Creation and maintenance of a living biobank how we do it.Introduction. Traditional biobanks commonly contain non-viable tissue and blood samples. Despite reflecting adequately the diversity of cancer patient populations and allowing genetic and histological analyzes, they are unsuitable for preclinical assays evaluating therapeutic strategies.
A biobank integrating also patient-derived models overcomes these limitations. Thus enabling functional testing for precision medicine. Sample acquisition.
For biobanking of colorectal and pancreatic cancer, elect cases with proven diagnosis as well as sufficient tumor size, informed concent of the patient is mandatory. Prior to the start of surgery, draw 20ml of blood using a heparinized syringe and additional 7.5ml with a serum monovette. Serum processing and PBL isolation.
After centrifugation at 1, 128 RCF for 15 minutes at four degrees the serum is aliquoted in a pre-labeled cryotube and directly submerged in liquid nitrogen. The heparinized blood is transferred to a polypropylene tube and diluted with 15ml PBS. Use a serological pipette to slowly underlay the mixture with 15ml Pancoll.
After density gradient centrifugation, the opaque layer containing the mononuclear cells is taken up with a serological pipette and transferred into a new PP tube. After washing with PBS and pelleting the mononuclear cells by centrifugation, discard the supernatant and resuspend the pellet in freezer medium and dispense into pre-labeled cryotubes. Transfer the tubes into a cooling container suitable for slow freezing with one degree per minute and store temporary at 80 degrees.
Tissue processing. As soon as the tissue specimen is resected by the surgeon, put it into a suitable container. Avoid under all circumstances that the tissue is covered in formalin.
Transport as fast as possible to the pathology for excision of tumor material not relevant for pathological evaluation of the resection margins. The tumor piece should be handled as aseptic as possible. Moreover, obtain a specimen of healthy tissue and place both in separate tubes prefilled with tissue storage solution on ice.
Return immediately to the lab to start tissue processing under sterile conditions. The tissue piece is placed on a sterile plastic dish filled with tissue storage solution to avoid desiccation. First of all, excise one or more pinhead-sized pieces for snap freezing depending on the size of the tissue specimen obtained.
Place the native tissue into pre-labeled cryotubes and submerge immediately in liquid nitrogen. Cut the remaining tissue into cubes of three by three by three cubic millimeters. Take into account that necrotic tissue must be dissected completely but should not be discarded.
Arrange the cubes in quadruples to determine the number of aliquots for vital storage. Label an adequate number of cryotubes and pre-fill them with 1.5ml freezer medium each. Since the DMSO in the freezer medium is cytotoxic, performed the next steps quickly and without interruption.
Use a scalpel blades to scoop up four tissue pieces per tube. Make sure that all pieces are submerged completely in freezer medium ideally at the bottom of the tube and rapidly freeze tubes using a freezing container. Proceed in the same way with the healthy specimen.
For long-term storage, store all aliquotes in a liquid nitrogen tank. Cell culture. Grind the remains of the tumor tissue processing, including the necrotic portions with the scalpel blades as small as possible.
Place a cell strainer at the top of a steril PP tube and aspirate the suspension with a serological pipette to pass through the cell strainer. Use the plunger of a single use syringe to press the tissue remains through the cell strainer to generate a single cell suspension. Rinse with PBS and repeat these steps until there is no material left.
Remove the cell strainer and centrifuge the suspension at 180 RCF for seven minutes. In the meantime, prepare a collagen pre-coated six-well plate with different media compositions to increase the probability of tumor outgrowth. Discard the supernatant and resuspend the pellet in PBS and dispense 500 microliters per well.
Afterwards, place the plate in a standard incubator. Generation of patient-derived xenograft. For the generation of patient-derived xenografts in immunocompromised mice, animals should be kept in a specific pathogen-free environment.
Wear personal protective equipment comprising of scrubs, apron, face mask, hair cover and gloves. After arranging your workspace, take the desire tumor specimen from the liquid nitrogen container. Fill a fresh PP tube with 35ml PBS then wash the thawing process carefully and tilt the cryo tube up and down.
As soon as the content becomes slushy, poured into the PBS and rinse the tumor pieces. Discard the majority of the PBS and empty the cubes into the lid. Place a sterile plastic dish on an ice pack and add a droplet of 100 microliter Matrigel.
Use forceps to place the tumor pieces in the Matrigel and incubate the tumor for 10 minutes. In the meantime, anesthetize two mice. Check the depth of anesthesia by pinching the foot of the animal.
Any movement indicates insufficient anesthesia and requires either some waiting or additional dosing. Apply eye ointment, pinch the mouse by the neck and inject a microchip subcutaneously. Conduct the following steps in parallel.
Disinfect the flanks of the mice and make a small skin incision with stripe Metzenbaum scissors and form a subcutaneous pocket by blunt preparation. Use anatomical forceps to insert the tumor pieces. Make sure that the piece is placed at the rear end of the skin pocket.
Aspirate the remaining Matrigel and distribute equally to all four pockets. Await curing of the gel and close the skin with single button sutures without damaging the tumor pieces with the needle. Cut the thread as short as possible above the knot and apply spray dressing to prevent noying of the sutures.
Scan the microchip and add the tumor information to the database for later identification of the animal. Prepare a cage with fresh bedding and nesting material, placed the mice in front of an infrared lamp and monitor the animal until the anesthesia has subsided. Explantation of PDX.
After the PDX tumor has reached the required size, the mouse is euthanized by CO2 asphyxiation and subsequent cervical dislocation. Carefully dissect the skin from the tumor and remove the PDX completely. Representative results.
Place a tumor on a sterile plastic dish and use sterile scalpel blades for further processing. Cut hole with the slices, place them in a histology cassette and submerge in formalin to generate paraffin embedded specimen for histological assessment at a later time. Transfer the rest of the tumor to tissue storage solution and create new vitally preserved and snap frozen specimen for the biobank.
Additionally, use the remains of the PDX tumor analogous to the chapter cell culture to establish secondary tumor cell lines. To generate further PDX, transfer two or more tumor pieces with Matrigel to a new recipient mouse as shown previously.Conclusion. By means of the presented protocol, we so far managed to establish nine pancreatic and more than 100 colorectal patient-derived cancer cell lines, as well as 19 pancreatic and over 150 colorectal PDX models.
