Tumor recurrence is a very prominent problem of delivery resection. In this study, we are trying to understand more about the tumor growth in a regenerative environment. Tumor organoids have been shown to recapitulate the patient tumor histologically and genetically, although in vivo models are scarce and lack stability.
We've successfully in graft the patient derived organoid into the liver of the mouse. We've shown this for two example cases. The histology immunohistochemical stainings show that the organoids and the xenograft resemble the original patient tumor.
The development of organoids from the original tumor resembles a more accurate model of patient tumor compared to other possibilities like 2D cell cultures. Embedding this model into the complex environment of liver regeneration will help to understand more about the existing interactions between tumor cells and regenerative environment. We hope that we gain knowledge to our field regarding the understanding of the recurrence of HCC of the liver resection.
Therefore, with this model, we want to create the basis for future research projects. To begin, use a scalpel and forceps to cut the harvested liver cancer tissue into small fragments of one to two square millimeters. Transfer the tissue pieces into a 15 milliliter tube containing five milliliters of dissociation buffer.
Place the tube on a rotation device at 37 degrees Celsius. After incubating, pass the cell mixture over a 100 micrometer cell strainer placed on top of a 50 milliliter tube. Use a plunger of a five milliliter syringe to smash the tissue fragments through the filter.
Now add five milliliters of supplemented advanced DMEM over the filter to wash it. Transfer the filtrate into a 15 milliliter conical tube. After aspirating the supernatant, resuspend the pellet in five milliliters of red blood cell lysis buffer.
Centrifuge the suspension again for five minutes at 300g. Then resuspend the pellet in one milliliter of PBS before counting. Prior to cell plating, remove the supernatant from the centrifuge cell suspension.
Resuspend with about one milliliter of basement membrane matrix to the tube and place on ice. Pipette 20 microliters of cell suspension into the wells of a prewarm six-well plate to create drops. After waiting for two to three minutes, place the plate in an inverted position in a flow cabinet for five minutes.
Then transfer the plate to an incubator to solidify. Finally, add two milliliters of organoid culture medium into each well. The patient-derived organoid line presented morphological features of the original patient tumor.
To begin, place an anesthetized mouse in a supine position with the limbs taped down. Use forceps and microsurgical scissors to make an incision on the depleted surgical site along the linear alba from the lower abdomen to the xiphoid cartilage. Insert the retractors into the incision, adjusting the direction of traction to gain optimal exposure of the upper abdomen.
Place a clean napkin made wet with saline right next to the incision site. Now, exteriorize the small intestine and the cecum onto the wet napkin. Expose the right superior lobe until an injection is possible.
Draw the organoid mixture into a rinse marked Hamilton syringe. Steadily inject the suspension into the mouse until the mark without moving the needle. Apply pressure with a cotton swab onto the injection site.
Then check the abdomen for any lobe rupture or backflow. Rinse the abdomen with 0.5 milliliters of sterile saline. Place a running suture with a resorbable suture to close the abdomen.
The tumor developed in the mouse resembled both the patient-derived organoids as well as the original patient tumor. To begin minor liver resectioning, perform laparotomy on an anesthetized mouse in a supine position. With moist cotton swabs, push the medial lobe cranially to create more space in the surgical field.
Flip the left lateral liver lobe cranially to expose the caudate lobe and ligament. Cut the ligament completely. Then insert a ligature below the lateral liver lobe.
Now flip the left lateral liver lobe caudally and guide the left free end of the ligature around the left tip of the left lateral liver lobe. Guide the right free end around the other tip. Use a cotton swab to apply traction on the lobe, ensuring that the ligature is positioned centrally.
Then tie a double half knot. Secure this knot with another second half knot tied in the other direction without fully tightening it. When the ligature is positioned properly, hold the cranial free end of the ligature with blunt-tipped forceps and pull the caudal end with another pair of forceps.
Secure the knot with a second knot tied in the opposite direction. Cut close to the ligature without cutting it to resect the liver. After the minor resectioning of the left lateral liver lobe, transect the falciform ligament.
Flip the lobe cranially, then insert a ligature below it and flip it caudally. Now loop the free ens of the ligature around the labial. Once the ligature is placed correctly, slowly tie a double half knot.
Fully tighten a second half knot in the opposite direction, then add a third half knot in the opposite direction. Resect the lobe, then flush the abdomen with sterile saline solution. Place the intestines back in their anatomical place.
To close the abdomen, use forceps to grab the skin and peritoneum. Place a stitch with a resorbable suture on the top end of the incision line. Perform a second stitch on the other side of the incision exiting on the skin side.
Tie the knot tightly leaving the free end without the needle short. Leave the needle end of the stitch long. Finish the running suture with a knot at the bottom end of the incision.
Clean the abdomen of any remaining blood. The relative contribution of the liver lobes is comparable between the different mice of the same strain. The mean weight of the right inferior lobe increased to 0.82%of the total body weight after minor resection.
Major resectioning resulted in an increase of 0.99%of the total body weight. Major resectioning of the right superior lobe resulted in a pronounced increase in its weight relative to the right inferior lobe.
