Rapid and Sensitive Assessment of Human Hepatocyte Functions

Abstract

Transplantation of human hepatocytes (HTx) has gained recognition as a bridge to, or an alternative to, orthotopic liver transplantation for patients with acute liver failure or genetic defects in liver function. Although the quality of the hepatocytes used for cell transplantation is critical, no consensus exists on protocols to assess the function of hepatocytes prior to HTx. Application of this cell therapy in clinical practice could be aided by fast and reliable assays to evaluate the functional competence of isolated hepatocytes prior to clinical transplantation. Traditional assays for measuring metabolic functions in primary hepatocytes frequently involve highly technical equipment, time-consuming methods, and large numbers of cells. We describe a novel approach for the rapid assessment of the metabolic capabilities of human hepatocytes. This report details simple procedures to evaluate 11 endpoints from cells isolated from human liver that can be performed by a single operator within approximately 2 h of isolation. Longer term cultured hepatocytes were also analyzed to determine if the results from the 2-h tests were predictive of long-term hepatic function. The assays simultaneously measure five cytochrome P450 activities, one phase II activity, plating efficiency, and ammonia metabolism in addition to viability and cell yield. The assays require fewer than 20 million cells and can be completed using commonly available and inexpensive laboratory equipment. The protocol details methods that can be used in a time frame that would allow analysis of hepatic functions in freshly isolated hepatocytes prior to their use for clinical transplantation.

Clinical Hepatocyte Transplantation: Practical Limits and Possible Solutions

“…To assess and predict cell engraftment, hepatocytes are commonly seeded to a substratum (i.e. collagen) and left to adhere for a few hours; then, adhesion efficiency is quantified [40,49,53]. Engraftment in a suitable animal model would likely be more relevant; however, as the evaluation of transplant engraftment may take several weeks to months, these data would only be available retrospectively or if cells had previously been cryopreserved.…”

“…Novel approaches have been proposed in order to assess the quality of hepatocytes prior to transplantation [53,54]. One report describes assays that simultaneously measure up to 11 different hepatic functions, within approximately 2 h of isolation, using commonly available and inexpensive laboratory equipment and very few cells.…”

“…One report describes assays that simultaneously measure up to 11 different hepatic functions, within approximately 2 h of isolation, using commonly available and inexpensive laboratory equipment and very few cells. As a result, hepatocyte preparations can be screened and customized to better fit the recipient's needs [53,54]. …”

Role of Chromatin Structural Changes in Regulating Human CYP3A Ontogeny

“…Four of these liver samples were from pediatric donors between 1 and 6 years of age (Table 2). To ensure mature CYP3A4 expression was present in each of these samples, as well as all postnatal samples, activities were determined using a commercial, cell-based assay (P450-Glo CYP3A4 Assay, Luciferin-IPA; Promega Corporation, Madison, WI) with some modifications, as previously described (Gramignoli et al, 2014). All but one of the postnatal samples exhibited CYP3A4 activity within one standard deviation of the mean activity previously determined for 75 primary adult hepatocyte preparations (Gramignoli et al, 2014).…”

“…To ensure mature CYP3A4 expression was present in each of these samples, as well as all postnatal samples, activities were determined using a commercial, cell-based assay (P450-Glo CYP3A4 Assay, Luciferin-IPA; Promega Corporation, Madison, WI) with some modifications, as previously described (Gramignoli et al, 2014). All but one of the postnatal samples exhibited CYP3A4 activity within one standard deviation of the mean activity previously determined for 75 primary adult hepatocyte preparations (Gramignoli et al, 2014). All four pediatric samples exhibited CYP3A4 activity greater than the mean, with the youngest sample exhibiting the highest activity (greater than one standard deviation from the mean) (see Supplemental Fig.…”