WO2009061725A2 - Réparation vasculaire et cellules endothéliales - Google Patents

Réparation vasculaire et cellules endothéliales Download PDF

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Publication number
WO2009061725A2
WO2009061725A2 PCT/US2008/082342 US2008082342W WO2009061725A2 WO 2009061725 A2 WO2009061725 A2 WO 2009061725A2 US 2008082342 W US2008082342 W US 2008082342W WO 2009061725 A2 WO2009061725 A2 WO 2009061725A2
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WIPO (PCT)
Prior art keywords
cells
endothelial cells
fdecs
ecs
endothelial
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PCT/US2008/082342
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English (en)
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WO2009061725A3 (fr
Inventor
Robert D. Simari
Allan B. Dietz
Barry A. Boilson
Harald Froehlich
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Publication of WO2009061725A2 publication Critical patent/WO2009061725A2/fr
Publication of WO2009061725A3 publication Critical patent/WO2009061725A3/fr
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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
    • C12N5/069—Vascular Endothelial cells

Definitions

  • This document relates to endothelial cells (e.g., endothelial cells derived from omental fat) and methods of using endothelial cells for to repair vascular tissue.
  • endothelial cells e.g., endothelial cells derived from omental fat
  • methods of using endothelial cells for to repair vascular tissue e.g., endothelial cells derived from omental fat
  • PTCA Percutaneous transluminal coronary angioplasty
  • PTA percutaneous transluminal angioplasty
  • CAD coronary artery disease
  • PTA peripheral artery disease
  • BMS Bare metal stents
  • This document relates to methods and materials involved in obtaining endothelial cells (ECs) and repairing vascular tissue.
  • ECs endothelial cells
  • FDECs fat derived ECs
  • ECs vascular repair
  • one aspect of this document features an enriched population of fat (e.g., omental fat) derived endothelial cells.
  • the cells can be positive for vWF, eNOS, VEGFR-2, and Griffonia lectin, and can be negative for SM-actin.
  • this document features a method for isolating an enriched population of fat derived endothelial cells.
  • the method comprises obtaining DiO acetylated LDL positive endothelial cells from a population of cells.
  • this document features a method for repairing vascular tissue in a mammal.
  • the method comprises administering an amount of an enriched population of fat derived endothelial cells to the mammal under conditions effective to repair vascular tissue.
  • FIG. 1 Fat derived cells after one day (A) and 7 days (B) in culture showing cobblestone morphology.
  • the FDECs were highly proliferative and did not exhibit a difference in proliferation in use of FBS or autologous rabbit serum (C). After 14 days, almost 5 x 10 7 cells could be cultured (D).
  • FIG. 3 Fat derived endothelial cells from rabbit exhibited two times longer tube formations than rabbit carotid artery endothelial cells.
  • FIG. 4 Reendothelialisaton after 48 hours.
  • FIG. 5 Cells stained for DiI acetylated LDL are covering the whole vessel lumen (A: 10Ox), (B: 5Ox), (C: 20Ox). D reveals the vessel wall of a untreated vessel.
  • FIG. 6. Results on endothelial dependent vasoreactivity in organchamber plotting relaxation in percent.
  • FIG. 7. Intima media ratio of 28 day survivors. Unselected FDECs did not reveal a significant improvement in morphology, whereas the DiO sorted cells exhibited a significant improvement.
  • FDECs e.g., FDECs
  • methods and materials for using ECs e.g., FDECs
  • FDECs e.g., FDECs
  • enriched populations of FDECs means that the population has at least a two fold increase (e.g., at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 500, 750, 1000, 2500, or 5000 fold increase) in ECs from the crude population of cells present in the fat tissue from which the ECs are isolated.
  • the ECs can be isolated from mammals, including rodents such as mice and rats, farm animals such as pigs, cattle, sheep, and goats, and humans or other primates.
  • ECs can be obtained from fat tissue of a mammal.
  • fat tissue For example, omental, arterial, kidney, or subcutaneous fat tissue can be used to obtain FDECs. Once obtained, the fat tissue can be treated to that a population of ECs are obtained.
  • the methods and materials described herein can be used to obtain an enriched population of FDECs.
  • an enriched population of FDECs can be obtained directly from a source of fat.
  • EC isolation techniques can be used to obtain an enriched population of FDECs.
  • in vitro culture techniques can be used to obtain an enriched population of FDECs.
  • ECs for fat tissue can be cultured as described herein to obtain an enriched population of FDECs.
  • EC isolation and in vitro culture techniques can be used to obtain an enriched population of FDECs.
  • endothelial cell markers can be use to increase the purity of a population of ECs.
  • DiO acetylated LDL can be used to obtain an enriched population of ECs.
  • ECs e.g., FDECs
  • the ECs (e.g., FDECs) provided herein can be used to treat vascular conditions such as acute injury, chronic endothelial injury, hypertension, pulmonary hypertension, response to angioplasty or stenting, atherosclerosis, and endothelial dysfunction.
  • vascular conditions such as acute injury, chronic endothelial injury, hypertension, pulmonary hypertension, response to angioplasty or stenting, atherosclerosis, and endothelial dysfunction.
  • the ECs (e.g., FDECs) provided herein can be injected into the vascular system upstream of acute or chronic injury or endothelial dysfunction under conditions that result in vascular repair.
  • Omental-Fat was harvested from New Zealand White rabbits (3.5-4.5 kg) under deep anesthesia with Isoflurane. Through a one-inch incision in the epigastric region right below the sternum along the linea alba, about 3-5 g of omentum was exposed with a hook, and the visible vessel were tight before the fat was removed. Following mechanical disaggregation, the omentum was washed with Ca 2+ - and Mg 2+ -free PBS (Cellgro). A 1% solution of HBSS with Ca 2+ and Mg 2 (Cellgro) and Collagenes Type I (Worthington, 142u/mg) was used to digest the tissue.
  • EGM-2 is a product of Lonza and EBM-2 with 2% serum, hydrocortisone, recombinant Fibroblastic Growth Factor (hFGF), Vascular Endothelial Growth Factor (VEGF), recombinant Insuline-Like Growth Factor (R-IGF), ascorbic acid, recombinant Epidermal Growth Factor (hEGF), heparin, antimicrobial (penicillin and streptomycin), and antimicotic (amphotericin B) agents. Cells were passaged before reaching confluence, and the medium was changed daily until harvest. EGM-2 medium was slightly modified using 2% autologous serum (AS) instead of 2% Fetal Bovine Serum (FBS).
  • AS autologous serum
  • FBS Fetal Bovine Serum
  • VWF von Willebrand Factor
  • eNOS endothelial nitric oxide synthase
  • SM-actin smooth muscle actin
  • Griffonia lectin vascular endothelial growth factor receptor-2
  • vWF was visualized using donkey anti-sheep biotin followed by Streptavidin fluorescein (Amersham Biosciences Corp, Piscataway, NJ). Hoescht staining was used to identify the nuclei. DiI acetylated LDL was also used to co- stain with vWF, eNOS, and SM-actin as described elsewhere (Fraser et al., Nat. Clin. Pract. Cardiovasc. Med., 3 Suppl. l :S33-37 (2006)) for coexpression studies. Fluorescence-activated cell sorting (FACS) detection of DiO acetylated LDL was performed to detect the purity of the cultured cells. For a long-term group sub-study of 28 days, FACS selection was used to select only strongly positive cells who had a high uptake for DiO acetylated LDL. This was used to raise the purity of delivered cells.
  • FACS Fluorescence-activated cell sorting
  • FDECs from 12 different rabbits were cultured. Six cultures were grown with EGM-2 with 2% FBS, and six cultures with EGM-2 and 2% autologous serum. The cultures were detached using 0.05% Trypsin and 0.53 mmol/L EDTA in HBSS (Cellgro) every other day, and the cell-numbers were determined with a hemocytometer. Afterwards, the cells were plated again on a freshly f ⁇ bronectin coated well.
  • Tube forming Assays To determine the angiogenetic potential of the fat derived ECs, tube forming assays were performed using Matrigel (Invitrogen, Geltrex Reduced Growth Factor Basement Membrane Matrix). 96 well plates were coated with 150 ⁇ L of Matrigel. After detaching the ECs with 0.05% Trypsin and 0.53 mmol/L EDTA in HBSS (Cellgro), cells were plated in a density of about 1000 cells per 96 well plate. As a control rabbit carotid artery endothelial cells were used. After 24 hours, pictures of each well were taken, saved, and imported into Image Pro Plus software program. The total length of each tube or the long axis of single cells or groups of adjacent cells was measured. The assay was tested in triplicate and repeated three times.
  • New Zealand White rabbits weighting 3.5 to 4.5 kg were anesthetized with Isoflorine.
  • the left and the right common carotid artery were exposed from the suprasternal notch to just below the internal/external bifurcation.
  • vessel clamps were used to isolate the vessels.
  • 8-0 purse-string suture was placed through which a small arteriotomy was created.
  • a 2F Fogarty balloon catheter (Baxter) was introduced antegrade into the vessel lumen. The balloon was inflated to cause a visible distension and withdrawn three times to denude a 3 -cm length of artery as described elsewhere (Miranvill et al, Circulation, 110(3):349-55 (2004)).
  • a 24-gauge catheter was placed in the vessel lumen.
  • 250 ⁇ L of Ca 2+ and Mg 2+ PBS was injected, and in the left carotid artery, 250 ⁇ L Of Ca 2+ and Mg 2+ PBS with FDECs (2.0 to 3.0 x 10 6 cells).
  • the instillation time was 20 minutes for both sides.
  • the arteriotomy was closed with a purse string suture, and the vessel clamps were removed to restore blood flow.
  • 81 mg ASA was administered per os daily to the rabbit.
  • the slides were manually traced and analyzed with Image ProPlus. Endo luminal, internal elastic laminar, and external elastic lamina were used to calculate intimal and media areas.
  • a rabbit model of acute balloon injury of both carotid arteries was used. Rabbits were treated with 2-3 x 10 6 DiI acetylated LDL labeled cells, and after 48 hours intraluminal coverage of endothelial cells was measured with the absence of Evans-Blue staining as a macroscopic method and with cross-sections of the vessels and the identification of labeled cells. 48 hours after treatment, the vessels in which cells were delivered exhibited almost complete exclusion of Evans- Blue, while the control vessels were stained entirely blue. A mean coverage of 82.2 % ( ⁇ 26.9) of the treated vessel against 4.2 % ( ⁇ 3.0) of the untreated vessel (p ⁇ 0.001) was detected (Fig. 4).
  • results provided herein demonstrate that autologous microvascular endothelial cells derived from omental fat can be used to enhance re-endothelialization in 48 hours almost completely. Moreover, FDEC delivery was associated with an increase of endothelium dependent vasoreactivity and a decrease in neointimal formation compared to the controls, which were treated with saline. The results provided herein also demonstrate that only a very low amount of fat (3-5 g) is necessary to obtain a very high number of phenotypical and functional microvascular endothelial cells. In addition, the results provided herein demonstrate that autologous serum can be used to culture those highly proliferative cells.
  • DiO acetylated LDL was used as a functional marker to select positive cells with FACS to enhance the purity of FDECs. Even though most of the cultures were highly positive for DiO acetylated LDL, we did gate only for 75% of the highest positive cells to improve the purity of the cells. Using this method, we could highlight next to the 4 times increase of endothelium dependent vasoreactivity also a 60% decrease in neointimal formation.
  • FDECs provide a extremely fast reendothelialization and an improvement in vascular function and morphology after vessel injury.
  • results provided herein demonstrate that FDECs are highly proliferative, have a high angiogenic potential, and can be cultured and used with autologous serum.
  • FDECs can be used to treat vessel injuries and arterial occlusive disease.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Vascular Medicine (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des matériaux impliqués dans l'obtention de cellules endothéliales (EC) et la réparation de tissu vasculaire. Par exemple, des procédés et des matériaux pour obtenir des EC dérivées de corps gras ainsi que des procédés et des matériaux pour utiliser des EC pour une réparation vasculaire sont prévus.
PCT/US2008/082342 2007-11-05 2008-11-04 Réparation vasculaire et cellules endothéliales Ceased WO2009061725A2 (fr)

Applications Claiming Priority (2)

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US197507P 2007-11-05 2007-11-05
US61/001,975 2007-11-05

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WO2009061725A2 true WO2009061725A2 (fr) 2009-05-14
WO2009061725A3 WO2009061725A3 (fr) 2009-08-13

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CA2644115C (fr) * 2006-03-07 2015-02-24 Humacyte Procede d'extraction de cellules endotheliales de tissus adipeux

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