An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Mouse primary hepatocytes are important in liver studies. Especially for glucose metabolism, and hepatic drug testing. Time wise the isolation process can be time consuming and the energy costly.

This protocol can provide an efficient way to isolate mouse primary hepatocytes. It is time and labor friendly. Requiring very few steps with all regions commercially available.

Begin by using the peristaltic pump to start pumping warmed up double distilled water at a speed of 4 mL per minute, for five minutes. Then change the pumping tube from water to warmed up perfusion medium. Monitoring the air bubble between the water and perfusion medium.

Will help track the flow of the perfusion medium through the system. Next disinfect the abdomen of an anesthetized eight week old C57 black 6J female mouse with 70%ethanol. And using a scissor, cut open the abdomen to expose the liver, portal vein and inferior vena cava.

Then stop the peristaltic pump. After ensuring that the pumping tube contains perfusion medium and not water. Insert a 24 gauge catheter into the inferior vena cava.

Switch the pump back on and cut the portal vein open. While the perfusion medium is being pumped, press the portal vein every minute to let the liquid reach every corner of the liver. Continue pumping for approximately three to five minutes or until the flushed out liquid is clear.

Next change the pumping tube from the perfusion medium to the collagenase dispase medium. And press the portal vein every minute to let the liquid reach every corner of the liver. Continue pumping until all 25 mL of the collagenase dispase medium are depleted.

Next isolate the whole liver without the gallbladder. And placed it in 30 mL of wash medium in a Petri dish on ice. Using forceps tear the liver into pieces to release primary hepatocytes into the solution.

At this stage, the wash medium turns into a cloudy solution containing released primary hepatocytes and small liver pieces. Filter this cloudy solution through a 70 micron cell strainer, into 20 mL of 1X per call HBSS in a 50 mL tube on ice. And mix the solution by inverting the tube 20 times.

Next centrifuge the solution. Then in the tissue culture hood, aspirate the supernatant. And wash the pellet with 30 mL of cold wash medium.

Pellet the cells once again with centrifugation. Remove the supernatant, and re-suspend the pellet in 25 mL of culture medium. Count the cells and plate them on desired culture plates.

According to the experimental design. After plating, isolated primary hepatocytes were firmly attached by one hour and fully expanded after 12 hours. In the isolated hepatocytes, mRNA levels of hepatocyte markers, such as transthyretin, CD95, ASGR1, and ASGR2 were significantly increased compared to the whole liver.

In contrast the presence of immune cells, stellate cells, and endothelial cells was lower. As evidenced by a sharp decrease in CD45, collagen type 1 alpha 1, and endothelial cell 2 kinase 2 mRNA levels. Suggesting that this protocol can greatly reduce interference from other hepatic cells.

After plating ASGR1 and ASGR2 levels decreased with time, but remain comparable to the whole liver until the 12 hour time point. Especially for ASGR1. The expression level of membrane bound CD81 was consistent for up to 48 hours.

In comparison, toll-like receptor 4 expression generally increased and became higher than in vivo levels after 48 hours. Insulin sensitivity assays demonstrated that insulin significantly promoted phosphorylation of both AKT at serine 473. And forkhead box 01 at serine 256, in the hepatocytes.

Indicating the sensitivity of primary hepatocytes to insulin. Phosphoenolpyruvate carboxykinase protein levels significantly increased after glucagon treatment. Suggesting that the glucose production pathway was activated.

This activation was further confirmed by increased glucose production. And with other glucose production stimulators like Forskolin+IBMX. This protocol can save both time and energy for studies using mouse primary hepatocytes.

Following this protocol, related experiments such as the hypothetical glucose metabolism and pharmaceutical biomarker assays can be carried out.