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New technique for isolating human liver cells may help in future models for liver disease

In a new study published in Experimental Biology and Medicine (MayWood), researchers in Berlin, Germany found a way to simultaneously isolate human hepatocytes and non-parenchymal cell fractions, which could be useful for future liver disease models.

“[Primary human hepatocytes] mono-cultures are considered to be the gold standard for the investigation of hepatic metabolism and toxicity of xenobiotics,” Georg Damm, PhD, of the department for general, visceral and transplantation surgery at Charité University Medicine in Berlin, Germany, said in a press release. “However, detailed morphological and functional studies have demonstrated that these models are limited due to hepatocyte dedifferentiation and loss of functions within a few days. Reconstruction of the in vivo tissue architecture consisting of a 3D environment including [non-parenchymal cell fractions] is a promising approach to solve some of these problems.”

Georg Damm

Damm and colleagues isolated primary human hepatocytes (PHH) and non-parenchymal cell (NPC) populations in parallel from the same human tissue obtained through a partial liver resection. The cells were then purified by Percoll density gradient centrifugation and the Kupffer cells (KC), liver endothelial cells (LEC) and hepatic stellate cells (HSC) were separated using differences in cellular adherence properties and magnetic cell sorting (MACS) Technology (Miltenyi Biotec).    

Analyses showed KC dominated the NPC fraction and yielded a 1.9 × 10⁶ ± 0.2 × 10⁶ cells per gram of liver tissue (n=5), with an average viability of 92.8±3.5%. NPC fraction yielded 2.7 × 10⁵ LEC and 4.7 × 10⁵ [GD1] HSC per gram of liver tissue, according to the research. The viabilities of the cells were greater than 90%. The yield for PHH per gram of liver tissue was 14.8 × 10⁶ ± 6.6 × 10⁶ (n=12), with an average viability of 76.6 ± 4.2%.

The cells were identified by morphological features and by using immunofluorescent staining of cell type specific antigens , according to the research. PHH cells showed a purity of 92.3 ± 3.2%, KC had a purity of 81 ± 5.4%, LEC had a purity of 81 ± 1.7% and HSC had a purity of 93 ± 1.7%. 

“Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate,” the researchers wrote. “The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture linked to a loss in viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts.”

The researchers also stated that the different culture methods tested for HSC showed that these were able to “attenuate, but not prevent dedifferentiation in vitro.”

Damm concluded: “We present a new method for simultaneous extraction of human PHH and different NPC populations from the same donor tissue. The cells were clearly identified and characterized on the basis of morphological properties, specific marker expression and functional analysis.” – by Melinda Stevens

Disclosures: Damm reports no relevant financial disclosures. The study was supported by the German Federal Ministry of Education and Research (BMBF) project Virtual Liver: 0315741 and 0315739.