A Pulmonary Vascular Model From Endothelialized Whole Organ Scaffolds

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back—at least partially—toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Whole Heart Engineering: Advances and Challenges

Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach – recellularization of a decellularized cadaveric organ scaffold with human cells – is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field’s 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ.

Abstract 14125: An in vitro Pulmonary Vascular Platform From Endothelialized Whole Lung Scaffolds

Introduction: The development of an in vitro system is critical to studying human diseases including the novel coronavirus disease-19 (COVID-19), and is typically centered on recapitulating native physiology. In the case of the pulmonary vasculature, the endothelium is critical for establishing the fluid-tight barrier in the alveolar compartment, and displays significant phenotypic and functional heterogeneity along vascular tree. Objective: Here, we developed an experimental platform that could mimic physiological functions and cellular phenotypes of pulmonary vasculature, during homeostatic and diseased states. Methods and Results: Lymphatic low pulmonary microvascular endothelial cells (Lymph low PMECs) were isolated from Prox1-GFP rodent lungs from regions of having low amounts of lymphatic tissue. Single-cell RNA-sequencing (scRNAseq) data revealed that these cells were becoming phenotypically homogenous over several passages while losing some markers of native differentiation during passaging. Intriguingly, after culturing in decellularized lung scaffolds, the phenotype of the lymph low PMEC changed back toward native lung endothelium. Vascular barrier function was partially restored in engineered lungs repopulated with endothelium, while small capillaries with patent lumens were appreciable. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, the pro-inflammatory signal was significantly increased and the vascular barrier was severely impaired in the repopulated lung. Conclusions: Taken together, these results show a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Whole Organ Engineering: Approaches, Challenges, and Future Directions

End-stage organ failure remains a leading cause of morbidity and mortality across the globe. The only curative treatment option currently available for patients diagnosed with end-stage organ failure is organ transplantation. However, due to a critical shortage of organs, only a fraction of these patients are able to receive a viable organ transplantation. Those patients fortunate enough to receive a transplant must then be subjected to a lifelong regimen of immunosuppressant drugs. The concept of whole organ engineering offers a promising alternative to organ transplantation that overcomes these limitations. Organ engineering is a discipline that merges developmental biology, anatomy, physiology, and cellular interactions with enabling technologies such as advanced biomaterials and biofabrication to create bioartificial organs that recapitulate native organs in vivo. There have been numerous developments in bioengineering of whole organs over the past two decades. Key technological advancements include (1) methods of whole organ decellularization and recellularization, (2) three-dimensional bioprinting, (3) advanced stem cell technologies, and (4) the ability to genetically modify tissues and cells. These advancements give hope that organ engineering will become a commercial reality in the next decade. In this review article, we describe the foundational principles of whole organ engineering, discuss key technological advances, and provide an overview of current limitations and future directions.

Irreversible Electroporation in Liver Cancers and Whole Organ Engineering

Liver cancers contribute significantly to cancer-related mortality worldwide and liver transplants remain the cornerstone of curative treatment for select, early-stage patients. Unfortunately, because of a mismatch between demand and supply of donor organs, liver cancer patients must often wait extended periods of time prior to transplant. A variety of local therapies including surgical resection, transarterial chemoembolization, and thermal ablative methods exist in order to bridge to transplant. In recent years, a number of studies have examined the role of irreversible electroporation (IRE) as a non-thermal local ablative method for liver tumors, particularly for those adjacent to critical structures such as the vasculature, gall bladder, or bile duct. In addition to proving its utility as a local treatment modality, IRE has also demonstrated promise as a technique for donor organ decellularization in the context of whole-organ engineering. Through complete non-thermal removal of living cells, IRE allows for the creation of an acellular extra cellular matrix (ECM) scaffold that could theoretically be recellularized and implanted into a living host. Here, we comprehensively review studies investigating IRE, its role in liver cancer treatment, and its utility in whole organ engineering.

Development of perfusion bioreactor for whole organ engineering — a culture system that enhances cellular engraftment, survival and phenotype of repopulated pancreas

Whole organ engineering has emerged as a promising alternative avenue to fill the gap of donor organ shortage in organ transplantation. Recent breakthroughs in the decellularization of solid organs and repopulation with desired cell populations have generated neo-organ constructs with promising functional outcomes. The realization of this goal requires engineering advancement in the perfusion-based bioreactors to (i) efficiently deliver decellularization agents, followed by (ii) its reconstruction with relevant cell types and (iii) maintenance of viability and function of the repopulated organ. In this study, we report the development and assembly of a perfusion bioreactor with the potential to enable regenerative reconstruction of pancreas. The assembled bioreactor is versatile to efficiently decellularize multiple organs, as demonstrated by complete decellularization of pancreas, liver and heart in the same set-up. Further, the same system is amenable to support organ repopulation with diverse cell types. Using our in-house bioreactor system, we demonstrate pancreas repopulation with both immortalized MIN-6 beta cells and differentiating human pluripotent stem cells. Importantly, we show the significant advantage of perfusion culture over static culture in enhancing cell engraftment, viability and phenotypic maintenance of the repopulated pancreas. In addition, this study is a significant step forward for whole organ engineering as it will facilitate cost-effective and easy assembly of perfusion bioreactors to enable rapid advancement in regenerative organ reconstruction.

Towards a Bioengineered Kidney: Recellularization Strategies for Decellularized Native Kidney Scaffolds

Patients with end-stage renal disease often undergo dialysis as a partial substitute for kidney function while waiting for their only treatment option: a kidney transplant. Several research directions emerged for alternatives in support of the ever-growing numbers of patients. Recent years brought big steps forward in the field, with researchers questioning and improving the current dialysis devices as well as moving towards the design of a bioengineered kidney. Whole-organ engineering is also being explored as a possibility, making use of animal or human kidney scaffolds for engineering a transplantable organ. While this is not a new strategy, having been applied so far for thin tissues, it is a novel approach for complex organs such as the kidneys. Kidneys can be decellularized and the remaining scaffold consisting of an extracellular matrix can be repopulated with (autologous) cells, aiming at growing ex vivo a fully transplantable organ. In a broader view, such organs might also be used for a better understanding of fundamental biological concepts and disease mechanisms, drug screening and toxicological investigations, opening new pathways in the treatment of kidney disease. Decellularization of whole organs has been widely explored and described; therefore, this manuscript only briefly reviews some important considerations with an emphasis on scaffold decontamination, but focuses further on recellularization strategies. Critical aspects, including cell types and sources that can be used for recellularization, seeding strategies and possible applications beyond renal replacement are discussed.