Impact of Percoll purification on isolation of primary human hepatocytes

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.


Supplementary information (2) -Literature Research -Evaluation of the benefit of Percoll purification in primary human hepatocyte isolation Search strategy and exclusion criteria
On May 28th, 2017, a systematic literature search was performed in PubMed (MEDLINE) using the following two search terms: MeshTerm 1: ("Hepatocyte* Isolation" OR "primary hepatocyte*" OR "human hepatocyte*" OR ("Liver" AND "cell isolation")) AND ("plating efficiency" OR "density purification" OR Percoll OR Purification OR "Gradient Centrifugation" OR "density gradient" OR "culture purity")

MeshTerm 2:
"Hepatocyte* isolation" All papers (n=352) where exported into Endnote X9 (Thomson Reuters, New York, US) and screened for duplicates (n=16). The resulting 326 papers were then screened by reading the title and abstract. After reading the abstracts, 170 papers were excluded because their abstract revealed that they fulfil exclusion criteria, including: -Paper did not work with primary isolated hepatocytes but with cell lines like HuH7 or HepG2 -Paper focused on pharmaceutical drug-safety testing and gave no detailed information about the hepatocytes they used -Paper used non-mammalian primary hepatocytes After removal of papers which met the exclusion criteria, 156 papers were cross read. During this process publications were categorized for second exclusion: Of all the publications that were identified to deal with primary hepatocytes and meet aforementioned criteria, 29 were not available with the literature licenses of Charité and 2 could not be retrieved in English or German. The remaining publications (n=125) were studied with special emphasis on the papers' methods part. 85 papers that gave a good explanation on the isolation were identified, while 22 were excluded because they did not state thoroughly enough, how they obtained hepatocytes and how they purified cell suspensions. Another 18 publications were excluded because the focus on another issue, so that hepatocyte isolation was just a tool to get their experimental materials.
Additionally, 16 papers were included after manual search. These papers were either suggested in references of other publications or recommended at the journals website. 6 Figure 1 illustrates the literature search strategy and workflow.

Quality assessment
Resulting papers' quality was assessed using a numeric scoring system. Since an established, published and peer-reviewed quality assessment lacks for evaluation of experimental studies, following questions were considered: -Is there an adequate description of statistic methods used and power to meet significance enough?
-Are there any errors in the paper concerning logic or facts?
-Is all the relevant information given to ensure reproducibility?
For each question, 1 point was accounted resulting in a score from 1 (poor quality) to 5 (excellent quality).

Results
101 publications were assessed for this review. Of these, 41 do not use density gradient centrifugation in their protocol, but end with the low speed centrifugation (mostly 50g for 5 minutes).

Percoll for purification of Non-Parenchymal cells
Of the 60 publications that mention purification with percoll density gradient in their methods part, 12 According to Schröder et al. in 1994 (74), who performed purification through a 50% concentrated Percoll-suspension for 10min at 50-250g, the reached viability was 94% and the cell culture reached to a high purity. Culture purity was assessed using immunohistological staining and revealed low contamination level of 1%, consisting of fibrocytes. However low yield after percoll purification is reported and in 7 out of 32 experiments, no cells could be recovered after the percoll step. Yet, these authors do not reflect on the direct comparison on cells before and after Percoll purification regarding cell culture purity and plating efficiency, which are determining parameters for the hepatocytes' subsequent use. Non-perfusion-technique. Compare before and after 60% percoll -metabolic activity higher -When Percoll purification was performed, only larger cells were selected with a lower yield. These cells were not as confluent but metabolically more active. Percoll density 1.07g/ml (55%) and 1.02g/ml (12.5%), 30000g, 90min, was performed and two separate populations of hepatocytes were obtained this way. "One denser than the other with significantly greater trypan blue dyeexclusion capabilities (P < 0.05)." Purified cells through the higherconcentrated Percoll layer performed better metabolically and had a better membrane integrity than the less dense cells. Used Percoll concentrated 45%, 50g, 10min; separated different subpopulations of hepatocytes ("normal" vs "small hepatocytes") and NPCs. Assessed effects of different co-cultures and growth potential and purity with FACS and immunohistochemistry. Focus is not on one population of parenchymal cells before and after density centrifugation.