The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention—indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS), and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound bFGF and VEGF. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained three hours of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering.

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model parenchymal cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (DP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that DP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

Spontaneous assembly of human mesenchymal stem cells (hMSCs) into 3D aggregates enhances stem cell properties and enables formation of heterotypic organoids, which has significant implication in cell therapy and tissue engineering. While metabolic reprograming towards glycolysis is a salient feature of multicellular aggregates and it has commonly been attributed to oxygen diffusion limitations; however, recent studies have instead observed a limited decline in oxygen tension, in hMSC aggregates, challenging this view. Although aggregation of a dispersed cell population involves both changes in the physical and molecular environment most studies to date have focused on molecular gradients with limited investigation in biomechanical stress on the fate of aggregated cells. The objective of this study is to investigate how the mechanical gradient arising from aggregation induced cortical compaction alters the metabolic profile of human adipose derived mesenchymal stem cells (hASCs) by testing the hypothesis that size-dependent compaction in aggregates leads to differential cortical stress which induces metabolic reprogramming. Herein, we show that aggregation of multiple sizes covering a wide range of interest does not lead to hypoxic core formation but instead varying levels of cortical compaction, indicated by the balance between stress fiber formation and the deposition of extracellular matrix proteins, resulting in corresponding levels of metabolic reconfiguration. Increased glycolytic metabolism, increased mitochondrial fission, and increased release of aldolase A were all observed as a result of cortical compaction. Chemical inhibition with Gleevec, Wortmannin, and Y27632 almost completely abolishes the cortical stress induced enhancement in glycolytic properties. Our findings demonstrate that aggregation-induced biomechanical stress plays a central role in driving metabolic reprogramming.