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Bioartificial proximal tubule systems and methods of use

A bioartificial, renal proximal tubule technology, applied in the field of bioartificial proximal tubule devices, can solve the problems of inability to measure the influence of renal tubules, inability to measure flow changes, physiological and dynamic conditions, laborious separation techniques, species differences, etc.

Inactive Publication Date: 2014-06-04
DEPUY SYNTHES PROD INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latter is problematic due to laborious separation techniques and species differences that need to be considered
Another disadvantage of these alternative methods is that they only measure the transport of exogenous compounds applied to the culture medium into the renal tubular lumen
The effect of these compounds on normal tubular function (such as glucose reabsorption or albumin uptake) could not be determined because no reliable method existed for introducing labeled test substances into the lumen or sampling the luminal fluid of the tubules for to measure
Furthermore, changes in flow and the influence of physiologically dynamic conditions that may occur in various in vitro scenarios cannot be determined

Method used

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  • Bioartificial proximal tubule systems and methods of use
  • Bioartificial proximal tubule systems and methods of use
  • Bioartificial proximal tubule systems and methods of use

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0097] Example 1: Seeding and Differentiation of hKDCs on Extracellular Matrix Scaffolds

[0098] This experiment tests the attachment, growth and differentiation of human kidney-derived cells ("hKDC") on various configurations of extracellular (ie, acellular) matrix scaffolds as well as traditional culture on collagen-coated cell chambers.

[0099] Four passages of hKDC were seeded onto three different scaffold configurations and cell chambers (Coming, Coming NY). use REGM TM Kidney epithelial cell growth medium (Lonza, Walkersville MD) cells were cultured for a period of more than three weeks. Each scaffold configuration was tested with three different cell concentrations: 2.5 × 10 3 , 5×10 3 , and 1×10 4 cells. The configurations tested were: 1) collagen sandwich culture; 2) collagen-SIS sandwich culture; 3) SIS monolayer culture; and 4) collagen coated cell chambers.

[0100] Collagen sandwich culture

[0101] hKDCs were cultured between two collagen gel layers ...

example 2

[0114] Example 2: Optimization of Cell Seeding Concentration for hKDCs on Decellularized Scaffolds

[0115] Example 1 shows that cells seeded onto a collagen-free two-dimensional decellularized scaffold form a confluent monolayer of epithelial cells that express proximal tubule markers after three weeks in culture. Perform the following experiments to optimize cell seeding density and attempt to shorten the culture period required to form a monolayer.

[0116] Scaffolds were prepared by decellularizing certain fragments of the small intestinal submucosa (SIS) as described in Example 1 . at three different concentrations (1×10 4 , 5×10 4 and 1×10 5 cells / well) hKDCs of 4 passages were seeded on SIS scaffolds and treated with REGM TM Kidney Epithelial Cell Growth Medium (Lonza, Walkersville) was used to culture them for three weeks. Samples were removed for histological analysis after two and three weeks and fixed with Bouin's fixative for 1 hour. Then, they were washed ...

example 3

[0124] Example 3: Immunohistochemistry of p-glycoprotein-1

[0125] Scaffolds were prepared by decellularizing certain fragments of the small intestinal submucosa (SIS) as described in Example 1 . Take 5×10 4 Four passages of hKDCs were seeded onto SIS scaffolds at a concentration of 3 cells / scaffold and treated with REGM TMKidney Epithelial Cell Growth Medium (Lonza, Walkersville) was used to culture them for three weeks. Samples were removed for histological analysis after three weeks and fixed with Bouin's fixative for 1 hour. Afterwards, the samples were washed in water for at least 4 hours and embedded in paraffin. Subsequently, 3 μm thick sections were prepared. Immunohistochemistry (IHC) was performed to confirm the expression of p-glycoprotein-1 (pgp-1, also known as MDR1), an efflux transporter expressed by proximal tubule cells. Target retrieval of dewaxed segments was achieved by enzymatic treatment with proteinase K, heating in citrate buffer pH 6 (Dako, #S2...

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Abstract

This application invention discloses bioartificial proximal tubule device, constructed by preparing a decellularized biological matrix, seeding the biological matrix with mammalian kidney-derived cells and optionally mammalian endothelial cells. The device may then be cultured statically or matured using bioreactor culture to allow differentiation of the kidney cells into functioning proximal tubule cells. The device is capable of carrying out proximal tubule functions. The application also describes various methods of making the proximal tubule devices. The application also further describes methods of use of bioartificial proximal tubule devices for e.g. in vitro studies of tubule cell transport, toxicity effects of various compounds or pharmaceutical compound screening.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 490,890, filed May 27, 2011, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present invention generally relates to a bioartificial proximal tubule device comprising a bioscaffold and one or more progenitor cells (e.g., mammalian kidney-derived cells), the one or more progenitor cells Cells differentiated on the scaffolds into renal proximal tubular cell monolayers. The invention also relates to methods for manufacturing and culturing said devices in bioreactors. The present invention also provides methods for in vitro nephrotoxicity or drug compound screening using the device. Background technique [0004] Chronic kidney disease (CKD) and end-stage renal disease (ESRD) are defined by the failure of kidney function, primarily by glomerular filtration rate. This causes the kidneys to be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2533/54C12N2503/00C12N2533/92C12N5/0602A61L27/38
Inventor C.卡札内基D.C.科特J.香兹A.霍彭萨克J.汉斯曼恩H.瓦勒斯
Owner DEPUY SYNTHES PROD INC
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