Recellularization of Well-Preserved Acellular Kidney Scaffold Using Embryonic Stem Cells

Abstract

For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Microcomputerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and glomeruli. This experimental setup may open the possibility to obtain differentiation of stem cells with long lasting in vitro perfusion.

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization

“…Both kidney and liver bioreactors described here represent organ-specific refinements to improve recellularization and monitoring of cell growth and function based on the original cardiac perfusion model described by Langendorff 18,19 and later modified for the kidney 7,10,15,16 and liver 2,9,11,12,30 bioengineering. The modified kidney bioreactor design described here improves upon these models by incorporating both ureteral and arterial routes for recellularization allowing for a direct comparison, and we show improved recellularization using high-pressure arterial infusion compared to ureteral infusion of renal epithelial cells.…”

“…Specifically, given the inherent complexity of the renal architecture, kidney scaffold recellularization has been particularly challenging. 7,10,15,16 To overcome this limitation, we provide new detail on bioreactor features that allow for comparison of alternative seeding strategies and demonstrate improved renal epithelial cell dispersion throughout the tubular compartment of decellularized scaffolds.…”

“…Placing this finding in context of other methods of cell delivery into the scaffold, this method of tubular repopulation is more efficient compared to infusion of cells through the ureter or artery at flow rates (e.g., <1 mL/min) which others have shown to preferentially restrict cells to glomeruli and nearby arterioles. 7,10,15,16 We additionally infused primary CD133/1 + human renal progenitor cells recovered from the renal papillae 23 using the arterial seeding method to validate the bioreactor system using primary cells. We observed similar dispersion of the cells throughout the renal cortex over a 3-and 7-day culture period (Fig.…”

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

“…The goal of the described decellularization protocol is to produce an acellular renal ECM that can be used as a 3D scaffolding system for regeneration of kidney structures, including nephron tubules that are repopulated in the present example with human renal cortical tubular epithelial (RCTE) cells. We previously described our rigorous evaluation of an optimal, detergent-based rat kidney decellularization protocol 7 , which is more rapid (approximately one day) than other methods previously reported (Ross et ), and exposes the organ to a considerably lower concentration (0.1%) of the denaturant sodium dodecyl sulfate (SDS) during decellularization than prior reports [12][13][14][15] .…”

Bioengineered functional brain-like cortical tissue

“…Recent advances in stem and progenitor cell technology have induced cells to differentiate into and produce tissue-appropriate cell compositions and ECM components and form biomimetic tissues with nascent functions, including the cerebral organoids (9). These technologies show self-organization capability of cells in tissue-mimetic environments, such as native tissue-derived decellularized scaffolds (10)(11)(12)(13). However, densely packed brain tissue with an architecture defined by neuronal connectivity (14,15) presents a unique challenge to define modular structures with specific functions.…”