Abstract
Hemoglobin (Hb) based oxygen carriers (HBOCs) are designed to minimize the toxicity of extracellular Hb, while preserving its high oxygen-carrying capacity for oxygen delivery to cells. Polymerized human Hb (PolyHb) is a novel type of nanosized HBOC synthesized via glutaraldehyde-mediated crosslinking of free Hb, and which preserves the predominant quaternary state during the crosslinking reaction (low oxygen affinity tense (T) quaternary state PolyHb is synthesized at 0% Hb oxygen saturation, and high oxygen affinity relaxed (R) quaternary state PolyHb is synthesized at 100% Hb oxygen saturation). Major potential applications for PolyHbs, and HBOCs in general, include oxygenation of bioreactor systems containing large liver cell masses, and ex-vivo perfusion preservation of explanted liver grafts. The toxicity of these compounds toward liver cells must be evaluated before testing their use in these complex systems for oxygen delivery. Herein, we characterized the effect of PolyHbs on the hepatoma cell line HepG2/C3A, used as a model hepatocyte and as a cell line used in some investigational bioartificial liver support devices. HepG2/C3A cells were incubated in cell culture media containing PolyHbs or unmodified Hb at concentrations up to 50 mg/mL and for up to 6 days. PolyHbs were well tolerated at a dose of 10 mg/mL, with no significant decrease in cell viability; however, proliferation was inhibited as much as 10-fold after 6 days of exposure at 50 mg/mL. Secretion of albumin, and urea, as well as glucose and ammonia removal were measured in presence of 10 mg/mL of PolyHbs or unmodified Hb. In addition, methoxy- and ethoxy-resorufin deacetylase (MROD and EROD) activities, which reflect cytochrome P450 metabolism, were measured. R-state PolyHb displayed improved or intact activity in 3 out of 7 functions compared to unmodified Hb. T-state PolyHb displayed improved or intact activity in 4 out of 7 functions compared to unmodified Hb. Thus, PolyHbs, both in the R-state and T-state, are safer to use at a concentration of 10 mg/mL as compared to unmodified Hb in static culture liver-related applications.