Polymerized hemoglobin for enhanced oxygen transport and metabolic zonation in a mini-bioartificial liver device
Creators: name: Chen, Nuozhou
Publisher: name: No Publisher Supplied
Year: 2023

description: It is envisioned that bioartificial livers (BALs) could support liver function of patients with fulminant acute liver failure or acute-on-chronic liver failure while waiting for a liver transplant. BALs incorporate living hepatocytes or hepatoma cells to provide a wide range of liver-specific functions. The impact of oxygen gradient as a potential modulator of zonation of liver function in such devices is understudied. It is expected that application of novel Hemoglobin (Hb) Based Oxygen Carriers (HBOCs), such as Polymerized Hemoglobin (PolyHb), can further assist creation of in vivo-like oxygen gradient and metabolic zonation in BALs by improving oxygen transport. This study evaluated the biocompatibility of PolyHb in a hepatic model cell line (HepG2/C3A cells) for BALs, and developed a hollow fiber-based mini-BAL system that can replicate the in vivo oxygen gradient. This system represents a small functional unit that can be scaled up to a full-size BAL, akin to a liver lobule that is replicated many times in a whole liver. PolyHb was synthesized via glutaraldehyde-mediated crosslinking of free Hb, 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). We characterized the effect of PolyHbs on a model hepatoma cell line HepG2/C3A, and found that PolyHbs were well tolerated (based on viability assessment) at a dose of 10 mg/mL. Liver-related functions in HepG2/C3A cells were measured in the presence of 10 mg/mL of PolyHbs or unmodified Hb. We determined PolyHbs, both in the R-state and T-state, are safer to use at a concentration of 10 mg/mL compared to unmodified Hb in static cultures of HepG2/C3A cells.Mini-BALs containing two hollow fibers were seeded with HepG2/C3A cells, and cultured in the device for 3 days, while media was flown in via a syringe pump. Furthermore, a gas exchange set-up consisting of a thin silicone tubing in a controlled atmosphere was used to adjust the oxygen tension of the inlet to the desired value. Four distinct oxygen gradient profiles were established: (1) Normoxia condition (typical of that seen in previous BALs described in the literature); (2) In vivo condition (mimicking a liver sinusoid); (3) Zone 1 condition (representing the periportal oxygen gradient in a liver sinusoid); and (4) Zone 3 condition (representing the perivenous oxygen gradient in a liver sinusoid). Due to cell growth in the mini-BAL, flow rate was adjusted daily to maintain the desired oxygen gradient. Effluents from mini-BALs under each condition were collected for metabolite analysis. The data suggest that the In vivo condition could trigger an in vivo-like metabolic zonation in L-lactate production, cholesterol synthesis, and bile acid synthesis. Overall, the production levels were enhanced for albumin secretion, L-lactate production, and bile acid synthesis while under an in vivo oxygen gradient, compared to the Normoxia group, although the pattern of albumin secretion in mini-BAL is Zone 3 &gt; Zone 1, which is opposite to the albumin secretion pattern in in vivo metabolic zonation.Future work could incorporate the use of PolyHbs in the mini-BAL, which would allow decreasing flow rate during operation. The mini-BAL developed in this study could serve as a platform for investigating BAL operational conditions that can be scaled up to a clinically useful size. The mini-BAL may also be useful as an ex vivo drug metabolism evaluation system.