How do you isolate hepatocytes?
Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable.
How do you isolate mouse hepatocytes from the liver?
This protocol is aimed at isolating hepatocytes from mouse liver. Following anesthesia, the vena cava is cannulated and the liver is perfused to chelate calcium and wash out blood. Then, collagenase is perfused to the liver in order to dissociate extracellular matrix.
How do you culture primary hepatocytes?
Hepatocyte Culture Techniques. One common technique to culture primary hepatocytes is to seed the cells on a single layer of collagen gel. Under these conditions, hepatocytes secrete albumin and urea, and show minimal cytochrome P450 (CYP450) activity.
How do you perfuse a mouse liver?
Use the scissors to cut the abdominal skin vertically, to allow drainage of blood and perfusion liquid. Then squeeze the effluent blood vessel a few times with the straight forceps to inflate the liver, ensuring that all of the blood has drained out, and perfuse the liver with PBS until the liver tissue blanches.
Which of the following enzyme is used for hepatocyte isolation in cell culture laboratory?
Recent studies have used the enzyme liberase to isolate hepatocytes from normal resected liver tissue and have reported higher cell viabilities and cell yields .
What are primary human hepatocytes?
Hepatocytes directly isolated from liver tissue are called primary hepatocytes. The image on the left side is a schematic that shows the procedure for the isolation of primary human hepatocytes, and their cellular morphology in suspension and on plates.
How many hepatocytes does a mouse liver have?
For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139+/-25, 114+/-20, 117+/-30 and 135+/-10 million cells per gram of liver, respectively.
Do hepatocytes proliferate in vitro?
The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful.
How do you perfuse a mouse?
Place mouse in isofluorane chamber and lightly anesthetize the animal. For mouse anesthesia, administer 0.8 ml/20 g (of mouse body weight) Avertin through intraperitoneal injection with 30 ½ gauge needle. Wait for 3 minutes or until the mouse no longer responds to painful stimuli, such as paw pinch before proceeding.
What is primary rat hepatocytes?
Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation.
How to use Percoll for density separation of hepatocytes?
Note: Percoll is used for density separation of the viable hepatocytes from dead hepatocytes and cell debris. 28. Aspirate most supernatant, leave ∼1 mL and resuspend the cells by swirling the tube. 29. Add 10 mL plating media and resuspend by gentle swirling. 30. Add 10 mL Percoll solution and mix thoroughly by inverting the tube several times.
What is the lipid-filled hepatocytes protocol?
This protocol enables simultaneous isolation of normal and lipid-filled hepatocytes. Lipid-filled hepatocytes can be used in cell culture systems to study drug metabolism, hepatotoxicity, or glucose and lipid metabolism. For complete details on the use and execution of this protocol, please refer to Sharabi et al. (2017) and Kegel et al. (2016).
How do you isolate hepatocytes from primary mouse hepatocytes?
A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development. Cytotechnology. 2012;64:187–195.
How are hepatocytes separated from the liver during cell culture?
The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.