Evaluation of encapsulated liver cell spheroids in a fluidised-bed bioartificial liver for treatment of ischaemic acute liver failure in pigs in a translational setting

Liver failure is an increasing problem. Donor-organ shortage results in patients dying before receiving a transplant. Since the liver can regenerate, alternative therapies providing temporary liver-support are sought. A bioartificial-liver would temporarily substitute function in liver failure buying time for liver regeneration/organ-procurement. Our aim: to develop a prototype bioartificial-liver-machine (BAL) comprising a human liver-derived cell-line, cultured to phenotypic competence and deliverable in a clinical setting to sites distant from its preparation. The objective of this study was to determine whether its use would improve functional parameters of liver failure in pigs with acute liver failure, to provide proof-of-principle. HepG2cells encapsulated in alginate-beads, proliferated in a fluidised-bed-bioreactor providing a biomass of 4-6×10 10 cells, were transported from preparation-laboratory to point-of-use operating theatre (6000miles) under perfluorodecalin at ambient temperature. Irreversible ischaemic liver failure was induced in anaesthetised pigs, after portal-systemic-shunt, by hepatic-artery-ligation. Biochemical parameters, intracranial pressure, and functional-clotting were measured in animals connected in an extracorporeal bioartificial-liver circuit. Efficacy was demonstrated comparing outcomes between animals connected to a circuit containing alginate-encapsulated cells (Cell-bead BAL), and those connected to circuit containing alginate capsules without cells (Empty-bead BAL). Cells of the biomass met regulatory standards for sterility and provenance. All animals developed progressive liver-failure after ischaemia induction. Efficacy of BAL was demonstrated since animals connected to a functional biomass (+ cells) had significantly smaller rises in intracranial pressure, lower ammonia levels, more bilirubin conjugation, improved acidosis and clotting restoration compared to animals connected to the circuit without cells. In the +cell group, human proteins accumulated in pigs' plasma. Delivery of biomass using a short-term cold-chain enabled transport and use without loss of function over 3days. Thus, a fluidised-bed bioreactor containing alginate-encapsulated HepG2cell-spheroids improved important parameters of acute liver failure in pigs. The system can readily be up-scaled and transported to point-of-use justifying development at clinical scale

Biochemistry, liver function tests and haematology in pigs with ischaemic acute liver failure.

<p>Group 1  =  animals to be treated with a Cell-Bead BAL (bold) and Group 2  =  animals to be treated with a control empty bead non functional BAL (plain text). Data are from blood samples taken at the start of the BAL addition, representing baseline acute liver failure (normal range in italics).</p

Human proteins in pig plasma after BAL treatment.

<p>Pig blood samples were assessed by ELISA for specific human protein content during treatment; Group 1 cell bead treated BAL (solid symbols), compared with Group 2 empty bead control treated BAL (open circles) (<b>A</b>) Albumin (<b>B</b>) Fibrinogen, (<b>C</b>) Alpha-1-acid glycoprotein (A1AGP) and (<b>D</b>) Alphafoetoprotein (AFP). Results expressed as µg/ml pig plasma: mean±SEM n = 6. No human protein was detected in Group 2 animals, establishing the specificity of the assays for human proteins.</p

Amino acid concentrations in pig plasma before and after treatment.

<p>Amino acids, measured in pig plasma by ion exchange HPLC were quantified in Group 1 and Group 2 animals. Results expressed as µmol/L, mean±SEM. Differences between start and end of treatment for each group (Group 1 cell-bead BAL n = 6, and Group 2 empty bead non-functional control BAL n = 5), are show with statistical differences: *p<0.05; **p<0.02, t-test, paired, 2-tailed. Three amino acids showed differences in response to treatment with the BAL comparing Group 1 and Group 2.</p

Thromboelastography (TEG) measurements of coagulation.

<p><b>A&B) Example of TEG traces</b>: in cell-bead treated BAL Group 1 <b>(A)</b> and empty bead control treated BAL Group 2 <b>(B). Clotting parameters quantified (C-F):</b> Thromboelastography of blood during treatment with either Cell bead treated BAL (solid symbols) or empty bead control treated BAL (open symbols); n = 3: results for each pig are shown as a change from the start of ischaemia (normalized and denoted as 0 on Y axis), demonstrate changes in R-time <b>(C)</b>, K-time <b>(D)</b>, angle <b>(E)</b> and maximum amplitude (MA) <b>(F)</b>. BAL was added ~2.5h later. The four parameters describe the overall clotting reaction. The R-time (min) reflecting fibrin formation rate, is functionally dependent on clotting factors, notably fibrinogen; reported as the reaction time from placement in the cup to 2mm amplitude on the tracing, thus, the higher the number the longer it takes for formation of clot. In both groups initially the time taken for fibrin formation is increased during liver failure; as the BAL effects an improvement in Group 1, the time taken for fibrin to form is reduced; in contrast in Group 2 the time continues to increase <b>(C)</b>. The K time, reflecting viscoelasticity, is the time taken from the R time to the point where the trace amplitude reaches 20mm, indicating intrinsic clotting factor activity, as well as fibrinogen and platelet function. Similar to the R time, the longer the time the less intrinsic clotting factor activity; pigs in Group 1 show a decrease in K time with continued treatment whereas those in Group 2 continue to increase <b>(D)</b>. The alpha angle (angle of slope of r to k denotes rate of clot formation); in Group 1, pigs had an increased angle with BAL treatment, in Group 2 animals the angle decreased with time <b>(E)</b>. MA is the maximum amplitude on the trace, reflecting the absolute strength of the clot; Group 1 pigs showed a stronger clot compared with Group 2 animals <b>(F)</b>.</p

Protocol of ischaemic acute liver failure model.

<p>(<b>A</b>) Pigs were treated with the bioartificial liver machine 2–3 h after establishing ischaemic damage. After anaesthesia, brain monitoring catheters were inserted, prior to establishment of porta-caval shunt and arterial, venous and urine catheter placement. (<b>B</b>) <b>Schematic of the bioartificial liver machine:</b> The fluidised-bed bioreactor chamber containing either cell bead biomass (Group 1) or empty beads for control (Group2), was attached to the pig via a plasmapheresis machine. Blood was obtained from the pig via the splenic vein at 90 ml/min and separated from cellular components with a Cobe Spectra Plasma separator, providing plasma at a flow rate of ∼45 ml/min in a primary circuit, feeding into the BAL secondary circuit at ∼400–600 ml/min. Whole blood was returned to the pig via the plasma-separator at 90 ml/min, combining the “treated ”plasma with the cellular component.</p

Efficacy of HepG2-Fluidised-bed bioreactor treatment in pigs with ischaemic acute liver failure: biochemical response.

<p><b>A) Bilirubin conjugation in pigs with ischaemic acute liver failure.</b> Conjugated bilirubin concentrations (µmol/L) increased and unconjugated bilirubin concentrations decreased in Group 1 pigs, when attached to the cell-bead BAL; in contrast there was neither increase in conjugated bilirubin nor decrease in unconjugated bilirubin in Group 2 control animals, Group 1 n = 6; group-2 n = 5; p-value, unpaired 2-tailed t-test, mean±SEM. <b>B). Change in acidosis in pigs with ischaemic acute liver failure, after BAL treatment.</b> pH values dropped after ischaemic insult to a nadir in both groups. Blood pH was assessed in a blood gas analyser. Pigs in the Cell-bead BAL (Group 1 solid line) showed an increased pH towards normal at the end of BAL treatment whereas pigs treated with the control empty bead BAL (Group 2 -----) showed little improvement; inset shows group averages (n = 4, mean±SEM unpaired, one-tailed t-test). <b>C) Blood ammonia levels in pigs 4 h after BAL treatment.</b> values show a change in ammonia from 0.5 h after ischaemia (normalised to 100%) during BAL treatment. Concomitant with a decrease in ICP in Group 1, ammonia levels decreased (cell BAL) cf. group 2 (Control empty bead BAL); Group 1 n = 6, mean 120±8; Group 2 n = 7, mean 150±11. Statistics: two-tailed unpaired t-test, p = 0.035.</p

Brain parameters in pigs with ischaemic acute liver failure.

<p><b>A</b>) Intracranial Pressure (ICP) using a Codman catheter was recorded every 15 min, in mmHg. The rise after established acute ischaemic liver failure was halted when pigs were attached to the cell bead BAL (Group 1, n = 5, closed circles ·), whilst it continued to increase in Group 2 (empty bead BAL open circles o), n = 7. <b>B</b>) Brain oxygenation, normalised to 100% at time of ischaemia. There was an increase in brain oxygenation in Group 1 cell bead treated pigs (solid circles ·) compared with Group 2 (open circles o). Statistics compared the slopes of the line between groups with 95% confidence limits.</p

Parameters of brain homeostasis during BAL treatment of pigs with ischaemic liver failure: Brain metabolites.

<p>Microdialysate samples (18 µl) were collected from catheters on each side of the brain. Fig 4 <b>A&B</b> show glucose levels in the brain during the course of treatment using a cell-bead BAL (<b>4A</b>) or an empty bead BAL control (<b>4B</b>). Left and right refer to left and right brain hemispheres. 30 minute microdialysates were collected; measurements made hourly. Fig <b>4C&D</b> show the lactate:pyruvate ratio indicating ischaemic damage in cell-bead BAL (<b>4C</b>) and empty bead BAL control (<b>4D</b>); note that 4D shows different results on each side of the brain, perhaps indicating patchy brain ischaemia. These results are representative of the 4 pigs in which microdialysis was performed, two in Group 1 and two in Group 2.</p