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Methods and compositions of bioartificial kidney suitable for use in vivo or ex vivo

a bioartificial kidney and composition technology, applied in the field of bloartiflcial kidney, can solve the problems of insufficient time for primary cells to be used in bioengineering, immortalized cells do not possess the full range of function of primary renal cells,

Inactive Publication Date: 2005-10-27
RGT UNIV OF MICHIGAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, immortalized cells do not possess the full range of function of primary renal cells.
Prior to the contributions of the present inventors, primary cells could not be maintained for a sufficient time to make their use in bioengineering applications useful.

Method used

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  • Methods and compositions of bioartificial kidney suitable for use in vivo or ex vivo
  • Methods and compositions of bioartificial kidney suitable for use in vivo or ex vivo
  • Methods and compositions of bioartificial kidney suitable for use in vivo or ex vivo

Examples

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example 1

[0080] Endothelial Cell Culture. A permanent human endothelial cell line, designated ECV 304 (Takahashi et al, In vitro Cell Dev. Biol. 25:265-274, 1990), was used for the initial studies for ease of use. To extend the gene transfer and tissue engineering constructs, endothelial cells in primary culture from rabbit vasculature, was also developed.

[0081] ECV-304 Cell Isolation and Culture

[0082] Isolation: ECV-304 (Human Endothelial, transformed) permanent cell line was obtained from American Type Culture Collection (Rockville, Md.). Culture Conditions: ECV-3.04 cultures were maintained on tissue culture plates in a humidified, 37°, 5% CO2 incubator, and grown in media M 199 supplemented with 10% heat inactivated fetal calf serum plus 0.1 % fungi-bact, with media changed twice a week. Cells were subcultured at a ratio of 1:6 by using a solution of 1° endothelial cells 0.25% trypsin, 1 mM EDTA.

[0083] Mesangial Cell Isolation and Culture

[0084] Isolation: Mesangial cells were isolate...

example 2

[0087] Gene transfer of hirudin into endothelial cells. A full length cDNA encoding for the hirudin variant HV-2 (Johnson et al, Seminars in Thrombosis 15:302-315, 1989) has been constructed utilizing dual asymmetric polymerase chain reaction (PCR) in which four adjacent oligonucleotides of 76 to 89 bases in length having short overlaps of 14 bases were used as primers in a PCR mixture (Sander et al, Biotechniques 12:14-16, 1992). In constructing this cDNA, a signal sequence for von Willebrand factor (vWF) was—incorporated in frame 5′ to the hirudin gene to ensure secretion of hirudin from transduced cells; protein coding sequences were selected based upon optimal codon usage in rabbit and human genetic sequence data (Wada et al, Nucl. Acids Res. 19:1981-1986, 1991), and appropriate restriction enzyme cut sites 5′ and 3′ to the cDNA encoding the vWF signal peptide and hirudin HV-2 for ease of transfer into the retroviral vector. The sequence of the constructed cDNA was confirmed to ...

example 3

[0090] Improved Retroviral Gene Transfer for Hirudin Production. To improve hirudin protein production and secretion by endothelial cells, a different retroviral construct was developed, as detailed in FIG. 4. In this regard, introduction of a drug-selectable marker that is coexpressed with the hirudin gene was planned. Previous strategies have been employed for the coexpression of drug-selectable genes with a second nonselectable gene with the utilization of independent promoters, such as a retroviral LTR and another promoter (Emerman et al, Cell 39:459467, 1984). With this approach, a replication-defective retroviral vector was constructed capable of efficient constitutive expression of the hirudin gene in mammalian cells by using the LTR promoter. A selectable neomycin (neo) gene, conferring G418 resistance, is under the control of the SV40 promoter. The optimal codon usage in human genetic sequence data was selected to design the hirudin cDNA sequence. A signal sequence for plas...

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Abstract

A novel cell seeded hollow fiber bioreactor is described as a potential bioartificial kidney. Endothelial cells along with pericyte, vascular smooth muscle, and / or mesangial cells or any mesenchymally derived support cells are seeded along a hollow fiber in a perfused bioreactor to reproduce the ultrafiltration function and transport function of the kidney. Maintenance of tissue specific function and ultrastructure suggest that this bioreactor provides an economical device for treating renal failure.

Description

[0001] Applicants hereby claim as priority U.S. Provisional Application Ser. No. 60 / 027,495 filed on Sep. 30, 1996.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention provides a BlOARTIFlCIAL kidney comprising (1) a filtration device comprising endothelial cells and pericyte cells and (2) a tubule processing device. [0004] 2. Background of the Invention [0005] An implantable epithelial cell-system derived from immortalized cells grown as confluent monolayers along the luminal surface of impermeable polymeric hollow fibers has been described as a first step for tubule functional replacement (Ip and Aebischer, Artificial Organs 13:58-65, 1989, incorporated herein by reference). Unfortunately, immortalized cells do not possess the full range of function of primary renal cells. Critical to development of functional renal tissue is the isolation and growth in vitro of primary cell lines. Primary cell lines should possess characteristics such that the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K45/00A61M1/16A61M1/34A61M1/36B01D67/00B01D69/00B01D69/14C12N5/02C12N5/071C12N5/077
CPCA61M1/16A61M1/1678A61M1/3687B01D67/0088B01D69/00B01D69/141A61M1/3489C12N5/0697C12N2502/1347C12N2502/256C12N2502/28C12N2510/00C12N2510/02C12N5/0686C12M21/08C12M25/10A61M1/28A61M1/3689A61K35/14
Inventor HUMES, H. DAVID
Owner RGT UNIV OF MICHIGAN
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