Emma Moran

Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations.

Background:It is important to identify new sources of transplantable organs because of the critical shortage of donor organs. Tissue engineering holds the potential to address this issue through the implementation of decellularization–recellularization technology. Objective:To produce and examine acellular renal extracellular matrix (ECM) scaffolds as a platform for kidney bioengineering. Methods:Porcine kidneys were decellularized with distilled water and sodium dodecyl sulfate–based solution. After rinsing with buffer solution to remove the sodium dodecyl sulfate, the so-obtained renal ECM scaffolds were processed for vascular imaging, histology, and cell seeding to investigate the vascular patency, degree of decellularization, and scaffold biocompatibility in vitro. Four whole renal scaffolds were implanted in pigs to assess whether these constructs would sustain normal blood pressure and to determine their biocompatibility in vivo. Pigs were sacrificed after 2 weeks and the explanted scaffolds were processed for histology. Results:Renal ECM scaffolds were successfully produced from porcine kidneys. Scaffolds retained their essential ECM architecture and an intact vascular tree and allowed cell growth. On implantation, unseeded scaffolds were easily reperfused, sustained blood pressure, and were tolerated throughout the study period. No blood extravasation occurred. Pathology of explanted scaffolds showed maintenance of renal ultrastructure. Presence of inflammatory cells in the pericapsular region and complete thrombosis of the vascular tree were evident. Conclusions:Our investigations show that pig kidneys can be successfully decellularized to produce renal ECM scaffolds. These scaffolds maintain their basic components, are biocompatible, and show intact, though thrombosed, vasculature.

Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies

In the United States, more than 2600 kidneys are discarded annually, from the total number of kidneys procured for transplant. We hypothesized that this organ pool may be used as a platform for renal bioengineering and regeneration research. We previously showed that decellularization of porcine kidneys yields renal extracellular matrix (ECM) scaffolds that maintain their basic components, support cell growth and welfare in vitro and in vivo, and show an intact vasculature that, when such scaffolds are implanted in vivo, is able to sustain physiological blood pressure. The purpose of the current study was to test if the same strategy can be applied to discarded human kidneys in order to obtain human renal ECM scaffolds. The results show that the sodium dodecylsulfate-based decellularization protocol completely cleared the cellular compartment in these kidneys, while the innate ECM framework retained its architecture and biochemical properties. Samples of human renal ECM scaffolds stimulated angiogenesis in a chick chorioallantoic membrane assay. Importantly, the innate vascular network in the human renal ECM scaffolds retained its compliance. Collectively, these results indicate that discarded human kidneys are a suitable source of renal scaffolds and their use for tissue engineering applications may be more clinically applicable than kidneys derived from animals.

Whole-organ bioengineering: current tales of modern alchemy

End-stage organ disease affects millions of people around the world, to whom organ transplantation is the only definitive cure available. However, persistent organ shortage and the resulting widespread transplant backlog are part of a disturbing reality and a common burden felt by thousands of patients on waiting lists in almost every country where organ transplants are performed. Several alternatives and potential solutions to this problem have been sought in past decades, but one seems particularly promising now: whole-organ bioengineering. This review describes briefly the evolution of organ transplantation and the development of decellularized organ scaffolds and their application to organ bioengineering. This modern alchemy of generating whole-organ scaffolds and recellularizing them with multiple cell types in perfusion bioreactors is paving the way for a new revolution in transplantation medicine. Furthermore, although the first generation of bioengineered organs still lacks true clinical value, it has created a number of novel tissue and organ model platforms with direct application in other areas of science (eg, developmental biology and stem cell biology, drug discovery, physiology and metabolism). In this review, we describe the current status and numerous applications of whole-organ bioengineering, focusing also on the multiple challenges that researchers have to overcome to translate these novel technologies fully into transplantation medicine.

Human Liver Bioengineering Using a Whole Liver Decellularized Bioscaffold

As a result of significant progress made in the last years in developing methods of whole organ decellularization techniques, organ bioengineering may now look more feasible than ever before. In this chapter, we describe in detail the necessary steps in human liver bioengineering. These include ferret liver decellularization by detergent perfusion, human liver progenitor and endothelial cell isolation, and finally, liver bioscaffold recellularization in a perfusion bioreactor.

Multiscale computational model of fluid flow and matrix deformation in decellularized liver

Currently little is known about the biomechanical environment in decellularized tissue. The goal of this research is to quantify the mechanical microenvironment in decellularized liver, for varying organ-scale perfusion conditions, using a combined experimental/computational approach. Needle-guided ultra-miniature pressure sensors were inserted into liver tissue to measure parenchymal fluid pressure ex-situ in portal vein-perfused native (n=5) and decellularized (n=7) ferret liver, for flow rates from 3-12mL/min. Pressures were also recorded at the inlet near the portal vein cannula to estimate total vascular resistance of the specimens. Experimental results were fit to a multiscale computational model to simulate perfusion conditions inside native versus decellularized livers for four experimental flow rates. The multiscale model consists of two parts: an organ-scale electrical analog model of liver hemodynamics and a tissue-scale model that predicts pore fluid pressure, pore fluid velocity, and solid matrix stress and deformation throughout the 3D hepatic lobule. Distinct models were created for native versus decellularized liver. Results show that vascular resistance decreases by 82% as a result of decellularization. The hydraulic conductivity of the decellularized liver lobule, a measure of tissue permeability, was 5.6 times that of native liver. For the four flow rates studied, mean fluid pressures in the decellularized lobule were 0.6-2.4mmHg, mean fluid velocities were 211-767μm/s, and average solid matrix principal strains were 1.7-6.1%. In the future this modeling platform can be used to guide the optimization of perfusion seeding and conditioning strategies for decellularized scaffolds in liver bioengineering.

Self‐assembled liver organoids recapitulate hepatobiliary organogenesis in vitro

Several three‐dimensional cell culture systems are currently available to create liver organoids. In gneral, these systems display better physiologic and metabolic aspects of intact liver tissue compared with two‐dimensional culture systems. However, none reliably mimic human liver development, including parallel formation of hepatocyte and cholangiocyte anatomical structures. Here, we show that human fetal liver progenitor cells self‐assembled inside acellular liver extracellular matrix scaffolds to form three‐dimensional liver organoids that recapitulated several aspects of hepatobiliary organogenesis and resulted in concomitant formation of progressively more differentiated hepatocytes and bile duct structures. The duct morphogenesis process was interrupted by inhibiting Notch signaling, in an attempt to create a liver developmental disease model with a similar phenotype to Alagille syndrome. Conclusion: In the current study, we created an in vitro model of human liver development and disease, physiology, and metabolism, supported by liver extracellular matrix substrata; we envision that it will be used in the future to study mechanisms of hepatic and biliary development and for disease modeling and drug screening. (Hepatology 2018;67:750‐761).

Fluid Flow Regulation of Revascularization and Cellular Organization in a Bioengineered Liver Platform.

OBJECTIVE Modeling of human liver development, especially cellular organization and the mechanisms underlying it, is fundamental for studying liver organogenesis and congenital diseases, yet there are no reliable models that mimic these processes ex vivo. DESIGN Using an organ engineering approach and relevant cell lines, we designed a perfusion system that delivers discrete mechanical forces inside an acellular liver extracellular matrix scaffold to study the effects of mechanical stimulation in hepatic tissue organization. RESULTS We observed a fluid flow rate-dependent response in cell distribution within the liver scaffold. Next, we determined the role of nitric oxide (NO) as a mediator of fluid flow effects on endothelial cells. We observed impairment of both neovascularization and liver tissue organization in the presence of selective inhibition of endothelial NO synthase. Similar results were observed in bioengineered livers grown under static conditions. CONCLUSION Overall, we were able to unveil the potential central role of discrete mechanical stimulation through the NO pathway in the revascularization and cellular organization of a bioengineered liver. Last, we propose that this organ bioengineering platform can contribute significantly to the identification of physiological mechanisms of liver organogenesis and regeneration and improve our ability to bioengineer livers for transplantation.

Porohyperviscoelastic Model Simultaneously Predicts Parenchymal Fluid Pressure and Reaction Force in Perfused Liver

Porohyperviscoelastic (PHVE) modeling gives a simplified continuum approximation of pore fluid behavior within the parenchyma of liver tissue. This modeling approach is particularly applicable to tissue engineering of artificial livers, where the inherent complexity of the engineered scaffolds prevents the use of computational fluid dynamics. The objectives of this study were to simultaneously predict the experimental parenchymal fluid pressure (PFP) and compression response in a PHVE liver model. The model PFP matched the experimental measurements (318 Pa) to within 1.5%. Linear regression of both phases of compression, ramp, and hold, demonstrated a strong correlation between the model and the experimental reaction force (p<0.5). The ability of this PVE model to accurately predict both fluid and solid behavior is important due to the highly vascularized nature of liver tissue and the mechanosensitivity of liver cells to solid matrix and fluid flow properties.

Shear Stress Upregulates Regeneration-Related Immediate Early Genes in Liver Progenitors in 3D ECM-like Microenvironments†

The role of fluid stresses in activating the hepatic stem/progenitor cell regenerative response is not well understood. This study hypothesized that immediate early genes (IEGs) with known links to liver regeneration will be upregulated in liver progenitor cells (LPCs) exposed to in vitro shear stresses on the order of those produced from elevated interstitial flow after partial hepatectomy. The objectives were: (1) to develop a shear flow chamber for application of fluid stress to LPCs in 3D culture; and (2) to determine the effects of fluid stress on IEG expression in LPCs. Two hours of shear stress exposure at ∼4 dyn/cm2 was applied to LPCs embedded individually or as 3D spheroids within a hyaluronic acid/collagen I hydrogel. Results were compared against static controls. Quantitative reverse transcriptase polymerase chain reaction was used to evaluate the effect of experimental treatments on gene expression. Twenty‐nine genes were analyzed, including IEGs and other genes linked to liver regeneration. Four IEGs (CFOS, IP10, MKP1, ALB) and three other regeneration‐related genes (WNT, VEGF, EpCAM) were significantly upregulated in LPCs in response to fluid mechanical stress. LPCs maintained an early to intermediate stage of differentiation in spheroid culture in the absence of the hydrogel, and addition of the gel initiated cholangiocyte differentiation programs which were abrogated by the onset of flow. Collectively the flow‐upregulated genes fit the pattern of an LPC‐mediated proliferative/regenerative response. These results suggest that fluid stresses are potentially important regulators of the LPC‐mediated regeneration response in liver.

Chapter 3 – Whole-Organ Bioengineering—Current Tales of Modern Alchemy

End-stage organ disease affects millions of people around the world, to whom organ transplantation is the only definitive cure available. However, persistent organ shortage and the resulting widespread transplant backlog are part of a disturbing reality and a common burden felt by thousands of patients on waiting lists. Several alternatives and potential solutions to this problem have been sought in the past decades, but one seems particularly promising now, whole-organ bioengineering. This modern alchemy of generating whole-organ scaffolds and recellularizing them with multiple cell types in perfusion bioreactors is paving the way for a new revolution in transplantation medicine. In this chapter, we describe the present status and numerous applications of whole-organ bioengineering, focusing also in the multiple challenges that researchers have to overcome to fully translate these novel technologies into transplantation medicine.