Abstract
Research and therapeutic applications create a high demand for primary human hepatocytes. The limiting factor for their utilization is the availability of metabolically active hepatocytes in large quantities. Centrifugation through Percoll, which is commonly performed during hepatocyte isolation, has so far not been systematically evaluated in the scientific literature. 27 hepatocyte isolations were performed using a two-step perfusion technique on tissue obtained from partial liver resections. Cells were seeded with or without having undergone the centrifugation step through 25% Percoll. Cell yield, function, purity, viability and rate of bacterial contamination were assessed over a period of 6 days. Viable yield without Percoll purification was 42.4 × 106 (SEM ± 4.6 × 106) cells/g tissue. An average of 59% of cells were recovered after Percoll treatment. There were neither significant differences in the functional performance of cells, nor regarding presence of non-parenchymal liver cells. In five cases with initial viability of <80%, viability was significantly increased by Percoll purification (71.6 to 87.7%, p = 0.03). Considering our data and the massive cell loss due to Percoll purification, we suggest that this step can be omitted if the initial viability is high, whereas low viabilities can be improved by Percoll centrifugation.
Highlights
Primary human hepatocytes serve as the gold-standard model for in vitro testing of drugs that are metabolized in the liver[1,2]
The aim of this work is to investigate whether purification of primary human hepatocytes with density separation by Percoll has a beneficial effect on the performance of isolated human hepatocytes; e. g. yield, viability, plating efficiency, metabolic function, purity, bacterial contamination levels[28] and purity of cell cultures
Using Percoll density centrifugation is a double-edged sword: Is the tremendous loss of cells worth the better performance? In all publications that provide detailed information regarding the Percoll purification step during isolation of human liver cells[22,23,33,34,35,36], the cell yield was diminished by this procedure, while the viability improved
Summary
Primary human hepatocytes serve as the gold-standard model for in vitro testing of drugs that are metabolized in the liver[1,2]. Hepatocyte suspensions are purified through density separation with Percoll, which is a low-density fluid containing colloidal silica particles coated with polyvinylpyrrolidone[13,14]. This additional purification step is included to the isolation protocol in almost 50% of published studies on primary human hepatocytes in the literature reviewed for this study (see supplementary data). Performed to purify parenchymal human hepatocytes themselves[22,23,24,25,26], adding about 45 minutes to the 3 hours isolation procedure Systemic evaluation of this cell- and time-consuming step in hepatocyte isolation is not currently available in the literature, not for human primary liver cells. The aim of this work is to investigate whether purification of primary human hepatocytes with density separation by Percoll has a beneficial effect on the performance of isolated human hepatocytes; e. g. yield, viability, plating efficiency, metabolic function, purity, bacterial contamination levels[28] and purity of cell cultures
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