Engineering a bioartificial liver prototype using cell loaded macroporous hydrogel scaffolds

Student thesis: Doctoral Thesis

Abstract

Bioartificial Liver (BAL) devices are extracorporeal systems designed to support or recover hepatic function in patients with liver failure. However, the design of an effective BAL remains an open challenge due, in part, to the conflicting requirements to simultaneously increase blood/hepatocyte mass exchange and prevent intradialytic haemolysis. One approach to the design of a BAL cell scaffold material is the use of cryogels which possess tunable properties allowing the creation of interconnected, macroporous matrices with a pore size of up to 150 µm. This is required for supporting both hepatocyte growth and allowing flow recirculation. Whilst poly(NiPAAm)-chitosan cryogels have previously been investigated for BAL application, no consideration of non-protein fouling characteristics or fluid dynamics in prototype device design was made and early studies indicated loss of device function after 90 minutes exposure to liver failure patient plasma. Furthermore, these cryogels showed pore size only up to 90 µm. In this work, a range of HEMA based cryogels were synthesised by cryogelation technique. They were modified with alginate using different strategies to enhance the non-fouling surface properties and functionalised with an RGD containing peptide to increase cell adhesion. Cryogels were characterised with a range of physical assays to evaluate porosity, pore interconnectivity, mechanical strength and flow resistance. Protein adsorption and in vitro studies were conducted to investigate non-fouling properties, biocompatibility and cell adhesion. Internal fluid dynamics was investigated with a Particle Image Velocimetry (PIV) setup which allowed the visualisation of flow inside cryogels. A bioreactor was optimised in a multi-layered design to enhance mass exchange and compared to a column version in terms of hepatocyte colonisation and synthetic functions. An in-line circuit of carbon monolith cartridge with bioreactor was used to assess cytokine adsorption from spiked plasma and its effect on hepatocyte growth inside the BAL. The stacked cryogel BAL prototype without cells was assessed for safety using a bile duct ligation (BDL) in vivo model of liver fibrosis. RGD-alginate functionalised HEMA-based cryogels were successfully synthesised with pore size up to 150 µm, porosity of 88.76 % and 99 % interconnection of pores. Although pre and post synthesis incorporation of alginate did not affect porosity, the latter method was chosen because it did not affect the flow rate of the resultant cryogels. Alginate significantly reduced plasma protein adsorption to the modified cryogels (p< 0.001). RGD surface modification significantly increased cell adhesion (p< 0.001) over time within in vitro studies using Human Healthy Liver (HHL) hepatocytes. PIV analysis showed laminar flow with little recirculation inside the cryogel pores and was used to refine the prototype to a multi layered bioreactor design. The optimised BAL produced significantly more albumin and urea (p< 0.001) comparing to the column version with cell colonisation throughout the device length. Carbon monolith incorporation reduced cytokine levels in spiked plasma perfused through the cryogels in the first hour of perfusion (p< 0.001). Cytokine levels rose subsequently due to cell production on stimulation by the toxin-rich environment. Assessment of the no-cell multilayered cryogel prototype using a BDL in vivo model indicated that the device was safe to use with no significant effect on vital parameters and blood biochemistry. This work has developed and characterised a new surface engineered cryogel-based BAL prototype with a multi-layered design to improve mass exchange in the bioreactor, extend device life and improve efficacy. An optical setup was used for the first time to allow visualisation of flow inside the macroporous scaffold. The work extends understanding of routes to optimise scaffolds for BAL. Further work to refine this model includes optimisation of the cell component and design parameters to improve hepatocyte loading for further improvement in maintained metabolic function.
Date of AwardApr 2021
Original languageEnglish
Awarding Institution
  • University of Brighton
SupervisorSusan Sandeman (Supervisor), Cyril Crua (Supervisor) & Irina Savina (Supervisor)

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