Dipen Vyas

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.

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.

Liver Regeneration and Bioengineering - The Emergence of Whole Organ Scaffolds

Every year an estimated two million people die of advanced liver disease. The World Health Organization estimates that over six hundred and fifty million people worldwide are affected by some form of liver disease, including thirty million Americans. On a worldwide base, the bleak cenario of one to two million deaths are accounted to liver related diseases annually. From all the countries, China has the world’s largest population of Hepatitis B patients (approx. 120 million) with five hundred thousand people dying of the liver disease every year(1, 2). In the US alone, there are around five hundred thousand critical episodes of liver problems every year requiring hospitalization with a huge burden to the patients and an enormous cost to the health care system. In the European Union and United States of America alone, over eighty one thousand and twenty six thousand people died of chronic liver disease in 2006, respectively(1, 3). For these patients, liver transplantation is currently the only therapy proven to extend survival for end-stage liver disease, as it is also the only treatment for severe acute liver failure and the some forms of inborn errors of metabolism. However, the waiting list for liver transplants is extensive and many on the list will not receive an organ due to a dramatic shortage of donors or not being eligible(1).

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.

Translational Regenerative Medicine—Hepatic Systems

The liver is the largest gland in the body and performs hundreds of vital functions, most of which are carried out by the hepatocytes that constitute about 80% of the livers total cell population. Besides being the main organ for a variety of vital functions, the liver is the best example of an organ that possesses major regenerative capacity following partial hepatectomy or toxic injury. The adaptive response and robustness of liver regeneration is critical to ensure the bodys metabolic functions, and liver disease may have a profound negative impact on metabolism and homeostasis of the body. As a result, therapies that ensure some degree of recovery from liver disease are greatly sought worldwide. Herein, we describe some of the most common liver diseases and numerous novel regenerative therapies in development or already translated into the clinic, that rely on liver regeneration.

Tu2053 Precise Replication of Human Liver Fetal Development with Human Bioengineered Liver Organoids: A Novel Model to Test the Teratogenesis of New Drug Candidates?

Introduction Bioengineering of a fully functional tissue requires precise recapitulation of normal tissue development. Specifically for the liver, one may use bipotent human liver progenitor cells (hFLCs) capable of differentiation into hepatocytes and cholangiocytes. The goal of the current study was to develop a system that would efficiently recapitulate embryonic development of hepatic parenchymal tissue and bile ducts, using decellularized liver extracellular matrix (ECM) as scaffolds.Materials andMethods hFLCs were seeded on decellularized liver ECM discs (300 μm thickness, 8 mm diameter) and were cultured for up to 3 weeks in presence of hepatic differentiation medium. Immunofluorescence microscopy was used to determine the extent of progenitor cell differentiation into hepatocytes and cholangiocytes. Urea, albumin and drug metabolism were quantified as paramaters of liver function. Furthermore, a γ-secretase inhibitor was added to the culture media and bile duct and hepatocyte development was monitored. Results hFLCs seeded on acellular liver ECM discs differentiated into hepatocytes and cholangiocytes. The cells showed predominant albumin expression along with loss of α-feto protein (AFP) expression at 3 weeks (Fig. 1D,E). The cells also expressed other mature hepatocyte markers like HNF-4α, α-1-antitrypsin and cytochrome P450 1A2, 2A and 3A (Fig. 1B-E). The cells in the ductular structures expressed bile duct specific markers like CK19, SOX9, EpCAM, ASBT, β-catenin and the presence of apical primary cilia (stained with α-acetylated tubulin), thus demonstrating differentiation towards cholangiocyte lineage along with maintaining apico-basal polarity (Fig. 1B-E). Urea and albumin secretion was higher in the liver disc organoids compared to control hFLCs cultured in petri dishes. Several metabolites of the drugs diazepam and 7-ethoxycoumarin were also detected by LC-MS/MS, showing broad cytochrome P450 activity in these organoids. The addition of a γ-secretase inhibitor severely impacted the number and maturation of bile ducts formed, mirroring a biliary atresia model. Discussion and Conclusions Our results demonstrate the efficient generation of bioengineered human liver tissue with hFLC that recapitulates stepwise development of hepatocyte and bile duct formation (Fig. 1A). Altogether, this study demonstrates the potential of this technology to study and mimic human liver development. These models provide novel approaches for liver bioengineering, drug discovery and toxicology (including drug teratogenesis evaluation in vitro), and ultimately for the treatment of liver disease.

Liver Regeneration and Bioengineering: The Role of Liver Extra-Cellular Matrix and Human Stem/Progenitor Cells

Although the extracellular matrix (ECM) is only a minor constituent of the liver, it has a fundamental role, providing a structural framework and maintaining the differentiated state of all liver resident cells. The important role of the hepatic ECM was demonstrated in vitro, indicating that hepatocyte phenotype is dependent on the nature of the ECM upon which it is cultured [1]. The ECM modulates repair in many tissues, including the liver. Therefore, defining the ECM distribution in the normal liver, its phenotypic expression in various regenerative states, and the cells responsible for its synthesis in vivo, is an important step in understanding its role in homeostasis and repair. In this chapter, we sought to provide a thorough description of the liver ECM and its intrinsic association with the cells that constitute the hepatic tissue, as well as their location (zonation) and associations in order to maintain hepatic function. Hepatic mechanobiology and ECM contribution is also described. Furthermore, we list important breakthroughs and shortcomings of current human liver cell therapies and how new approaches involving matrix carriers could impact the field.

Liver Regeneration: the Role of Bioengineering

An estimated two million people die of terminal liver disease every year. The World Health Organization calculates that over six hundred and fifty million people worldwide suffer from some form of liver disease, including thirty million Americans. On a worldwide base, approximately one to two million deaths are accounted to liver related diseases annually. Around the globe, China has the world’s largest population of Hepatitis B patients (approximately 120 million) with five hundred thousand people dying of liver illnesses every year(1, 2). In the US alone, five hundred thousand critical liver problem episodes are reported every year requiring hospitalization with great burden to the patients and a huge cost to the health care system. In the European Union and United States of America alone, over eighty one thousand and twenty six thousand people died of chronic liver disease in 2006, respectively(1, 3). For these patients, liver transplantation is presently the only proven therapy able to extend survival for end-stage liver disease. It is also the only treatment for severe acute liver failure and to some forms of inborn errors of metabolism. Nevertheless, the waiting list for liver transplantation is long and many patients will not survive long enough to receive an organ due to the dramatic shortage of donors or lack of eligibility(1). A good example of this is that in 2007 there were almost seventeen thousand candidates on the US waiting list for liver transplantation. From those, only 30% were actually transplanted by the end of the year, with an average waiting time of more than 400 days. In the same year, nearly one thousand and three hundred people died while waiting for a suitable donor, with no real therapeutic alternative available to save their lives. Moreover, for those patients with fulminant hepatic failure, a severe liver disease with 60-90% mortality, depending on the etiology, only 10% received a transplant. Altogether, liver transplantation still has a relatively high mortality of 30-40% at 5-8 years with 65% of the deaths occurring in the first 6 months. Patients who have undergone transplantation have to also use lifelong immunosuppressive therapy, with sometimes severe side effects(4). There are innumerous etiologies of end-stage chronic liver disease that lead to transplantation and approximately 80% of the candidates in the liver transplantation waiting list have a primary diagnosis of liver cirrhosis. Fortunately, some of the causes of these diseases are currently preventable. An excellent example is the successful vaccination programs in many countries around the world against Hepatitis B virus, which have