to increase glucose metabolism and restore insulin sensitivity in two animal models of congenital generalized lipodystrophy with what is established the critical role of the hormone in the pathophysiology of these disorders. Currently being carried out in various clinical studies that patients who have both congenital hormone deficiency (Farooqi IS, Jebb, SA, Langmack, G., Lawrence, E., Cheetham, is OH., Prentice, AM, Hughes, IA, McCamish, MA and O'Rahilly S. (1999) Effects of recombinant leptin therapy na child with congenital leptin deficiency. N Engl J Med 341, 879-884) and patients suffering from diabetes with lipodystrophic daily injections of recombinant leptin. The results are being obtained are very encouraging and stress the therapeutic potential of leptin for the treatment of these pathologies.

Systemic gene therapy is a medical intervention in which cells from the patient, or another appropriate source, are genetically modified to treat or cure any condition, regardless of etiology, resulting from a deficiency in circulating levels of a particular protein . Therefore it can be considered to systemic gene therapy as a method of in situ production and delivery of proteins. For diseases in which the protein should be administered for the life of the patient, it is necessary to ensure maintenance of both functionality incorporated as the survival of the target cell genetic modification gene. Approaches currently available for systemic and continuous gene therapy make use of viral vectors and adenovirus type AAV normally quiescent cells such as hepatocytes and skeletal muscle. Administration in these cases is in vivo, that is, the viral vector is injected directly into the tissue or into the bloodstream. These approaches have serious limitations restricting their therapeutic applicability, such as the possibility of generating undesired immune responses (adenovirus), a very limited capacity for cloning into the viral vector (adenovirus associated virus: AAV). Or difficulty accessing the tissue genetically modified if an adverse reaction to the introduced gene patent document ES 2106891 describes a technique using transfected with an exogenous DNA encoding a therapeutic protein cells. Genetic information by nonviral methods type transfers: electroporation, microinjection or homologous recombination, but these methods are much less efficient than using vectors retrovlrales. This is particularly true when working with the skin, where it is necessary to achieve high percentages of cells genetically engineered to ensure transfer also epidermal stem cells. Obtaining modified epidermal stem cells is the must when it comes to generating a permanent production from the skin, the therapeutic protein in the patient. So far, the genetic modification of epidermal stem cells has been achieved only with the use of retroviral vectors.

Skin keratinocytes represents a very attractive system exporter exogenous proteins (Fenjves, ES, Smith, J., Zaradic, Taichman LB. S. and (1994) Systemic delivery of secreted protein by grafts of epidermal. Prospects for keratinocyte gene therapy Hum Gene Ther. 5, 1241-1248). Furthermore, the patient's own skin are epithelial stem cells (stem) whose genetic manipulation ex vivo (expression of the deficient protein) and subsequent reimplantation would ensure regeneration in the patient of his own modified skin, now secreting the therapeutic protein, which production would be maintained throughout life (De Luca, M. and Pellegrini, G. (1997) the Importance of epidermal stem cells in keratinocyte-mediated gene therapy. Gene Therapy 4, 381-383). Epithelial skin cells, including stem or stem cells can be isolated, grown and genetically modify using retroviral vectors and transplanted to the patient again. Epithelium regenerated from a genetically modified stem cells would result in a skin that expresses the factor consistently across all strata and for unlimited time. Finally the skin is our outermost covering thus represents an organ that can be monitored clinically and atraumatically removed little if adverse effects. It would therefore very valuable to have a method in which the production and delivery in vivo leptin is carried out from an autologous keratinocytes to include epithelial stem cells genetically modified secretory protein for the treatment / healing disease course with deficient levels of leptin.

In patent documents P9701533 and P200100494 two methods for the preparation of artificial skins, in which keratinocytes are grown on a dermal fibrin matrix obtained by bioprecipitado or directly from the plasma, resulting to be the fibrin matrix optimum substrate described for the proliferation of keratinocytes. Proliferation (amplification of keratinocytes) is a critical step when trying to use this skin for the treatment of major burns, but not in the case of gene therapy. The important thing here is that fibrin retains all the properties of the genetically modified stem cell, ensuring reimplantation of functional stem cells that perpetuate the production of the therapeutic protein. The use of a type of artificial skin in the present case essentially exploits the ability of fibrin to maintain functional status in genetically altered epidermal cells mothers, instead of looking for great expansions of autologous biopsy. The object of the invention is, consequently, have an autologous artificial skin capable of secreting and maintain in vivo after transplant, therapeutic levels of leptin acting in the receiver of the transplant individual, as an alternative source of lost hormone, perpetuating the effect for the rest of the patient's life. DESCRIPTION OF THE INVENTION

Autologous artificial skin used in the present invention consists of a dermal epidermal component and a component that has been transferred the gene coding for leptin. A basic aspect of this invention is the development of an efficient process for obtaining and maintaining in vivo genetically modified epidermal stem cells. This procedure involves the efficient transfer (> 50% transfected cells) of the gene coding for leptin by use of a retroviral vector followed by an enrichment culture cells that produce the therapeutic protein and a recovery culture enriched in a matrix fibrin and fibroblasts that protects epidermal stem cell differentiation and possible loss of property. Incorporating into the artificial skin to be transplanted, epidermal stem cells secreting leptin ensures permanent correction of the defect with a single intervention. BRIEF DESCRIPTION OF THE DRAWINGS To complement the foregoing description and in order to aid a better understanding of the characteristics of the invention, be a detailed description of a preferred embodiment, based on a set of drawings that accompany this specification and where with a merely illustrative and non-limiting character memory has been represented: figure 1 shows the effect of transplantation of human skin secreting leptin on blood glucose levels in ob / ob mice.

Figure 2 shows the effect of transplantation of human skin secreting leptin on daily intake in ob / ob mice. Figure 3 shows the effect of transplantation of human skin secreting leptin on body weight in ob / ob mice.

Figure 4 shows the effect of transplantation of human skin secreting leptin on leptin levels in ob / ob mice. DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT The embodiment described relates to the development of a method for the treatment of pathologies characterized by a permanent impairment of protein hormone leptin. The method involves transplantation of autologous artificial skin acting in the individual receiving the transplant, as an alternative source of lost hormone and that this effect is perpetuated by the rest of the patient's life.

Obtaining the principal component of the artificial skin, keratinocytes genetically modified to produce and secrete leptin, described below.

Keratinocytes are obtained from a skin biopsy by incubation with the enzyme trypsin. Once isolated are seeded on 3T3-J2 cells or

Swiss 3T3 lethally irradiated. Viral transduction is performed on keratinocyte colonies 8 to 16 cells of the first pass of a primary culture. Keratinocytes then incubated well with retroviral supernatants produced by transient transfection in 293T line or from a stable producer such as PG13 or PA317 line line. Infection with retroviral supernatants generated from 293T lines,

PG13 or PA317 is performed on two consecutive days in each of which the cells are exposed to two cycles of infection. The genetic modification may also be performed by co-culture with PG13 or PA317 after lethal irradiation of the same lines.

Genetic modification of keratinocytes, comprises the following steps:

1. First cycle of genetic modification, starting from colonies with a number of cells between 8 and 16 of culture of keratinocytes derived from skin biopsy, and seeded on 3T3-J2 or Swiss 3T3 cell line lethally irradiated, comprising centrifugation at 3,500 rpm and 30 ° C for one hour in the presence of a retroviral vector carrying the gene encoding human leptin and in the presence of a solution of polybrene concentration 60 ug / ml. In this first contact of keratinocytes with the retrovirus is followed by an incubation time of 4 hours in a fresh medium free of retrovirus. Surprisingly or centrifugation at speeds higher than the usual or high concentrations of polybrene used in the first cycle alter the viability or functionality of epidermal stem cells.

2. Second cycle genetic modification, comprising an incubation for 2 hours keratinocytes in the presence of a retroviral vector carrying the gene encoding human leptin and in the presence of a solution of polybrene concentration 8 mg / ml, medium change culture by another free retrovirus and incubation for a time of 16 hours.

3. Third cycle genetic modification, comprising an incubation for 4 hours keratinocytes in conjunction with a retroviral vector carrying the gene encoding human leptin and in the presence of a solution of polybrene concentration 8 mg / ml, medium change culture by another free retrovirus and incubation for a time of 4 hours,

4. Fourth cycle genetic modification, comprising an incubation for 4 hours keratinocytes in conjunction with a retroviral vector carrying the gene encoding human leptin and in the presence of a solution of polybrene concentration 8 ug / ml, and change culture medium free of other retroviruses.

Among the viral vectors may be employed in addition to vectors carrying a DNA sequence that encodes and expresses Leptin also carry a sequence encoding a marker gene (eg pLZRS-leptin-IRES

EGFP wherein the marker is the gene for green fluorescent protein (EGFP) or pLZRS-leptin-IRES-GHR wherein the marker is receptor growth hormone (GHR).

When keratinocytes reach 60-80% confluence, are detached from the culture flasks by trypsinization, resuspended in PBS / 2% fetal bovine serum, they are analyzed by flow cytometry and selected for example based on fluorescence green protein. This manipulation does not alter the functionality of epidermal stem cells transduced population between virus. The thus purified population consists of only cells receiving the marker gene and the gene encoding for leptin are now seeded on a fibrin matrix as described in the patent document ES 200100494. Example 1. Animal models deficient in leptin are tools invaluable for monitoring induced various protocols such as the administration of recombinant protein or other possible agonistic effects. For monitoring of the present invention they were carried out transplants artificial fur secreting human leptin in ob / ob immunosuppressed or immunodeficient able to accept the xenograft mice. The ob / ob mouse is deficient mutant mouse leptin (Coleman, DL (1978) Obese and diabetes: two mutant genes Causing diabetes-obesity syndromes in mice Diabetologia 14, 141-148.). In these animals the lack of leptin leads to a phenotype characterized by severe obesity, due to a significant increase in daily intake, hipergiucemia with hipeinsulinemia (type II diabetes) and infertility.

A population of ob / ob mice deficient in leptin transplantation artificial skin made in accordance with the present invention was applied. Circulating glucose levels and leptin as well as daily intake and weight they were monitored in relation to a control population that received genetically skin transplant unmodified observed progressive normalization thereof and maintenance over time, as shown in figures 1 to 4.

Claims

What is claimed
1. Method for obtaining an autologous artificial skin secreting leptin, characterized by comprising the following steps: • obtaining plasma with platelets by gentle centrifugation of whole blood of a human and subsequent freezing
• culturing skin cells from a skin biopsy by known methods
• gelation of plasma with platelets, previously thawed by adding calcium salts to obtain a fibrin matrix, seeding the cultured dermal cells inside the fibrin malriz
• keratinocyte culture from a skin biopsy by known methods • genéíica modification queraíinocilos; so that they are capable of expressing leptin permanently
• cultivation of genetically modified keratinocytes until they are confluent
• selection of genetically modified keratinocytes based on a marker gene
• seeding of keratinocytes selected on the artificial dermis obtained after the first three stages described.
2. Method of obtaining a leptin auíóloga producing artificial skin according to claim 1, caracíerizado because genéíica queraíinocitos modification, comprising the following steps:
• first cycle of genetic modification, starting from colonies with a number of cells enire 8 and 16 of culture of keratinocytes derived from skin biopsy, comprising a cenírifugación at 3,500 rpm and 30 ° C duranle one hour in conjunction with a retroviral vector carrying the gene encoding human leptin and in the presence of a solution of polybrene concentration 60 ug / ml, change the culture medium by other free retrovirus and incubation for a time of 4 hours,
• second cycle of genetic modification, comprising an incubation for 2 hours of keratinocytes together with a carrier retroviral vector of the gene encoding human leptin and in the presence of a solution of polybrene concentration 8 ug / ml, change medium crop for another free reírovirus and incubation for 16 hours, • third cycle of genetic modification, comprising an incubation for 4 hours of keratinocytes together with a carrier retroviral vector of the gene encoding human lepíina and in the presence of a solution of polybrene conceníración 8 .mu.g / ml, change medium cullivo other free refrovirus and incubation for a íiempo 4 hours, • fourth cycle of genetic modification, comprising an incubation for 4 hours keratinocytes in binding a retroviral vector carrying the gene encoding human lepíina and in the presence of polybrene solution with centration 8 mcg / ml, and changing the culture medium by OIRO free retrovirus.
3. Artificial skin aulóloga secreting lepíina, characterized by incorporating genetically modified to express leptin keratinocytes susceptible permanently.
4. Artificial skin auíóloga secreting leptin, characterized by genetically modified seed keratinocytes through its infection with a retroviral vector carrying the gene encoding human lepíina.
PCT/ES2002/000320 2001-06-29 2002-06-28 Artificial autologous skin secreting leptin and method for obtaining the same WO2003002154A1 (en)

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ESP200101526 2001-06-29

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Cited By (12)

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EP1653807A2 (en) * 2003-05-01 2006-05-10 Medgenics, Inc. Dermal micro organs, methods and apparatuses for producing and using the same
WO2006123004A1 (en) 2005-05-16 2006-11-23 Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañon Endothelized artificial matrix comprising a fibrin gel, which is a superproducer of proangiogenic factors
US7358397B2 (en) 2003-09-09 2008-04-15 Lanxess Deutschland Gmbh Preparation of halogenated 4-aminophenols
EP2165678A1 (en) * 2001-03-01 2010-03-24 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Artificial dermis and production method therefor
US8088568B2 (en) 2001-11-05 2012-01-03 Medgentics, Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8142990B2 (en) 2001-11-05 2012-03-27 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
EP2452691A3 (en) * 2005-08-29 2012-09-05 HealOr Ltd. Methods and compositions for prevention of diabetic and aged skin
US8501396B2 (en) 2001-11-05 2013-08-06 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8507431B2 (en) 2003-08-07 2013-08-13 Healor Ltd. Methods for accelerating wound healing by administration of a preadipocyte modulator or an adipocyte modulator
US8877175B2 (en) 2006-09-14 2014-11-04 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9127084B2 (en) 2006-09-14 2015-09-08 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9155749B2 (en) 2006-09-14 2015-10-13 Medgenics Medical Israel Ltd. Long lasting drug formulations

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ES2551143B1 (en) * 2014-05-13 2016-07-01 Fundación Tekniker artificial skin, artificial skin, methods for their preparation and their uses

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EP2165678A1 (en) * 2001-03-01 2010-03-24 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Artificial dermis and production method therefor
US8088568B2 (en) 2001-11-05 2012-01-03 Medgentics, Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8530149B2 (en) 2001-11-05 2013-09-10 Medgenics Medical Israel Ltd Dermal micro-organs, methods and apparatuses for producing and using the same
US8501396B2 (en) 2001-11-05 2013-08-06 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8293463B2 (en) 2001-11-05 2012-10-23 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US8142990B2 (en) 2001-11-05 2012-03-27 Medgenics Inc. Dermal micro-organs, methods and apparatuses for producing and using the same
US9468667B2 (en) 2001-11-05 2016-10-18 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US9107896B2 (en) 2001-11-05 2015-08-18 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US9101595B2 (en) 2002-11-05 2015-08-11 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8685635B2 (en) 2002-11-05 2014-04-01 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
US8771291B2 (en) 2002-11-05 2014-07-08 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
EP2377402A1 (en) * 2003-05-01 2011-10-19 Medgenics, Inc. A genetically modified dermal micro-organ expressing interferon
EP2936984A1 (en) * 2003-05-01 2015-10-28 Medgenics, Inc. Apparatus and method for harvesting a dermal micro-organ
EP1653807A4 (en) * 2003-05-01 2009-05-27 Medgenics Inc Dermal micro organs, methods and apparatuses for producing and using the same
EP1653807A2 (en) * 2003-05-01 2006-05-10 Medgenics, Inc. Dermal micro organs, methods and apparatuses for producing and using the same
EP2377404A1 (en) * 2003-05-01 2011-10-19 Medgenics, Inc. A dermal micro-organ which is an explant of living tissue
US9572593B2 (en) 2003-05-01 2017-02-21 Medgenics Medical Israel Ltd. Dermal micro-organs, methods and apparatuses for producing and using the same
EP2377403A1 (en) * 2003-05-01 2011-10-19 Medgenics, Inc. A genetically modified dermal micro-organ expressing factor VIII
EP2377401A1 (en) * 2003-05-01 2011-10-19 Medgenics, Inc. A genetically modified dermal micro-organ expressing erythropoietin
US8507431B2 (en) 2003-08-07 2013-08-13 Healor Ltd. Methods for accelerating wound healing by administration of a preadipocyte modulator or an adipocyte modulator
US7358397B2 (en) 2003-09-09 2008-04-15 Lanxess Deutschland Gmbh Preparation of halogenated 4-aminophenols
WO2006123004A1 (en) 2005-05-16 2006-11-23 Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañon Endothelized artificial matrix comprising a fibrin gel, which is a superproducer of proangiogenic factors
ES2263382A1 (en) * 2005-05-16 2006-12-01 Fundacion Para La Investigacion Biomedica Del Hospital Gregorio Marañon Endothelized fibrin gel matrices, useful for forming vacular bridges between transplanted skin and receptor sites, contain embedded transfected endothelial cells overexpressing proangiogenic factor
US8367606B2 (en) 2005-08-29 2013-02-05 Healor Ltd. Method and compositions for prevention and treatment of diabetic and aged skin
EP2452691A3 (en) * 2005-08-29 2012-09-05 HealOr Ltd. Methods and compositions for prevention of diabetic and aged skin
US9155749B2 (en) 2006-09-14 2015-10-13 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9127084B2 (en) 2006-09-14 2015-09-08 Medgenics Medical Israel Ltd. Long lasting drug formulations
US8877175B2 (en) 2006-09-14 2014-11-04 Medgenics Medical Israel Ltd. Long lasting drug formulations
US9687564B2 (en) 2006-09-14 2017-06-27 Medgenics Medical Israel Ltd. Long lasting drug formulations

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ES2184623A1 (en) 2003-04-01 application

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