The overall goal of this procedure is to demonstrate a reliable protocol to generate viable hepatocytes in large numbers. This is accomplished by first perfusing collagenase solution through the rat portal vein. Then the dissociated cells are isolated and purified using mesh nylon filter, and the dead cells are separated from viable cells using per call solution.
The final step is to culture the hepatocytes in an incubator. Ultimately, this protocol generates up to 100 million cells per preparation with 90%cell viability. Visual demonstration of this method is critical because this procedure requires surgical and ster cultural steps that can be better understood by video than by text demonstrating the procedure will belying a technician from my laboratory, The various required perfusion, buffers, and Williams complete medium should all be prepared one day in advance using sterile technique prior to their use.
The buffer should be warmed for 30 minutes in a 42 degrees Celsius water bath. This corresponds to an optimal outlet temperature at the cannula of 37 degrees Celsius. The perfusion system consists of the pump autoclavable, cy elastic tubing and a water bath preset the flow rate of the peristaltic perfusion pump to 10 milliliters per minute.
Next, anesthetize an adult rat with an intraperitoneal injection of ketamine and xylazine. When the rat no longer responds to a pain pinch, shave the abdominal hair and clean the surgical area with Betadine and ethanol. Enter through a midline incision and expose the hepatic portal vein.
Carefully move the viscera to the right outside of the abdominal cavity and insert an 18 gauge angio cath into the hepatic portal vein. Connect the PERFUSE eight tubing to the needle and initiate the infusion in C two at a low flow rate with prewarm perfusion buffer one. If performed properly, the liver should instantly begin to blanch.
Once a successful cannulation is confirmed, cut open the inferior vena CVA to allow flx. Another test for successful cannulation is to apply light pressure on the IVC. Using a swab, all of the liver's lobes should quickly begin to swell.
Next, increase the buffer flow to 25 milliliters per minute. The liver should become pale in color. Once the liver goes pale, switch the perfusion solution to buffer two with collagenase two for six minutes of digestion, five to 10 times during the digestion, apply pressure to the IVC with a swab for five seconds.
The liver will swell leading to enhanced hepatic cell dissociation. In turn, reducing total digestion time and increasing final yield. After collagenase perfusion, the liver should begin to look mushy.
Dissect the liver free. Place it in a pre chilled sterile beaker with 40 milliliters of medium, and transfer it to a tissue cell culture hood for cell isolation at the cell culture hood. Transfer the liver to a Petri dish with 20 milliliters of Williams'complete medium and use a cell scraper to gently disperse the liver cells to remove connective tissues and undigested tissue fragments.
Filter the cell dispersion through a 100 micron pore size cell strainer into a 50 milliliter conical tube. Next, suspend the cells in 40 milliliters of medium centrifuge. The suspension at 50 times G for three minutes at four degrees Celsius.
Aspirate the sup, natin and gently resuspend cells. In 40 milliliters of cold medium to wash the cells, centrifuge the cells, again, aspirate the snat and gently resuspend cells. With 25 milliliters of medium pellet the cells and aspirate the dead cells From the top of the perol gradient, the viable cells remain at the bottom.
Suspend the viable cell pellet in 30 milliliters of warm medium and repeat the centrifugation. Resuspend the pellet in 20 milliliters of medium. Count the cells within the suspension using a hemo cytometer and determine the cell's viability.
Using trian blue staining, dilute the hepatocytes in Warm Williams, complete medium to the preferred concentration, and deposit them on uncoated culture plates at the desired volume. A typical prep has greater than 85%viable cells or a plated cell density of 60 to 70%confluence. This allows for cell cell contact while maintaining sufficient space for the hepatocytes to grow to their full size and yield a final confluence of 90 to 95%to grow an even monolayer of hepatocytes without cell aggregates.
In the central area of the well, allow the plates to remain at room temperature for 30 minutes before Incubation. Sometimes when you see the cells aggregating in the central area of the whale, this could be overcome by leaving the plate in cultural hood for 30 minutes before prac into the incubator. This ensures that cells will grow in their even mono layer.
After four hours of incubation, the medium can be replaced with serum free medium to maintain cell morphology with no adverse effects from hormones allow cells to recover and grow at least overnight prior to experimentation. Critical enzymes like P four 50 will still be functional within 24 hours, but not much longer. Replace the growth medium every other day if needed.
After four hours in culture, the hepatocytes aggregate and cluster most isolated cells flatten and spread in typical monolayer growth. By 24 hours, the cells spread in typical monolayer growth. Their edges are defined, their junctions are linear, their surfaces are fairly smooth, and lipid droplets are visible.
The cells have one to three nucleoli, which are round located at the center of the cells, and the nuclei display consistent size among cells. After watching this video, you should have a good understanding of how to generate viable hepato in large numbers in red.
