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Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Published on: October 23, 2018
Apoptosis occurs in isolated and banked primary mouse hepatocytes
1Department of Pediatrics, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614, USA.
Cell Transplantation
|April 11, 2001
Summary
Cryopreservation of liver cells leads to significant apoptosis, a form of cell death. Adding glucose to the freezing solution reduces apoptosis and improves cell recovery for hepatocyte transplantation.
Area of Science:
- Hepatology
- Cell Biology
- Cryobiology
Background:
- Hepatocyte isolation and cryopreservation are crucial for liver cell banking.
- Current methods yield insufficient viable cells for research and transplantation.
- Mechanisms of poor hepatocyte recovery post-cryopreservation remain unclear.
Purpose of the Study:
- To investigate the role of apoptosis in cell death of cryopreserved primary hepatocytes.
- To evaluate the efficacy of glucose supplementation in mitigating apoptosis and improving cell viability.
Main Methods:
- Primary hepatocytes from C57BL/6J mice were isolated and cryopreserved.
- Apoptosis was assessed using Annexin binding, TUNEL assay, flow cytometry, confocal microscopy, and DNA laddering.
- Cryopreservation solutions with varying glucose concentrations (10-20 mM) were tested.
Main Results:
- Cryopreserved hepatocytes showed a significant increase in apoptosis compared to unfrozen controls.
- DNA electrophoresis confirmed apoptosis in isolated and banked liver cells.
- Glucose addition (10-20 mM) reduced apoptosis by 84% and increased cell attachment fourfold.
Conclusions:
- Apoptosis significantly contributes to cell death in isolated and cryopreserved primary hepatocytes.
- Glucose supplementation in University of Wisconsin Solution enhances hepatocyte recovery post-cryopreservation.
- Findings suggest strategies to improve hepatocyte banking for transplantation and research.
