The overall goal of this procedure is to isolate human hepatocytes using a two-step collagenase perfusion procedure. This is accomplished by first cannulating the blood vessels in a piece of liver with irrigation cannula. This is followed by the perfusion and digestion of the liver piece using collagenase.
The third step is the removal of the glistens capsule, which releases the dissociated liver cells. The final step is a low speed centrifugation that isolates hepatocytes. Ultimately, hepatocytes can be identified with phase contrast microscopy or by albumin staining using immuno cyto chemistry with viability and yield of hepatocytes determined using a triam blue exclusion assay.
Visual demonstration of this method is critical as the cannulation and digestion steps are difficult to learn. This is because cannulation requires some finesse and the completion of digestion can be difficult to identify. For persons new to the technique, demonstrating the procedure will be Natalie Lun, a technician from my laboratory.
In this demonstration, we will use a piece of lamp liver in place of a piece from a human, Have the equipment for the perfusion of the liver piece set up as follows, set the water bath to an appropriate temperature such that the solutions are 37 degrees Celsius when they reach the liver.Piece. 41 degrees Celsius is good for solutions one, two, and three, and for the jacketed glass condenser solution four, however, should be at 37 degrees Celsius to reduce the loss of collagenase activity. Shortly before the profusion turn on the gas regulator For the oxygenation apparatus from a pathologist, obtain a liver piece that ideally has a mostly or fully intact glistens capsule and only one cut surface place.
This liver piece on the Buchner funnel that contains a perforated filter disc, the perfusion system must now be primed with solution one using a low flow rate insert curved irrigation cannula with olive tips into the larger blood vessels on the cut surface of the liver piece. As blood flushes out from the liver, the tissue becomes lighter in areas with good perfusion. The number of cannula used varies for differently sized livers.
In general, four to eight cannula should be used for livers ranging from below 20 grams to over 80 grams. Select the gauge of the cannula to have a snug and secure fit. Leave the smaller blood vessels open for the buffer to drain out from the tissue.
Now, increase the flow rate on the peristaltic pump to between 110 to 460 milliliters per minute. It is a good perfusion if the liver piece is a lighter color throughout the flow rate used. Depends on the size of the liver with higher perfusion rates used for larger livers.
Generally speaking, an average perfusion speed of 44 milliliters per minute per cannula is ideal across a range of different liver weights. The chosen flow rate should result in a slight plumping up of the liver piece. Keep the liver piece moist during the perfusion by covering it with a piece of gauze soaked in saline.
After perfusing the tissue with one liter of solution, one to flush out any remaining blood in the liver piece. Change the perfusion fluid to solution two and perfuse for 10 minutes. Next, switch the perfusion fluid to solution three and perfuse with a half liter.
Now change the perfusion fluid to solution four, which contains collagenase. Carry out this perfusion in a recirculating manner for nine to 12 minutes or until the liver is sufficiently digested. The liver tissue should appear to break apart slightly under the glisten capsule.
The liver should also feel softened when probed with the blunt side of a scalpel. It is important to digest the liver piece for the correct amount of time as over digestion can lead to low viability of hepatocytes and under digestion can lead to low yield of hepatocytes. Begin by turning off the peristaltic pump and removing cannula from the liver piece.
Then place the tissue in a crystallizing dish containing 100 to 200 milliliters of solution.Five. Remove the glistens capsule carefully and gently shake out the cells. If there are regions that are not well perfused, a scalpel can be used to cut through these regions to release cells contained within.
Add more solution five as needed during the process, and ultimately bring the volume of solution five to 500 milliliters. Now filter the cell suspension twice. First, filter the cells through a 210 micron nylon mesh into a one liter conical flask.
Then filter the cells through a 70 micron nylon mesh into a one liter conical flask. Pour out two equal volumes of the cell suspension into 200 milliliter centrifuge tubes. Then centrifuge the filtered cell suspension at 72 Gs for five minutes at four degrees Celsius.
Aspirate the snat and gently resuspend the cell pellet in about 200 milliliters of solution.Five. Repeat the centrifugation and resus suspension three times after the final centrifugation resuspend the cells in cold storage solution for a final concentration of two to 5 million hepatocytes per milliliter. A triam blue assay can be used to accurately measure the cell count using a hemo cytometer.
Shown here are trian blue stained hepatocytes from 648 isolations. The average viability of isolated human hepatocytes was 77 plus or minus 10%The average yield of hepatocytes was 13 plus or minus 11 million hepatocytes per gram of liver by means of immunofluorescence. Isolated hepatocytes that stained positively for albumin in green had a purity of 94 plus or minus 1%to determine hepatocyte purity.
Immunofluorescent staining was conducted four times with five replicates per trial. Phase contrast microscopy shows the morphological characteristics of hepatocytes such as their large cell size and polygonal shape. After watching this video, you should have a good understanding of how to isolate human hepatocytes using a two step collagenase perfusion procedure.
