Isolation of rat hepatocytes for pharmacological studies on metabotropic glutamate receptor (mGluR) subtype 5: a comparison between collagenase I versus collagenase IV

Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.

[1]  F. Nicoletti,et al.  Selective Blockade of the Metabotropic Glutamate Receptor mGluR5 Protects Mouse Livers in In Vitro and Ex Vivo Models of Ischemia Reperfusion Injury , 2018, International journal of molecular sciences.

[2]  R. McCarthy,et al.  Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets , 2017, Islets.

[3]  L. Hodson,et al.  The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus , 2017, Cell and Tissue Banking.

[4]  C. Berardo,et al.  Localization and role of metabotropic glutamate receptors subtype 5 in the gastrointestinal tract , 2017, World journal of gastroenterology.

[5]  M. Vairetti,et al.  Selective blockade of mGlu5 metabotropic glutamate receptors is protective against hepatic mitochondrial dysfunction in 6‐OHDA lesioned Parkinsonian rats , 2015, Clinical and experimental pharmacology & physiology.

[6]  Lijun Liu,et al.  Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy. , 2014, Journal of visualized experiments : JoVE.

[7]  Christopher M. Overall,et al.  Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen , 2014, Journal of proteomics.

[8]  M. Vairetti,et al.  Troubleshooting and improving the mouse and rat isolated perfused liver preparation. , 2013, Journal of pharmacological and toxicological methods.

[9]  Nicole Fenwick,et al.  The welfare of animals used in science: how the "Three Rs" ethic guides improvements. , 2009, The Canadian veterinary journal = La revue veterinaire canadienne.

[10]  C. Hendriksen,et al.  Reduction Strategies in Animal Research: A Review of Scientific Approaches at the Intra-experimental, Supra-experimental and Extra-experimental Levels , 2005, Alternatives to laboratory animals : ATLA.

[11]  F. Nicoletti,et al.  Selective blockade of mGlu5 metabotropic glutamate receptors is protective against acetaminophen hepatotoxicity in mice. , 2003, Journal of hepatology.

[12]  A. Dhawan,et al.  Progress in human hepatocytes: isolation, culture & cryopreservation. , 2002, Seminars in cell & developmental biology.

[13]  Chengwei Tang,et al.  Primary Culture of Porcine Pancreatic Acinar Cells , 2001, JOP : Journal of the pancreas.

[14]  T. Knöpfel,et al.  Selective blockade of mGlu5 metabotropic glutamate receptors protects rat hepatocytes against hypoxic damage , 2000, Hepatology.

[15]  S. Williams,et al.  Collagenase lot selection and purification for adipose tissue digestion. , 1995, Cell transplantation.

[16]  W. Sherman,et al.  Evidence that inositol 1-phosphate in brain of lithium-treated rats results mainly from phosphatidylinositol metabolism. , 1987, The Biochemical journal.

[17]  T. Serwold,et al.  Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments. , 2019, Methods in molecular biology.