In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds

Abstract

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.

Introduction

Orthotopic liver transplantation is a unique effective treatment for end-stage liver diseases [1]. However, due to severe donor-liver shortage, high cost and exacerbation of disease, many patients die before they can receive the operation. Therefore, bioartificial liver (BAL) has been proposed as a temporary liver support for patients awaiting liver transplantation [2].

Nevertheless, the clinical application of this new treatment strategy is not very optimistic [3]. One major reason is that hepatocytes rapidly lose liver-specific functions and viability when cultured in the bioreactor in vitro. Thus, to build a good bioreactor, it is crucial to mimic the microenvironment of hepatocytes in vivo and to afford the hepatocytes in a suitable environment [4].

In vivo, most cells adhere to the extracellular matrices (ECMs), which have extremely complex topography in the nanometer range. In order to mimic the topography of ECMs, various materials have been fabricated into nanometer materials [5] and they can affect cell migration, adhesion, proliferation and other cellular behaviors [6], [7]. Nanometer scaffolds have been widely used in different tissue engineering, such as bone [8], nerve [9] and bladder [10]. However, there have been few reports on the application of nanometer scaffolds in the liver tissue engineering. We reported the influence of chitosan nanofibers on hepatocytes in vitro in a previous study, in which we showed that nanofiber scaffolds mimic ECMs well and enhance cell adhesion [11].

Besides the nanometer topography of ECMs, there are many important biochemical groups on the ECMs, which are also essential for the growth of cells, such as the galactose ligands. Many studies have shown that the asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes selectively adhere to galactose ligands [12], [13], [14]. This interaction between ASGPR and galactose ligands can induce the formation of hepatocyte aggregates [15], [16], which exhibits a higher level of liver-specific functions [17]. Therefore, intensive efforts have been made in developing new scaffolds modified with galactose ligands to enhance hepatocyte adhesion as well as to maintenance of liver-specific functions and mechanical stability [18], [19], [20]. We grafted the galactose ligands onto the chitosan nanofibers in the previous study and observed its influence on hepatocytes cultured in vitro. Our results showed that compared with hepatocytes on chitosan nanofiber scaffolds and galactosylated chitosan film, hepatocytes cultured on galactosylated chitosan nanofiber scaffolds exhibit excellent cell bioactivity and higher levels of liver functions for a long time [21].

In this study, we developed a multi-layer radial-flow bioreactor. We introduced nanofiber scaffolds into the bioreactor to mimic the topography of ECMs, and grafted the galactose onto nanofibers to mimic the biochemical environment of ECMs. Moreover, we added red blood cells (RBCs) into the medium to mimic the arterial blood in order to meet the high oxygen demand of hepatocytes. We hypothesize that nanofiber scaffolds could enhance the cell adhesion by tight contact between cells and scaffolds; the grafted galactose group could enhance the hepatocellular functions by the formation of spheroids; and the RBCs added into the medium could improve the oxygen supply to hepatocytes.