Title: Hepatocyte and mesenchymal stromal cell co-encapsulation in alginate microbeads as therapy of acute liver failure.
Funder: Department of Health and Social Care

Abstract: Acute liver failure (ALF) carries a high mortality, especially for young infants, mainly because of the scarcity of timely suitable donor organs. However, hepatocytes isolated from unused or rejected livers can be cryopreserved so they are available at the time of clinical need. Human hepatocyte transplantation is emerging as a real alternative to liver transplantation in the acute setting. At King's College Hospital we have undertaken a NIHR-funded pilot study of alginate-encapsulated hepatocyte transplantation in 7 children with acute liver failure, showing the safety and feasibility of the technique. The encapsulated cells are delivered into the peritoneal cavity, a simple and safe procedure, with the added advantage of avoiding immunosuppression in a high-risk population. Alginate encapsulation allows nutrients, oxygen and metabolites to diffuse to and from the hepatocytes, but does not allow antibodies, complement or immune cells to enter the beads, thus protecting the liver cells from the host immune response. However, the major limitations are (i) the quality of cells isolated from marginal/fatty livers (ii) sustaining the viability and function of hepatocytes for long enough to allow liver regeneration or to buy time until a suitable organ becomes available: whilst the function of the transplanted beads is well established for the first few days, it declines rapidly. We have shown that 2D-coculture of hepatocytes and MSCs prevents apoptosis and has a long-lasting positive effect on hepatocyte function (40-fold increase in albumin synthesis after 23days). Subsequent studies, however, showed that this effect was lost after ultra-pure alginate cell encapsulation, potentially due to the lack of cell adhesion capacity in this alginate. Cell adhesion has indeed been shown to be necessary for the maintenance of cell viability and function. The aim of our study is to assess the effects of diverse alginates coupled with cell adhesion peptides on MSC and hepatocyte adhesion, viability, function and interaction, using classical function tests, as well as proteomics, lipidomics and metabolomics. Using an extensive array of metabolism analysis will help us understand the exact functions performed by the hepatocyte beads, bearing that the alginate chemistry will influence its physical properties and therefore the proteins and metabolites secreted owing to their size, but also their charge and polarity. Analysing earlier and late metabolism and viability markers will help us identify potential markers to be used as quality controls for our clinical bead production procedures, as well as suitable biomarkers to be used during the patients treatments. MSCs will be isolated from umbilical cords and grown under GMP to produce master cell banks and working banks. We have ~200 hepatocyte cell isolates consented for research and more will be collected during the time of this work. The cells will be encapsulated in the 4 alginates modified with anchoring-peptides and their function compared to the current alginate for (i) MSC beads to obtain an in depth picture of the trophic factors produced by the cells and released by the beads in different alginates; (ii) the hepatocyte beads to obtain a comprehensive measurement of cell functions; (iii) the co-culture of MSCs and hepatocytes to obtain QC data and potential biomarkers useful for the clinical trial. Within the study period we will undertake the first part of a clinical trial. Children with ALF (INR>3) will be recruited and will have infusion of the optimal alginate cell preperation determined from the in vitro work. Nine patients will be recruited in 18 months and the treatment will be in addition to standard of care. If, as anticipated, this pilot study shows safety, feasibility and non-futility we will be able to establish the confidence interval for a larger randomised trial to investigate efficacy.

Lay abstract: Acute liver failure (ALF) is a rare medical emergency that carries very high mortality without liver transplantation. Liver transplantation depends on the timely availability of a suitable donor organ, and whole organ replacement requires life-long suppression of the immune system with its associated complications. The liver has tremendous regenerative potential and if the patient's failing liver could be supported until regeneration has happened, whole organ replacement, and its associated complications, could be avoided. This has been demonstrated in our centre as the partial replacement of the patient s liver with a healthy donor liver served as a bridge to native liver regeneration. The transplanted liver thereafter became redundant in up to 70% of the patients. This experience demonstrated that a small mass of liver tissue may be sufficient to support the patient and that a transitory procedure can replace life-long whole liver replacement. This led to the idea of transplanting liver cells (the hepatocytes ) rather than liver tissue, with the added advantage that (i) isolated cells can be kept frozen for years and used off-the-shelf, something that is impossible with entire organs and (ii) cells isolated from one liver could treat more than one patient, therefore reducing the need for donor organs. Earlier clinical experiences with human hepatocytes in ALF have been only partly successful when cells were injected either in the liver increasing the bleeding risk or the abdominal (peritoneal) cavity increasing infection risk in patients who needed immunosuppression. We have developed a novel technique where human hepatocytes are encapsulated in beads made of clinical grade alginate gel (the beads ). This technique helps avoid the need for immunosuppression and the encapsulated cells can be safely deposited in the peritoneal cavity with relative ease, a technique which is much less invasive than organ transplantation. Our preliminary results are encouraging but are restricted by the limited availability of good quality cells and limited life span of even these. We and others have shown that mesenchymal stromal cells (MSCs) drastically improve the survival of hepatocytes and their liver-specific functions, in standard cell culture conditions. However, the beneficial effects of MSCs on primary human hepatocytes were lost when the cells were encapsulated in the standard alginate. In general, most cells need to be attached to survive and function properly, attached to other cells or to proteins that form the cement of cells in tissues (the extra-cellular matrix ). This attachment occurs at tissue-specific anchorage sites, which are not present in the standard alginate beads we currently use. We will therefore test whether the anchorage of MSCs in 4 new types of alginate with different anchorage sites will restore their effects on hepatocyte viability and function. We will also determine specific markers of cell function to help us determine how efficient the hepatocyte / MSC beads will be, when transplanted in patients. Finally we will start a pilot clinical trial to test the safety and efficacy of the optimised peptide-alginate beads in children with ALF.

Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: ORAL AND GASTROINTESTINAL