4.4 Article

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

期刊

出版社

JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/53271

关键词

Bioengineering; Issue 102; Bioreactor; cell seeding; decellularization; extracellular matrix; epithelial; kidney; perfusion; recellularization; renal; resazurin; scaffold; tissue engineering

资金

  1. Zell Family Foundation
  2. Northwestern Memorial Foundation Dixon Translational Research Grants Initiative
  3. American Society of Transplant Surgeon's Faculty Development Grant
  4. National Kidney Foundation of Illinois
  5. Robert R. McCormick Foundation
  6. NIDDK [K08 DK10175]
  7. Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC)
  8. Simpson Querrey Institute Equipment Core at Northwestern University
  9. Cancer Center Support Grant [NCI CA060553]
  10. [P30 CA118100]
  11. NATIONAL CANCER INSTITUTE [P30CA118100, P30CA060553] Funding Source: NIH RePORTER
  12. NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [F32DK010175, K08DK101757] Funding Source: NIH RePORTER

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This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a customdesigned perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 degrees C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (similar to 230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 mu M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

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