A Simple and Useful Predictive Assay for Evaluating the Quality of Isolated Hepatocytes for Hepatocyte Transplantation

Muneyuki Matsumura,Shigehito Miyagi,Akiko Inagaki,Susumu Satomi,Masafumi Goto,Michiaki Unno,Takashi Kamei,Ibrahim Fathi,Takehiro Imura,Hiroyuki Ogasawara,Kengo Fukuoka,Kazuo Ohashi

doi:10.1038/s41598-019-42720-x

Muneyuki Matsumura, Shigehito Miyagi + Show 10 more

Open Access

https://doi.org/10.1038/s41598-019-42720-x

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Journal: Scientific Reports	Publication Date: Apr 16, 2019
Citations: 10	License type: open-access

Affiliation: Tohoku University, Osaka University

Abstract

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P < 0.001). In the apoptosis model, the ADP/ATP ratio assay, PE, DNA quantification and an ammonia elimination test clearly distinguished the groups (P < 0.001, respectively). The ADP/ATP ratio, PE and DNA quantity were well-correlated and the ammonia elimination was slightly correlated with the transplant outcome. TBE could not distinguish the groups and was not correlated with the outcome. The ADP/ATP ratio assay predicted the transplant outcome. PE and DNA quantification may improve the accuracy of the retrospective (evaluations require several days) quality assessment of hepatocytes. The ADP/ATP ratio assay, alone or with a short-term metabolic assay could improve the efficiency of HTx.

Highlights

Hepatocyte transplantation (HTx) has been performed as an alternative to liver transplantation to treat lethal acute liver failure[3,4] and some liver-based inborn errors of metabolism[5,6]
Our findings revealed that the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio of isolated hepatocytes, but not the Trypan blue exclusion (TBE) viability—which is routinely used as quality assay—was correlated with the outcomes of HTx in an in vivo model
This is consistent with previous reports which showed that TBE viability measurements were not conclusive enough to predict the outcome of HTx, as there was no correlation between the in vitro TBE viability of hepatocytes and the graft function after HTx19,25

Summary

Introduction

Hepatocyte transplantation (HTx) has been performed as an alternative to liver transplantation to treat lethal acute liver failure[3,4] and some liver-based inborn errors of metabolism[5,6]. Hepatocytes isolated from steatotic livers show low viability and a poor function[17,18], the quality of hepatocytes isolated from older donors is highly variable[19], and the viability of hepatocytes from livers donated after cardiac death is low and is well-correlated with the warm ischemic time[20], clearly indicating that the quality of isolated hepatocytes is a limiting factor for HTx. Trypan blue exclusion (TBE) is the only established method for evaluating the hepatocyte quality before transplantation in the clinical setting[19,21]. Considering the fragility of isolated hepatocytes[29], these assays require relatively too much time to assess the quality of grafts before HTx in the clinical settings; optimal methods must be established. In the field of HTx, no quality assay that is correlated with the outcome of HTx in an in vivo model has been developed, whereas several novel assays have been correlated with TBE viability assays[23,26]

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