Patient-derived Xenograft Models and Primary Cell Lines are becoming an integral part of drug development and the initiation and progression of tumor biology research. Patient-derived Xenograft Models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. His models are for more accurate representations of human cancer then traditional cancer cell lines and have the potential to improve the preclinical evaluation of novel anticancer therapies.
In addition, these models may be useful in comparing molecular characteristics or tumor signatures between different subgroups of cancer patients. NSG mice are recommended as immunodeficient mouse model. Due to the severe immunodeficiency of the mice, sterility must be maintained in all experiments.
Under sterile conditions, use forceps and scissors to carefully dissect the tumor tissues and trim them into several small pieces. After anesthetizing the mouse, use sterile scissors to make a one centimeter incision on both dorsal flanks. Use sterile forceps to disrupt the subcutaneous tissue.
Then, clip a piece of the tumor tissue, and place it into the deep site. Close the implant area by subcutaneous suture with surgical suture needles. Sterilize the wound with iodine.
After this, gently place the mice in an empty cage while maintaining sternal recumbency. Pay close attention to the mice as they should wake up and begin walking after approximately three to four minutes. Place mice that underwent surgery in a new cage separated from those not subjected to surgery.
Assess the tumor size by palpation of the implantation site and measure them with a Vernier caliper twice a week. After euthanizing the mice, use sterile forceps and scissors to slowly isolate the tumor from the mice. Wash the tumor tissues in DPBS in a 10 centimeter dish.
You forceps and scissors to dissect and remove necrotic areas, fatty tissue, blood clots and connective tissue. Next, use a mold to cut the tumor tissues to a maximum thickness of one millimeter. Wash the tumor slices with DPBS in a 10 centimeter dish.
To begin the vitrification process, use forceps to transfer the slices into tube V1 and incubate at four degrees Celsius for four minutes. Roll and invert the tube briefly and incubate at four degrees Celsius for another four minutes. Next, pour the V1 solution and slices into a 10 centimeter dish and use forceps to transfer the slices into tube V2.Incubate at four degrees Celsius for four minutes.
Roll and invert the tube briefly and incubate it at four degrees Celsius for another four minutes. After this, pour the V2 solution and the slices into a 10 centimeter dish. Transfer the slices into tube V3 and incubate at four degrees Celsius for five minutes.
Roll and invert the tube briefly and incubate at four degrees Celsius for another five minutes. Make sure that all of the slices sink to the bottom of the tube. If several slices remain floating, roll and invert the tube briefly and incubate at four degrees Celsius until all of the slices sink completely.
Then, pour the V3 solution and slices into a 10 centimeter dish. Cut the tissue holders to the proper length and place them on sterile gauze. Transfer the slices onto the holders.
Wrap the holders with gauze. Using forceps, place the holders in liquid nitrogen and let them incubate for five minutes. After this, label the cryogenic vials with the tissue information.
Transfer the holders with tissue slices into the vials, which will be stored in liquid nitrogen. First, resect gastric cancer samples from resected specimens or harvested PDX tissues. Place the tissues on ice and transfer them to a 10 centimeter sterile culture dish.
Use forceps and scissors to remove necrotic areas, fatty tissue, blood clots and connective tissue. Wash the tumor tissues once with DPBS containing penicillin and streptomycin in a 10 centimeter dish. Next, cut the tumor into pieces on the lid of the dish.
The maximum thickness of each piece should be one millimeter. Transfer the pieces into a 50 milliliter centrifuge tube containing seven milliliters of a Type 1 collagenase and trypsin solution. Vortex the mixture briefly.
Incubate in a water bath at 37 degrees Celsius for 30 to 40 minutes, making sure to vortex the mixture every five minutes. After this, add an equal volume of RPMI-1640 medium supplemented with 10%FBS and vortex the mixture thoroughly. Transfer this mixture into a new 50 milliliter centrifuge tube by slow filtration through a 40 microliter filter.
Centrifuge the filtrates at a speed between 113 and 163 times g for five to seven minutes at room temperature. Carefully remove this supernatant. Wash the pellet with five milliliters of PBS and carefully remove the supernatant.
If the pellet is red, it contains erythrocytes. Gently resuspend the pellet with 500 microliters of red blood cell lysis buffer and incubate at room temperature for five minutes. Then, add five milliliters of PBS.
Centrifuge at a speed between 113 and 163 times g for five to seven minutes at room temperature. Carefully remove the supernatant and resuspend the pellet with culture medium and transfer the mixture into a sterile 10 centimeter dish. Incubate the culture at 37 degrees Celsius with 5%carbon dioxide, making sure to replace the medium with serum-containing medium every two to three days.
In this study, gastric cancer PDX models and primary cell lines are established. First-generation tumors are seen to grow more slowly than those in later generations, taking three weeks or longer to reach the appropriate size. The success rate of first-generation subcutaneous tumor formation is over 80%The identity of the cancer cells from PDX models is confirmed from PDX models by H&E staining.
The success rate of tumor formation from cryopreserved tumor tissue is seen to be approximately 95%Tumor cells can be easily recognized by differences in morphology. The primary cells are authenticated independently by two pathologists under a microscope. For further confirmation, H&E staining is used to observe cancer cell morphology after fixation.
The rate of successful isolation of primary cell lines is approximately 40%These models have been shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation by a marker identification, biological studies, and personalized medicine strategies.
