Date/Time
Date(s) - 07/17/2013
3:00 pm
Each year, there is a severe shortage of kidneys for organ transplant. One approach to alleviate this demand is to reconstruct kidneys using tissue engineering scaffolds. Such scaffolds may be produced through the decellularization of whole organs. Ideally, the resulting extracellular matrices would retain the organ-specific architecture and chemical composition to guide implanted cells into functional structures and eventually working organs. Pluripotent embryonic stem cells are highly proliferative, capable of differentiating into all cells of the kidney, and respond to tissue-specific extracellular matrix proteins. Examining the ability of these cells to proliferate and differentiate in a whole kidney extracellular matrix scaffold would serve as a valuable baseline for future studies in producing a functioning organ.
Decellularization of whole rat kidneys was achieved by perfusion of detergent-based solutions through the renal artery. The most effective 5-day protocol utilized Triton X-100, sodium dodecyl sulfate (SDS), salts, and deoxyribonuclease (DNase) and was determined by modifying detergent concentration and perfusion duration and testing an alternative ionic detergent. The resultant organ-shaped structure was spongy and translucent. Hematoxylin and eosin histochemical analysis and electron microscopy showed preserved glomerular, vascular and tubular structures without observed nuclear material and only trace amounts of residual cellular debris. In addition, immunohistochemistry (IHC) showed removal of cellular proteins and preservation of essential basement membrane (BM) proteins collagen IV and laminin.
Murine embryonic stem cells were seeded into the decellularized scaffolds and cultured up to 14 days with one of three protocols: whole scaffold, perfused scaffold, and sectioned scaffold culture. Cells delivered into the renal artery showed initial glomerular localization with subsequent expansion into adjacent vasculature, interstitium, and tubules. Over time, histological analysis revealed distinct cell morphologies, patterns of cell division and apoptosis, and cell arrangements with Pax-2 and Cytokeratin positivity. Cytokeratin is an epithelial cell marker and Pax-2 is necessary for kidney development. The evidence of cellular change of mouse stem cells in response to a rat scaffold is a promising step toward establishment of a xenogenic scaffold source for engineered kidneys.