Optimization of primary hepatocyte isolation for the pharmacological characterization of metabotropic glutamate receptor (mGluR) s ubtype 5: A study on Reduction

To minimize the number of animals used during experiments, it is important to choose the suitable enzyme according to the final goal. In our work, we demonstrated the superiority of collagenase IV in the maintenance of functional transmembrane receptor, thus the pharmacological activity, in isolated rat hepatocytes.

Recommended parameters for hepatocyte isolation-a review

<p>There is an increasing demand for primary human hepatocytes for toxico-pharmacological studies and for exploring their role in liver cell transplantation. During the whole process of isolation, culture and cryopreservation hepatocytes are subjected to various stress. All these factors will lead to the impairment in the metabolic capability of the hepatocytes post isolation. Hepatocyte isolation is an expensive and resource driven procedure. Thus, it is important to consider all the factors before we subject the specimen for isolation of cells. By considering the factors that influence hepatocyte isolation, hepatocytes of good quality can be obtained. With recommendations on the ideal parameters for isolation being made the researcher shall have a knowledge on what critical factors need to be taken care of during hepatocyte isolation. There will be an opportunity to modify critical factors if possible, during the procurement of specimen by working in tandem with the clinicians. The specimen which shall be deemed un fit when two or more factors have exceeded the critical limits can be rejected.</p>

Beneficial Role of ROS in Cell Survival: Moderate Increases in H2O2 Production Induced by Hepatocyte Isolation Mediate Stress Adaptation and Enhanced Survival

High levels of reactive oxygen species (ROS) can lead to impairment of cell structure, biomolecules’ loss of function and cell death and are associated with liver diseases. Cells that survive increased ROS often undergo malignant transformation. Many cancer cells tolerate high levels of ROS. Here we report a transiently increased production of H2O2 and concomitant upregulation of antioxidative enzymes triggered by hepatocyte isolation; the H2O2 levels revert in about two days in culture. Three-day survival rate of the isolated cells in the presence of 2.5-fold increase of H2O2 is almost 80%. Apoptosis activation through the mitochondrial pathway is meanwhile reduced by inhibition of caspase-9 triggering. This reduction depends on the amount of H2O2 production, as decreased production of H2O2 in the presence of an antioxidant results in increased apoptosis triggering. These stress adaptations do not influence urea production, which is unchanged throughout the normal and stress adapted phases. We conclude that hepatocytes’ stress adaptation is mediated by increased ROS production. In this case, high ROS improve cell survival.

Primary mouse hepatocyte isolation

This protocol is intended to isolate primary hepatocytes from mus musculus (mouse) for the purposes of culturing primary hepatocytes. Hepatocytes can be used for approximately 5 days from the time of isolation, and cannot be frozen for future use. A single isolation from a single mouse, including time to prepare reagents, takes approximately 2 hours.