This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.
This rat hepatocytes isolation protocol is based on the liver resection prior to digestion approach. The protocol introduces a compact and portable integrated perfusion system for cell isolation. This technique involves collagenase recirculation which reduces the volume of collagenase use, and provides more flexibility in extension of digestion time while ensuring high gravity yield and hepatocytes specific function.
Put the rat in the hood. After cutting the abdominal muscles, push the intestines to the right with the back of the curved forceps. Break the membrane underneath the bile duct with the tip of the curved forceps for looping the suture around the portal vein and the bile duct.
Use a silk surgical suture to make a very loose ligature around the portal vein close to the liver, just before the vein branches left and right into different liver lobes. About two to three centimeters upstream of the first ligature, just before the gastric vein branches off from the portal vein, create a hole through the tissue underneath the portal vein with the tips of two pairs of curved forceps. Then stretch the hole to support the portal vein during cannulation.
Prevent pressure buildup in the liver by reducing the pump speed to four RPM for a flow rate of approximately three milliliters per minute. And ensure that the outflow of the calcium free buffer from the intravenous catheter is reduced to a slow drip. While gently supporting the portal vein with forceps, hold the intravenous catheter with the bevel of the needle facing up.
And insert the needle into the portal vein at a 10 to 20 degree angle. Then slowly advance until the whole bevel is inside the vein. Correct insertion should result in blanching of the liver color.
Advance the cannula over the needle, and retract the needle behind the cannula by pulling back the wing with the index finger while holding the catheter with the thumb and middle finger. Secure the portal vein onto the catheter with a vein clip and clip below the portal vein to avoid perfusing flow disruption. Next, cut the infrahepatic inferior vena cava to prevent the pressure buildup in the liver, and observe for blood spurting impulses to ensure the correct cut.
Increase the pump speed to 38 RPM for a flow rate of approximately 33 milliliters per minute. And flush the liver three times by closing the infrahepatic inferior vena cava with forceps for two to three seconds, and then reopening it. In order to perfuse all liver lobes, adjust the position of the cannula tip and make sure that it is placed before the portal vein branches into different liver lobes.
Then, tighten the loose ligature around the portal vein, slightly upstream of the branching point, and secure the position of the cannula in the portal vein to prevent backflow of the buffer by making three knots at the point where the hard needle supports the cannula, in between the bevel and the side hole. After liver resection allow the liver to perfuse with the calcium free buffer for 12 minutes. Then, tighten the roller clamp of calcium free buffer and loosen the roller clamp of collagenase buffer to change the perfusion buffer.
After the collagenase buffer reaches the liver, flush the liver three times by closing the suprahepatic inferior vena cava for two to three seconds and reopening it. Carefully move the liver to the stage on top of the beaker for recirculation of the collagenase buffer by holding the diaphragm with forceps while supporting the catheter, taking care not to pull the catheter to prevent accidental detachment. Digest the liver for 12 minutes, and stop the perfusion when the liver loses its smooth brown texture and becomes mushy.
Extend the digestion time if necessary. After digestion of the liver cut the portal vein carefully remove the cannula. And then transfer the liver into cold DMEM to isolate the hepatocytes.
Gently tap the liver using the backside of the curved forceps to break and peel off the glistens capsule. And release the liver cells by gently swaying the liver in DMEM until the cells are fully dissociated in the media. The viability of hepatocytes was between 88 to 94.8%as determined by trip and blue counting.
The yield of hepatocytes from rats weighing 200 to 300 grams was up to five times 10 to the eighth cells per isolation. The representative images are showing unpurified liver cell suspension and purified hepatocyte suspension. The purity of the isolated hepatocytes was approximately 96.8%which was quantified by counting albumin positive fluorescent stained cells in relation to the total number of the cell nuclei.
In the representative images shown in false color, green represents the albumin, and blue indicates the cell nuclei with albumin. Cell nuclei without albumin are highlighted red. The hepatocytes in sandwich culture formed distinct bile canaliculi, and had good cell cell contact.
As shown by albumin, urea and cytochrome P450 assays, the hepatocytes exhibited high urea and albumin secretion. On day three, the hepatocytes exhibited high enzyme activities for CYP1A2, CYP2B1/2, and CYP3B2. Be mentally prepared before inserting the needle into the portal vein.
Upon insertion, a surgeon will need to continue without pulse until the pump speed was increased after cutting the IVC.
