Development of highly functional bioengineered human liver with perfusable vasculature

Abstract

Liver tissue engineering offers a promising strategy for liver failure patients. Since transplantation rejection resulting in vessel thrombosis is regarded as a major hurdle, vascular reconstruction is one of indispensable requirements of whole organ engineering. Here we demonstrated a novel strategy for reconstruction of a vascularized bioengineered human liver (VBHL) using decellularized liver scaffolds in an efficient manner. First we achieved fully functional endothelial coverage of scaffolds by adopting the anti-CD31 aptamer as a potent coating agent for re-endothelialization. Through an ex vivo human blood perfusion that recapitulates the blood coagulation response in humans, we demonstrated significantly reduced platelet aggregation in anti-CD31 aptamer coated scaffolds. We then produced VBHL constructs using liver parenchymal cells and nonparenchymal cells, properly organized into liver-like structures with an aligned vasculature. Interestingly, VBHL constructs displayed prominently enhanced long-term liver-specific functions that are affected by vascular functionality. The VBHL constructs formed perfusable vessel networks in vivo as evidenced by the direct vascular connection between the VBHL constructs and the renal circulation. Furthermore, heterotopic transplantation of VBHL constructs supported liver functions in a rat model of liver fibrosis. Overall, we proposed a new strategy to generate transplantable bioengineered livers characterized by highly functional vascular reconstruction.

Introduction

End-stage liver diseases, including chronic liver disease and cirrhosis, cause approximately 2 million of deaths worldwide each year [1]. Liver transplantation is a well-established therapeutic strategy for patients who suffer from hepatic failure. As the demand for organs has grown worldwide, a severe shortage of donor organs had necessitated the development of substitutes for human organs. In this regard, bioengineered liver constructs have currently been suggested as an alternative.

Decellularized extracellular matrix scaffolds, which are obtained by the removal of cells from donor tissue, are as a promising substrate for tissue engineering [[2], [3], [4], [5]]. Owing to preservation of their own complex microstructures and biological signals, the cells seeded into decellularized scaffolds can migrate to the appropriate microenvironment. Thus, decellularized scaffolds have been employed as compatible sources for the fabrication of bioengineered organs. For instance, Song et al. generated functional transplantable kidney grafts by engineering decellularized rat and human kidney matrix [6]. Subsequent work by the same group also demonstrated that decellularized lung scaffolds were repopulated with human cells and then displayed vascular maturation [7].

Likewise, in recent years, a number of studies have been conducted on establishing artificial liver using decellularized liver scaffolds [2,3,[8], [9], [10]]. Given that the liver is a vascular organ with intricate vessels, complete re-endothelialization of the scaffolds is the key to successful reconstruction of the bioengineered liver. To date, various coating agents, such as heparin-gelatin and REDV peptide, have been explored to improve the delivery efficiency of endothelial cells (ECs) [8,11,12]. However, since these chemicals do not specifically interact with ECs, to create a bioengineered liver composed of multiple cell types, there is a need to develop coating agents that interact specifically with ECs so that only ECs attach to the luminal surface of the vasculature within the scaffolds. Although the anti-CD31 antibody coating can selectively capture ECs to ensure better attachment [13], the limitations are that mass production of antibodies requires complicated manufacturing procedures, and the cost of production is very high. Moreover, antibodies derived from other species are possibly recognized as foreign bodies by the human immune system and induce an immune response, leading to undesirable outcomes [14]. These limitations, which obstruct the clinical applications of antibodies led us to develop a new method for efficient re-endothelialization of the liver scaffolds.

Nucleic acid aptamers are composed of synthesized single-stranded oligonucleotides with short sequences. Aptamers are considered alternatives to antibodies due to their low immunogenicity and strong binding affinity for specific proteins [15,16]. It is also beneficial that aptamers can be easily modified and produced cost-effectively in large quantities without much variability or contamination, compared to antibodies [17]. In general, various aptamers have been utilized for medical diagnosis and as therapeutic agents for diseases, such as cancer [18,19] and viral infections [20].

In this study, we aimed to efficiently produce bioengineered human livers with well-organized vascular structures that maintain functional integrity. Hence, we first utilized an anti-CD31 aptamer as a coating agent for the purposes of the re-endothelialization of decellularized liver scaffolds and identified the influence of anti-CD31 aptamers on ECs. To predict vascular thrombosis associated with the rejection in humans, we evaluated the thrombogenicity of the re-endothelialized constructs after continuous human blood perfusion. With these strategies, we fabricated vascularized bioengineered human liver (VBHL) constructs that retained the structural and functional properties of the liver. We also reported the in vivo functionality of VBHL constructs, including graft patency following blood transfusion using the renal circulatory system in rats and the ability to support liver functions by heterotopic implantation into the cirrhotic livers of rats. Eventually, we established a highly functional bioengineered liver with a perfusable vasculature that could feasibly be clinically applied as a substitute for liver transplantation.