Polymerized Hemoglobins for Facilitated Oxygen Transport in Hepatic Bioreactors

ABSTRACT Bioartificial liver assist devices (BLADs) typically consist of liver parenchymal cells (differentiated hepatocytes or hepatoma cells) cultured within an extracorporeal system (in this application, a hollow fiber (HF) bioreactor) in order to recapitulate a broad range of differentiated liver functions. Several challenges must still be overcome to realize a viable BLAD, including provision of appropriate oxygenation to the cells in the device. Our work in this area demonstrates that supplementation of hemoglobin-based O2 carriers (HBOCs) into the circulating culture medium of a HF-based BLAD represents a feasible strategy to improve O2 transport to both preserve hepatocyte differentiated functions and maintain a high cell density. However, our prior studies have shown that unmodified bovine hemoglobin (BvHb) is cytotoxic at high concentrations (>15 g/L, see preliminary data). BvHb is a tetrameric molecule (?2?2, Mw ~64 kDa) that exists in equilibrium (KD = 0.2 µM) with ?? dimers (Mw ~32 kDa) in aqueous solution. Because of the small size of the ?? dimers (32 kDa) in relation to the molecular weight cut-off (MWCO) of typical HF membranes (>35 kDa), these molecules are able to extravasate through the HF membrane and accumulate in the extracapillary space (ECS) of the HF bioreactor which contains the hepatocytes. In the ECS, ?? dimers eventually autoxidize, unfold and release free heme into solution which is cytotoxic. In light of this knowledge, we hypothesize that supplementation of circulating culture medium in a HF-based BLAD with polymerized BvHb (PolyBvHb) will provide the benefits of unmodified BvHb, namely enhance O2 transport, improve hepatocyte differentiated functions, and support a high cell density in the ECS, but without eliciting cytotoxicity. The presence of chemical cross-links within PolyBvHb will prevent cytotoxicity via two mechanisms: (a) by preventing dissociation of ?2?2 into ?? dimers and their subsequent extravasation into the ECS; (b) by preventing globin chains from unfolding and releasing free heme into solution. Three specific aims are proposed to investigate this hypothesis: Specific Aim 1: To characterize the stability of PolyBvHb in plasma and its effect on the viability, function, and proliferation of liver parenchymal cells. Specific Aim 2: To evaluate the function of a liver cell microbioreactor with zonated functions induced via controlled O2 tension. Specific Aim 3: To optimize the zonation and scale-up the HF liver bioreactor oxygenated with PolyBvHb. The proposed studies will generate proof-of-principle data that describe, both in vitro and in a clinically relevant model, the impact of using physiologically relevant oxygen tensions through the use of PolyBvHb on bioreactor performance. These studies will therefore provide the first stepping stones towards future clinical development, which would likely involve large animal (pig) and human studies.