We kindly acknowledge Jenny Burmeister, our graphical assistant, for the recording and editing of the video. Furthermore, we thank our colleagues of the surgical and pathological department for the longstanding collaboration. We would also like to thank Marcus M&#252;ller, production manager of the IT and Media Centre, University of Rostock, for supplying the audio recording equipment and refining the sound quality.
FUNDING: The German Cancer Aid Foundation (DKH e.V.), grant number 108446, and grant number TBI-V-1-241-VBW-084 from the state Mecklenburg-Vorpommern partly funded this research.

The following study has been approved by the institutional review board of the University Medical Center Rostock (II HV 43/2004, A 45/2007, A 2018-0054, A 2019-0187 and A 2019-0222). Furthermore, all veterinary relevant procedures have been approved by the Landesamt f&#252;r Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern under the registration numbers LALLF M-V/TSD/ 7221.3-2-020/17 and 7221.3-1-007/19.
1. Experimental Prerequisites
<ol>
	<li>Meet several important framework conditions to establish and maintain a biobank.
	<ol>
		<li>Use a clinic with a surgical department and sufficient number of oncological resections together with a well-equipped lab and sufficient academic staff. A good infrastructure and a firm liaison with a cooperating pathology department are further prerequisites.</li>
		<li>For in vivo research, use an animal facility with housing conditions appropriate to immunodeficient mice.</li>
		<li>Obtain authorization on any research on patient-derived material from a health care ethics committee. Obtain approval on any in vivo research from the competent authority according to local statutory regulations.</li>
	</ol>
	</li>
</ol>
2. Sample collection
<ol>
	<li>The day before surgery
	<ol>
		<li>Evaluate all patients with resectable colorectal or pancreatic cancer and/or corresponding metastases for biobanking eligibility. Avoid including cases with neoadjuvant pretreatment, very small tumors, tumors of uncertain dignity or lesions which have been partly resected endoscopically before.</li>
		<li>Obtain written approval of participation from the patient during the informed consent discussion about the surgical procedure. Inform timely all involved surgeons, the laboratory team as well as the pathologist.</li>
	</ol>
	</li>
	<li>Sample acquisition
	<ol>
		<li>Inform all attendants in the operation room (OR) about the tissue collection for the biobank immediately before the start of the surgical procedure. 
		NOTE: It is crucial that the tissue must not be fixed in formalin. If the tissue is submerged in formalin, it becomes unsuitable for integrated biobanking.</li>
		<li>Draw 40 mL of heparinized blood (2 x 20 mL syringe) as well as a standard 7.5 mL serum tube immediately after anesthetic induction and transfer quickly to the lab for PBL isolation and serum processing (see step 3-4).</li>
		<li>Obtain the resected specimen directly from the operating table, place it into an appropriate container and take it to the pathological department. Write down the time point of detachment from the circulation, resection and arrival at pathology. 
		NOTE: The suitability of the specimen for bio banking should be assessed by the cooperating pathologist who dissects a slice of tumor and non-malignant tissue. Do not excise any parts of the specimen by yourself which may compromise the subsequent pathological report.</li>
		<li>Place both tissue pieces in a separate 15 to 50 mL polypropylene tube with 10 to 30 mL tissue storage solution (or DPBS) on ice. Write down the time of receipt and transfer the specimens immediately to the lab. 
		NOTE: The following protocol steps 3-6 must be conducted in a laminar flow cabinet under strict sterile conditions. Use all liquids at room temperature.</li>
	</ol>
	</li>
</ol>
3. Serum processing
<ol>
	<li>Centrifuge the 7.5 mL serum tube at 1128 x g and 4 &#176;C for 15 min in a pre-cooled centrifuge.</li>
	<li>Aliquot 1 mL serum per tube in pre-labeled cryotubes and freeze in liquid nitrogen.</li>
</ol>
4. Isolation of PBL by density gradient centrifugation
NOTE: Work parallel with each of the two 20 mL syringes.
<ol>
	<li>Fill 20 mL of heparinized blood into a 50 mL polypropylene tube and add 15 mL of DPBS.</li>
	<li>Take 15 mL of Pancoll with a serological pipette, insert the pipette carefully all the way to the bottom of the polypropylene tube and release the Pancoll very slowly to form a layer beneath the blood/DBPS column.</li>
	<li>Centrifuge at 375 x g for 15 min without brake.</li>
	<li>Aspirate and transfer the opaque interphase layer between the mid and top column of both samples into a fresh 50 mL Polypropylene tube and fill up with DPBS to 50 mL.</li>
	<li>Centrifuge at 270 x g for 15 min with brake.</li>
	<li>Aspirate and discard the supernatant, resuspend the cell pellet in 4.5 mL of freezer medium.</li>
	<li>Aliquot 1.5 mL of the suspension per cryotube, close the tubes tightly and place them in a freezing container suitable for slow freezing and store at -80 &#176;C.</li>
</ol>
5. Tissue processing
NOTE: Start with the generation of snap frozen samples of tumor and healthy tissue to maintain the integrity of nucleic acids.
<ol>
	<li>Tumor tissue specimen
	<ol>
		<li>Transfer the tumor specimen with several mL of tissue storage solution from the polypropylene tube to a Petri dish. Rinse with DPBS if necessary. Avoid touching the specimen and use two sterile scalpels to handle the tissue. Avoid desiccation at any time.</li>
		<li>Weigh the tumor specimen on a convenient scale in a separate dish and note the tissue weight.</li>
		<li>Evaluate size, shape and tissue quality of the tumor tissue before cutting. Aim to obtain at least one piece about the size of a pinhead for snap freezing and four cubes of approx. 30 mm3 (edge length 3 x 3x 3 mm) each for vital cryopreservation. Generate as many 30 mm3 -cubes in quadruples as possible and generate one piece for snap freezing per 5 quadruples. Also take into account that necrotic portions must be cut off so that the cubes consist of vital tissue only.</li>
		<li>Generate slices of 3 mm thickness. Cut off necrotic portions, distinguishable as gel-like or liquid mass, and cut the slices into cubes of the two desired sizes. 
		NOTE: Do not discard any tissue at this point.</li>
		<li>Snap freezing
		<ol>
			<li>Label cryotubes accordingly (see step 7.7).</li>
			<li>Place one small tissue piece per pre-labeled cryotube. Submerge the samples immediately into liquid nitrogen for several minutes and store at -80 &#176;C subsequently.</li>
		</ol>
		</li>
		<li>Cryopreservation of vital tissue
		<ol>
			<li>Label cryotubes accordingly (see step 7.7) and fill each with 1.5 mL of freezer medium. Place the freezing container beside the bench. 
			NOTE: Follow the next steps as quickly as possible. Since the DMSO in the freezer medium has cytotoxic properties, the time of the tissue being submerged in freezer medium without proper cooling should not exceed 2 minutes.</li>
			<li>Arrange the 30 mm3 cubes in quadruples. Shove necrotic tissue and other remains to the edge of the dish, but do not discard them.</li>
			<li>Scoop the cubes with the scalpel blade and transfer 4 cubes per cryotube. Make sure that the tumor pieces are entirely submerged in freezer medium. Close the tubes tightly and place them in a freezing container suitable for slow freezing and store in a -80 &#176;C freezer.</li>
			<li>Transfer cryotubes into a suited storage system for long term storage at -140 &#176;C or lower. Documentation in the laboratory inventory management system is mandatory.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Healthy tissue specimen: Repeat steps 5.1.1. to 5.1.6.4 for the healthy tissue specimen.</li>
</ol>
6. Primary cell culture
<ol>
	<li>Disintegrate the remains of the tumor tissue, including the necrotic scrap, in the Petri dish with the scalpels to pieces as small as possible.</li>
	<li>Place a sterile cell strainer (100 &#181;m pore size) on top of a 50 mL polypropylene tube.</li>
	<li>Use a serological pipette to add 5-10 mL of DPBS to the Petri dish, float the tissue remains and pipette up and down to generate a suspension.</li>
	<li>Transfer the suspension with the pipette to the cell strainer.</li>
	<li>Repeat Steps 6.3-6.4 until all tissue remains are resolved from the Petri dish.</li>
	<li>Use the plunger of a 20 mL one-way syringe to squeeze the cell and tissue suspension through the cell strainer.</li>
	<li>Rinse with 5-10 mL of fresh DPBS, discard the cell strainer and close the tube properly.</li>
	<li>Centrifuge the suspension at 180 x g for 5-10 minutes.</li>
	<li>Prepare a collagen-I precoated 6 well plate with 1.5 mL of medium per well.</li>
	<li>Aspirate and discard the supernatant. Resuspend the pellet in 3 mL of DPBS or medium and add 500 &#181;L of the suspension to each well. Place the plate into the incubator (100% humidity, 5% CO2, 37 &#176;C)</li>
	<li>Monitor the plate daily for cell growth and contamination. 
	NOTE: Further cell culturing up to the point of establishment of a permanent cell line is not described here.</li>
</ol>
7. PDX Generation
<ol>
	<li>Conduct in vivo experiments only by appropriately qualified persons meeting the requirements of the competent authority of your jurisdiction.</li>
	<li>House immunodeficient mice under specific-pathogen-free (SPF) conditions satisfying the demands of the used mouse strain. The hygienic measures include individually ventilated cages, autoclaved food, water and nesting material as well as a safety air lock and the wearing of personal, protective equipment.</li>
	<li>Autoclave all instruments beforehand and use only one set of instruments for each tumor case to avoid cross-contamination. Handle the tumor tissue as aseptic as possible. All plastic items named below should be sterile, single-use and discarded after each surgery. 
	NOTE: Determined by the method of freezing four tumor tissue pieces per cryotube, the PDX generation requires always two mice per sample, ideally resulting in four PDX tumors.</li>
	<li>Choose the desired primary tumor for engraftment via the laboratory inventory management system and transfer the sample (vitally preserved tumor tissue) from the main storage tank to a portable liquid nitrogen container (Intermediate storage at -80 &#176;C on dry ice is also convenient).</li>
	<li>Put on personal protective equipment before entering the SPF-section (scrubs, clogs, apron, hair cover, surgical mask and overshoes), disinfect your hands and all equipment.</li>
	<li>Matrigel soaking
	<ol>
		<li>Remove the cryotube form the liquid nitrogen container and await thawing of the specimen.</li>
		<li>Label a 50 mL polypropylene tube and fill with 35 mL of DPBS.</li>
		<li>Tilt the cryotube up and down and transfer the content immediately to the polypropylene tube as soon as the tissue-medium-slush can be shifted. Gently rinse the tumor tissue pieces, discard the main volume from the tube in a separate vessel, close the lid and put the tube up-side-down, so that the four tissue pieces gather in the lid.</li>
		<li>Put a Petri dish on the cooling accumulator and place 100 &#181;L of Matrigel as a single droplet into the middle. Use anatomical forceps to transfer the tumor pieces into the Matrigel. Make sure that each piece is covered completely with Matrigel. Incubate for 10 minutes at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Mouse anesthesia (2 mice per sample, work in parallel)
	<ol>
		<li>Prepare a 3:1- stock of a ketamine (100 mg/mL) and xylazine (20 mg/mL) anesthetic solution. The recommended dose is 90/6 mg/kg body weight.</li>
		<li>Weigh the mouse and draw up the necessary anesthetic solution into a single use insulin syringe.</li>
		<li>Place the mouse on the grid of the cage, pull its tail gently with one hand to induce a forward movement and simultaneously crab the neck with a pinch grip of the other hand. Lift the mouse of the grid and turn the holding hand, so that animal&#39;s back rests on your palm. Immobilize one of the hind legs with your pinky and inject the narcotics intraperitoneally. Put the mouse back to its cage and await narcotic induction.</li>
		<li>Place the anesthetized mouse on the heating plate and cover the eyes with ointment to avoid corneal harm. Asses the depth of anesthesia by gently pinching the back foot of the mouse with surgical forceps. 
		NOTE: Absence of movement indicates deep narcosis. Any kind of movement either requires more time for reaching the desired narcotic depth or an additional dose of anesthetics.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Form a skin fold by pinching the neck of the mouse and inject the microchip subcutaneously with the applicator (See step 9 for programming details)</li>
		<li>Shave the flanks of the mouse if necessary (NMRInu/nu mice do not require shaving), apply povidone-iodine with a cotton swab and use surgical drape to create a sterile field.</li>
		<li>Lift the skin of the flank with surgical forceps, make a small incision of circa 4 mm and form a small subcutaneous pocket by blunt preparation with scissors.</li>
		<li>Put one tumor piece into each pocket and place it at the rear end.</li>
		<li>Clip the end of a 100 &#181;L pipette tip and aspirate the remaining Matrigel from the Petri dish and apply it equally into each skin pocket.</li>
		<li>Close the wounds with simple interrupted sutures and apply spray dressing.</li>
	</ol>
	</li>
	<li>Scan the microchip and check validity of mouse- and tumor-ID.</li>
	<li>Prepare a new cage with fresh bedding and nesting material, as well as a gnawing stick. Fold a &#34;cushion&#34; out of paper towels and lay down the mouse with elevated head under an infrared heat lamp.</li>
	<li>Mix 0.25 mL of trimethoprim/sulfamethoxazole (400 mg/80 mg) with 100 mL of drinking water and administer via the drinking bottle. Consider that one mouse consumes approximately 150 mL per kg body weight daily. 
	NOTE: Since the subcutaneous PDX-model is not associated with postoperative pain, neither during the wound healing process, nor during tumor outgrowth, postoperative analgesia is not required. Please note that the animal welfare guidelines of your institution/authority may differ.</li>
	<li>Monitoring of experimental animals
	<ol>
		<li>Monitor the mice daily for signs of distress. This can be delegated to qualified animal caretakers.</li>
		<li>Keep up the postoperative antibiotic treatment with the aforementioned dosage for 4 weeks. Replace the antibiotic mixture twice per week.</li>
		<li>Measure the tumor size at least once per week, ideally daily, with a caliper (tumor volume = 0.52 x length x width x height [mm3]) and record in the database.</li>
	</ol>
	</li>
</ol>
8. PDX harvesting and processing
<ol>
	<li>Harvest and process the PDX tumor, when: 
	The tumor size reaches the target volume of 1.500 mm3. 
	The tumor bearing animal shows signs of distress and/or disease and treatment is futile. 
	The tumor becomes ulcerated or penetrates the skin of the mouse.</li>
	<li>Read out the microchip to identify the correct PDX.</li>
	<li>Euthanize the mouse by a legal method (depending on national guidelines) as for example CO2-asphyxation or ketamine/xylazine injection followed by cervical dislocation.</li>
	<li>Lift the skin with surgical forceps at the flanks and incise with Metzenbaum scissors a few millimeters distant from the tumor.</li>
	<li>Detach the skin above the tumor by blunt preparation, then carefully grasp the tumor with anatomical forceps and detach the tumor from the superficial fascia of the body.</li>
	<li>Rinse the tumor with DPBS, put it into a Petri dish and remove adjacent connective tissue.</li>
	<li>At this point, perform one of the following:
	<ol>
		<li>Cut 30 mm3 cubes and create new PDX (Proceed with protocol at point 7.7.4).</li>
		<li>Cut the tumor into slices, which are then transferred to histology cassettes and preserved in 4% formaldehyde for later paraffin embedding.</li>
		<li>Preserve the tumor in a tube with tissue storage solution to add it to the biobank (Proceed with protocol at step 3.) and/or create PDX-derived cell lines (Proceed with protocol at step 4.)</li>
	</ol>
	</li>
</ol>
9. Biobank and data management
<ol>
	<li>Assign an internal ID to each tumor case according to Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Laboratory location/name</td>
			<td>cancer entity</td>
			<td>consecutive case number</td>
			<td>specification</td>
			<td>consecutive number</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td>C=colorectal</td>
			<td rowspan="2"></td>
			<td>_Met=Metastasis</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>P=pancreatic</td>
			<td>_Tu=Tumor</td>
		</tr>
		<tr>
			<td colspan="5">Example: HROC389_Met2 = Rostock, colorectal cancer, case 389, second metastasis</td>
		</tr>
	</tbody>
</table>
Table 1: Definition of the sample ID.
<ol start="2">
	<li>Store the patient consent in electronic and paper form together with the tumor-ID.</li>
	<li>Gather as much clinical data as possible and store them anonymized and separately.</li>
	<li>Use a data management software (e.g., Freezerworks) or other and create an interface with a label printing software to generate temperature-resistant, self-sticking bar code labels.</li>
	<li>Add a new sample by opening the data management software, define the specimen type and record the following information: tumor ID, tissue type, freezing method, date, responsible employee, passage number, mouse ID and mouse strain.</li>
	<li>Assign the samples to specific positions in the storage tank.</li>
	<li>Tracing and monitoring of PDX (Applies to step 7-8)
	<ol>
		<li>Use a MS Access data base (or a similar system) on a portable, Bluetooth-enabled device (laptop or tablet) to record tumor ID, date of implantation, date of euthanasia, mouse age and strain as well as tumor growth over time.</li>
		<li>Connect the microchip reader to the device and read out the microchip prior to implantation.</li>
		<li>Assign a specific ID to each mouse; we use the following scheme: (see Table 2 below)</li>
		<li>After implantation, record the ID together with the mouse characteristics in the data base.</li>
		<li>Re-read the microchip and check, if the specifications of the microchip, the data base and the cryotube label are consistent.</li>
		<li>Create a label for each mouse cage accordingly. 
		NOTE: To create a physical back up, stick the cryotube labels with the corresponding microchip labels into a booklet and note date and mouse strain.</li>
		<li>To monitor the tumor growth of the individual PDX, scan the microchip of the mouse with the reader connected to the data base device for identification and record the tumor size measured by caliper each week.</li>
		<li>Plan the ideal time point of PDX harvesting by analyzing the growth curve of the tumor.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tumor-ID</td>
			<td>Prior storage in N2 (=f)</td>
			<td>Passage (=T) number</td>
			<td>consecutive mouse (=M) number</td>
		</tr>
		<tr>
			<td colspan="4">Example: HROP12 fT0 M1 = Rostock, pancreatic cancer, case 12, generated from frozen primary tissue, first passage, mouse 1.</td>
		</tr>
	</tbody>
</table>
Table 2: Definition of the PDX ID.

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+murine+tumors+in+the+development+of+patient-derived+xenografts:+a+potential+pitfall.">Spontaneous murine tumors in the development of patient-derived xenografts: a potential pitfall.</a> Oncotarget. 10 (39), 3924-3930 (2019).</li><li>Prall, F., Maletzki, C., H&#252;hns, M., Krohn, M., Linnebacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+carcinoma+tumour+budding+and+podia+formation+in+the+xenograft+microenvironment.">Colorectal carcinoma tumour budding and podia formation in the xenograft microenvironment.</a> PloS one. 12 (10), 0186271 (2017).</li><li>Guenot, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+tumour+genetic+alterations+and+intra-tumoral+heterogeneity+are+maintained+in+xenografts+of+human+colon+cancers+showing+chromosome+instability.">Primary tumour genetic alterations and intra-tumoral heterogeneity are maintained in xenografts of human colon cancers showing chromosome instability.</a> The Journal of pathology. 208 (5), 643-652 (2006).</li><li>Mattie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+patient-derived+human+pancreatic+tumor+xenograft+models+for+preclinical+and+translational+development+of+cancer+therapeutics.">Molecular characterization of patient-derived human pancreatic tumor xenograft models for preclinical and translational development of cancer therapeutics.</a> Neoplasia. 15 (10), 1138-1150 (2013).</li><li>Cho, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer+patient-derived+xenografted+tumors+maintain+characteristic+features+of+the+original+tumors.">Colorectal cancer patient-derived xenografted tumors maintain characteristic features of the original tumors.</a> The Journal of surgical research. 187 (2), 502-509 (2014).</li><li>Maletzki, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+and+Drug+Response+of+Freshly+Established+Patient-Derived+Tumor+Models+with+CpG+Island+Methylator+Phenotype.">Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.</a> PloS one. 10 (11), 0143194 (2015).</li><li>Katsiampoura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+Patient-Derived+Xenografts+in+Colorectal+Cancer.">Modeling of Patient-Derived Xenografts in Colorectal Cancer.</a> Molecular cancer therapeutics. 16 (7), 1435-1442 (2017).</li><li>Wege, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+tumor+mice--a+new+model+to+study+and+manipulate+the+immune+response+in+advanced+cancer+therapy.">Humanized tumor mice--a new model to study and manipulate the immune response in advanced cancer therapy.</a> International journal of cancer. 129 (9), 2194-2206 (2011).</li><li>Herndler-Brandstetter, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mouse+model+supports+development,+function,+and+tissue+residency+of+human+natural+killer+cells.">Humanized mouse model supports development, function, and tissue residency of human natural killer cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (45), 9626-9634 (2017).</li><li>Capasso, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+immune+responses+to+anti-PD-1+mono+and+combination+immunotherapy+in+hematopoietic+humanized+mice+implanted+with+tumor+xenografts.">Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts.</a> Journal for immunotherapy of cancer. 7 (1), 37 (2019).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Vlachogiannis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+organoids+model+treatment+response+of+metastatic+gastrointestinal+cancers.">Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.</a> Science. 359 (6378), 920-926 (2018).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature reviews. Cancer. 18 (7), 407-418 (2018).</li><li>Abe, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+phosphoproteomic+analysis+for+phosphosignaling+and+active-kinome+profiling+in+Matrigel-embedded+spheroids+and+patient-derived+organoids.">Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.</a> Scientific reports. 8 (1), 11401 (2018).</li><li>Neal, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+Modeling+of+the+Tumor+Immune+Microenvironment.">Organoid Modeling of the Tumor Immune Microenvironment.</a> Cell. 175 (7), 1972-1988 (2018).</li></ol>

The generation of a living biobank presupposes, apart from complying with the legal regulations of privacy, medical law and animal welfare, a good infrastructure and a well-coordinated team. It has proven advantageous to directly involve a part of the surgical staff in the research procedures, since they can very well assess the suitability of the individual patient for tissue donation. Moreover, patients tend to consent with biobanking more frequently, when their written approval is obtained within the course of the surgical informed consent discussion. To save time and resources, cases that will presumably yield insufficient amounts of tumor tissue should not be selected for biobanking. When it comes to specimen acquisition, the maxim &#34;communication is key&#34; is a simple, but often overlooked truth. It only takes a single uninformed theatre nurse or surgical colleague to ruin the specimen right at the outset by proceeding as usual and adding formaldehyde to the resection specimen. Therefore, it is absolutely crucial that every single member of the involved staff gets acquainted with the SOP for biobanking. Surgeons should be noticed the day before and right at the start of the procedure about scheduled tissue collection. Furthermore, cases selected for biobanking, should be highlighted in the electronic OR plan. Tissue harvesting from the surgical specimen should be performed by a pathologist. First, this will ensure that the tissue harvesting does not interfere with the final pathological report. Second, this increases the probability of receiving tissue with adequate amounts of viable cancer tissue. Especially in pancreatic cancers with a pronounced desmoplastic reaction and frequent necrotic areas, viable parts are hard to identify macroscopically for the untrained eye. As an exception to this rule, tissue blocks from large hepatic or pulmonal metastases, may at times be excised &#34;back-table&#34; by the surgeon, if surgical margins can be defined macroscopically. Rectal cancer resected by total mesorectal excision (TME), might not be suitable for biobanking, since tissue harvesting from the resected specimen prior to paraffin embedding might interfere with the TME quality assessment. Alternatively, tissue for biobanking can be acquired by transanal biopsy of rectal cancer.
The establishment rates for primary cell cultures derived from the original tumor are generally low. PDX-derived, secondary cell cultures can more likely be successfully established.We recommend testing of different media for each case and use of antibiotic supplements for the first passages to reduce contamination to a minimum since the harvested tissue is rarely sterile. After successful propagation, each individual cell line should be confirmed as a cancer cell line by FACS analysis and regularly tested for mycoplasma contamination. To exclude cross-contamination, regular STR analysis is advisable. It should be noted, that the establishment protocol for primary and secondary cell lines is constantly subjected to optimization. Details concerning the composition and success rates of the single media are clearly beyond the scope of this work and will be published separately.
For PDX engraftment, tumor tissue can be either implanted directly after resection or cryopreserved in fetal calve serum with 10% DMSO or similar freezing media for delayed implantation. Implantation immediately upon tumor tissue harvesting puts a strain on logistics and laboratory staff, and xenografting results after cryopreservation are not inferior at all 10. Moreover, incubation of the tissue in Matrigel prior to tumor implantation, significantly increases engraftment rates12. We recommend delayed engraftment following definite pathological finding and immediate disposal of erroneously collected tissue specimens. Since the success rate of primary engraftment increases with immunodeficiency of the recipient mouse, we tend to use NSG mice for the very first PDX passage. After the first successful PDX engraftment, NMRInu/nu mice can and should be used for subsequent passages and tissue expansion. This strain is more robust, cheaper and easier to breed compared to NSG or similar immunodeficient strains, but still shows reasonable engraftment rates. Moreover, its nudeness facilitates implantation and tumor growth monitoring. To increase the engraftment rates in subsequent passages, we recommend direct transfer of freshly harvested PDX tissues to host mice whenever possible, especially for slow growing PDX and cases with a low primary engraftment success rate. Collins and Lang recently reviewed 14 studies of colorectal PDX establishment and reported engraftment rates varying from 14 to 100% with a median PDX establishment rate of 68%, the latter being consistent with our findings13. In line with the literature, we observed lower establishment rates of pancreatic compared to colorectal cancer PDX14. Regardless of the host mouse strain and tumor entity, the outgrowth of human, Epstein-Barr Virus (EBV)-associated B-cell lymphomas and murine lymphomas at the implantation side poses an important pitfall15,16. If unrecognized, such tumors can &#34;contaminate&#34; subsequent passages and thus confound consecutive results. Unusual fast PDX growth and swelling of cervical, axillar and inguinal lymph nodes are strong indicators of murine lymphoma growth, but regular histological examination of PDX is nevertheless advisable. Furthermore, genetic concordance between PDX and the corresponding donor patient should be tested regularly by STR-analysis. Ideally, the biobank should be linked to a clinical database comprising patients characteristics (general information, survival, relapse free survival, therapy, secondary neoplasia etc.). Due to legal regulations of privacy protection and lack of such an anonymized data base, our clinical data set is regularly administrated and updated manually by the cooperating physicians.
While conventional biobanks are limited to observatory research, a living biobank provides the opportunity for in vitro and in vivo interventions. Patient-derived cell lines are an important tool for fundamental research, high-throughput drug screenings and assessment of new pharmaceutical agents4. Corresponding PDX models, however, are of increasing importance, since they closely recapitulate the histology of the original tumor17,18 and show a high genetic stability over several passages19,20. Our PDX biobank has proven itself as an excellent platform for preclinical and fundamental research6,21. Moreover, since large PDX collections adequately reflect the inter-individual heterogeneity of the patient population, the PDX clinical trial (PCT) approach (one animal per model per treatment) has gained importance for drug development since it allows the faithful prediction of clinical response to new drugs and combinatorial regimen8. We also are currently evaluating new experimental drugs in small PCT trials.
Despite these promising results, the median establishment duration of 12.2 month, impedes the clinical applicability of PDX models as &#34;avatar mice&#34; for testing anticancer treatment options, at least for those patients in need of immediate adjuvant or even neoadjuvant treatment22. An additional disadvantage of standard PDX models is the lack of usability for immunotherapy testing due to host mice&#39;s immunodeficiency. To overcome these limitations, several &#34;humanized&#34; mouse strains have been developed. These mice are heavily immunocompromised, but can be reconstituted with various types of human bone marrow-derived cells or CD34+ hematopoietic stem cells subsequent to PDX outgrowth23, allowing the evaluation of lymphocyte-mediated cytotoxicity and of therapy response to immune checkpoint inhibitor treatment24,25.
In recent years, patient-derived organoids (PDO) emerged as important cancer models competing with PDX. Derived from intact tumor pieces and cultured in an extracellular matrix scaffold, these three-dimensional structures closely reflect the histologic and genetic properties of the original tumor. The possibility of long-term expansion and cryopreservation renders PDO an ideal supplement of a living biobank26,27. In addition to a relatively high establishment rate, reliable drug response prediction has been reported for PDO of several tumor entities28. Moreover, PDOs have even been generated from circulating tumor cells and also the simultaneous establishment of organoids from corresponding healthy tissue is possible, allowing assessment of therapy-related toxicity on a patient-individual basis29,30. However, compared to conventional 2D cell cultures, organoid culture is time and resource consuming and artificial extracellular matrix compounds can interfere with certain analytic procedures31. Moreover, cancer organoids are susceptible to overgrowth by faster growing, non-malignant organoids derived from healthy epithelium30. Due to a lack of stroma, blood vessels and immune cells, PDOs are mostly inapplicable for the testing of antiangiogenic immunotherapeutic agents. Yet, new culturing methods allow the modeling of tumor microenvironment in vitro, rendering PDOs a true contender for PDX models32. In the near future, patient-individual tumor models, combined with powerful genetic tools like next-generation sequencing, will hopefully pave the path to true precision medicine and tailored-treatment approaches.

In recent years, the accumulated knowledge of cancers&#39; morphologic, clinical and genetic properties led to the conception of cancer as a heterogeneous, individual disease. Consequently, mutational characterization of neoplasms, besides clinical and pathological features, has gained importance for clinical decision making and many targeted therapies were developed for various molecular alterations. For instance, the efficacy of cetuximab in colorectal cancer treatment can be predicted by the analysis of the KRAS and PIK3CA mutational status1. Precision medicine aims for a tailored approach to provide the highest treatment response in each patient and avoid toxicity of inefficacious therapies2. Biobanks contain tissue, blood and other biological materials of cancer patients, which are linked to the clinical data, and thus are an excellent tool for translational cancer research. Due to the large number of clinical samples, biobanks enable the detection of rare, but potentially druggable mutations, which provides new treatment opportunities for the individual patient3.
To cover as broad as possible an oncologic research spectrum, we did not restrain our activity on sample harvesting alone, but focused on the establishment of patient-derived cancer cell lines and xenografts (PDX). Traditional 2D cell lines remain the corner stone of in vitro research and are the prime choice for large scale drug screenings4,5. Moreover, cell line analysis is often easier, cheaper and more readily available. Additionally, since patient-derived peripheral blood lymphocytes (PBL) are available, also tumor immunology can be studied in vitro6. However, the majority of newly developed drugs with promising preclinical effectivity in cell based in vitro or in vivo experiments, have shown disappointing results in clinical trials7. In contrast, preclinical studies based on PDX in vivo studies have reflected the clinical activity of antineoplastic agents much more faithfully8. Since PDX tissue closely reflects the histological and molecular properties of the donor tumor, PDX models are a good way to propagate the often very limited amounts of viable tumor tissue to maintain the integrity of a biobank and to allow the exchange of samples between research groups and institutions. Moreover, cancer cell lines derived from PDX tissue can be established significantly easier than primary cancer cell lines9. In recent years, our working group has established a comprehensive integrated colorectal and pancreatic cancer biobank by stepwise standardizing and optimizing the work flow for all biological samples in question (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62065/62065fig01.jpg" /> 
Figure 1: Workflow and organization of the biobank <a href="https://www.jove.com/files/ftp_upload/62065/62065fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacillol&#174; AF; 1L</td>
			<td>Bode, Hartmann</td>
			<td>REF 973380</td>
			<td>desinfection</td>
		</tr>
		<tr>
			<td>PP centrifuge tube, 15ml; sterile</td>
			<td>Greiner Bio One</td>
			<td>GBO Cat. No.:188271</td>
			<td>centrifuge tube</td>
		</tr>
		<tr>
			<td>PP centrifuge tube, 50ml, sterile</td>
			<td>Sarstedt</td>
			<td>Order number: 62.547.254</td>
			<td>centrifuge tube</td>
		</tr>
		<tr>
			<td>BD DiscarditTM II Syringe 20ml</td>
			<td>BD</td>
			<td>REF 300296</td>
			<td>blood collection</td>
		</tr>
		<tr>
			<td>Serum 7,5ml Sarstedt Monovette</td>
			<td>Sarstedt</td>
			<td>Item number: 01.1601</td>
			<td>blood collection</td>
		</tr>
		<tr>
			<td>serological Pipette 10ml</td>
			<td>Sarstedt</td>
			<td>REF 86.1254.001</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>Pipetboy ratiolab&#174; accupetta</td>
			<td>Ratiolab</td>
			<td>Item number: RL3200300</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>PIPETBOY acu 2</td>
			<td>Integra Biosciences</td>
			<td>VWR Cat.No: 613-4438</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>DPBS; w/o Ca &#38; Mg</td>
			<td>Pan Biotech</td>
			<td>Cat. No.: P04-36500</td>
			<td>washing</td>
		</tr>
		<tr>
			<td>Pancoll human</td>
			<td>Pan Biotech</td>
			<td>Cat. No.: P04-60500</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr>
			<td>DMEM/F12 (Dulbecco&#8217;s Modified Eagle Medium)</td>
			<td>PAN Biotech</td>
			<td>Cat. No.: P04-41500</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>FBS Good Forte (Filtrated Bovine Serum)</td>
			<td>PAN Biotech</td>
			<td>Cat. No.: P40-47500</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM</td>
			<td>PAN Biotech</td>
			<td>Cat. No.: P04-80100</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>Trypsin / EDTA</td>
			<td>PAN Biotech</td>
			<td>Cat. No.: P10-023100</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl Sulfoxid for cell culture)</td>
			<td>PanReac AppliChem</td>
			<td>VWR Cat.No: A3672.0250</td>
			<td>cell freezing</td>
		</tr>
		<tr>
			<td>Freezer Medium (FCS with 10% DMSO)</td>
			<td>selfmade</td>
			<td>---</td>
			<td>cell freezing</td>
		</tr>
		<tr>
			<td>cryotube- CryoPure 2ml</td>
			<td>Sarstedt</td>
			<td>72380</td>
			<td>cell freezing</td>
		</tr>
		<tr>
			<td>6-Well cell culture plate; steril; with lid</td>
			<td>Greiner bio-one</td>
			<td>Cat.-No.: 657 160</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>Petri dish 92 x 16 mm, PS, without cams</td>
			<td>Sarstedt</td>
			<td>Cat. No.: 82.1472.001</td>
			<td>tissue preparation</td>
		</tr>
		<tr>
			<td>sterile surgical blades</td>
			<td>B.Braun (Aesculap)</td>
			<td>REF BB510</td>
			<td>tissue preparation</td>
		</tr>
		<tr>
			<td>BD DiscarditTM II Syringe 10ml</td>
			<td>BD</td>
			<td>REF 309110</td>
			<td>tissue preparation</td>
		</tr>
		<tr>
			<td>cell strainer; yellow; 100&#181;m</td>
			<td>Falcon</td>
			<td>REF 352360</td>
			<td>tissue preparation</td>
		</tr>
		<tr>
			<td>CoolCell</td>
			<td>biocision</td>
			<td>Item number: 210004</td>
			<td>cooling container with -1&#176;C/min</td>
		</tr>
		<tr>
			<td>Dewar&#160;transport vessel type 27 B, 2 l, 138 mm</td>
			<td>KGW</td>
			<td>Cat. No.: HT39.1</td>
			<td>transport system</td>
		</tr>
		<tr>
			<td>Pipette tip 200&#181;l</td>
			<td>Sarstedt</td>
			<td>REF 70.760.002</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>Filter tip 1000&#181;l</td>
			<td>Sarstedt</td>
			<td>REF 70.762.411</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>Pipette 200&#181;l, yellow</td>
			<td>Eppendorf</td>
			<td>Cat. No.: 3121 000.082</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>Pipette 1000&#181;l, blue</td>
			<td>Eppendorf</td>
			<td>Cat. No.: 3121 000.120</td>
			<td>liquid transfer</td>
		</tr>
		<tr>
			<td>incubator&#160; BB 6220 CU</td>
			<td>Heraeus</td>
			<td>Cat.-No.: 51012839</td>
			<td>cell cultivation</td>
		</tr>
		<tr>
			<td>heating plate PR&#196;ZITHERM</td>
			<td>Harry Gestigkeit GmbH</td>
			<td>---</td>
			<td>heating</td>
		</tr>
		<tr>
			<td>Microscope Zeiss Primo Vert</td>
			<td>Carl Zeiss MicroImaging GmbH</td>
			<td>Serial number. 3842000839</td>
			<td>imaging cell cultures</td>
		</tr>
		<tr>
			<td>Sterile bench Safe flow 1.8 nunc</td>
			<td>nunc GmbH &#38; Co. KG</td>
			<td>---</td>
			<td>sterile working bench</td>
		</tr>
		<tr>
			<td>freezer -80&#176;C</td>
			<td>Kryotec-Kryosafe GmbH</td>
			<td>---</td>
			<td>sample storage</td>
		</tr>
		<tr>
			<td>Electronic balance MP-300</td>
			<td>Chyo</td>
			<td>---</td>
			<td>Scale</td>
		</tr>
		<tr>
			<td>BD Micro-fine, U100 insulin syringe</td>
			<td>BD</td>
			<td>REF 324826</td>
			<td>injection anesthetic</td>
		</tr>
		<tr>
			<td>Rompun 2%; 25ml</td>
			<td>Bayer</td>
			<td>approval number: 6293841.00.00</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Ketamin 100 mg/ml, 25ml</td>
			<td>CP-Pharma GmbH</td>
			<td>approval number: 401650.00.00</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>GES3S Reader</td>
			<td>Datamars</td>
			<td>not available</td>
			<td>RFID reader</td>
		</tr>
		<tr>
			<td>ISO-Transponder FDX-B (1,4x8mm)</td>
			<td>Peddymark</td>
			<td>---</td>
			<td>RFID chip</td>
		</tr>
		<tr>
			<td>Cotrim-ratiopharm&#174;&#160;Ampullen SF 480&#160;mg/5&#160;ml</td>
			<td>Ratiopharm</td>
			<td>PZN-03928197</td>
			<td>antibiotic drinking water</td>
		</tr>
		<tr>
			<td>Heating plate #FM-20 42x28cm</td>
			<td>Dragon</td>
			<td>---</td>
			<td>heating</td>
		</tr>
		<tr>
			<td>Heating lamp</td>
			<td>Electric Petra, Burgau</td>
			<td>---</td>
			<td>heating</td>
		</tr>
		<tr>
			<td>Ointment for the eyes and nose (5% Dexpanthenol) Bepanthen</td>
			<td>Bayer</td>
			<td>PZN-01578675</td>
			<td>Eye protection</td>
		</tr>
		<tr>
			<td>anatomical tweezer</td>
			<td>B.Braun Aesculap</td>
			<td>BD21 OR</td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>surgical tweezer</td>
			<td>B.Braun Aesculap</td>
			<td>BD50 1 R</td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>B.Braun Aesculap</td>
			<td>BC05 6R</td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle&#160;holder</td>
			<td>B.Braun Aesculap</td>
			<td>BH1 1 OR</td>
			<td>surgical instruments</td>
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		<tr>
			<td>Prolene 5-0</td>
			<td>Ethicon</td>
			<td>XN8870.P32</td>
			<td>surgical suture material</td>
		</tr>
		<tr>
			<td>Opsite moisture vapour permable spray dressing</td>
			<td>Smith&#38;Nephew</td>
			<td>REF 66004978, PZN- 02063507</td>
			<td>surgical suture material</td>
		</tr>
		<tr>
			<td>Adhesive aperture drape</td>
			<td>Barrier</td>
			<td>REF 904622</td>
			<td>sterile OP tissue</td>
		</tr>
		<tr>
			<td>gauze swap Gazin&#174;; steril; 10x10 cm</td>
			<td>Lohmann&#38;Rauscher</td>
			<td>REF 18506</td>
			<td>sterile OP tissue</td>
		</tr>
		<tr>
			<td>Raucotupf cotton tipped applicators</td>
			<td>Lohmann&#38;Rauscher</td>
			<td>REF 11969</td>
			<td>applicator</td>
		</tr>
		<tr>
			<td>Corning&#174; Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>Cat.-No.: 354234</td>
			<td>Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>iodine solution Braunol (7,5g povidone iodine)</td>
			<td>B.Braun Melsungen AG</td>
			<td>Item number: 18839</td>
			<td>desinfection</td>
		</tr>
		<tr>
			<td>MACS&#174; Tissue Storage Solution</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>Order No.:130-100-008</td>
			<td>storage solution</td>
		</tr>
		<tr>
			<td>Formafix 4%</td>
			<td>Grimm med. Logistik GmbH</td>
			<td>Item number: F10010G</td>
			<td>fixation solution</td>
		</tr>
		<tr>
			<td>Software FreezerworksBasic</td>
			<td>Dataworks Development, Inc</td>
			<td>---</td>
			<td>sample organization</td>
		</tr>
		<tr>
			<td>Zebra TLP 2844 printer</td>
			<td>Zebra</td>
			<td>---</td>
			<td>label printer</td>
		</tr>
	</tbody>
</table>

creation and maintenance of a living biobank - how we do it

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In our hands, the establishment rate of primary cell cultures (Figure 2A &#38; B) was 12.9% in a large series9. The majority of attempts to isolate expandable tumor cells from fresh surgical resected specimens failed due to a lack of outgrowth or early contamination. Cell line establishment was considered successful after 3 passages with a steady growth under standard culture conditions (DMEM, 10% FCS, standard culture vessel) and validation of epithelial differentiation via FACS-analysis10. Cell lines derived from PDX tumors (Figure 2C &#38; D) showed a higher establishment rate of 23.6% which is also due to the possibility of repetitive attempts in contrast to primary resected tumors9. However, some mixed cultures (Figure 2E) cannot be freed of fibroblastic growth or are even lost due to fibroblastic overgrowth (Figure 2F).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62065/62065fig02.jpg" /> 
Figure 2: Cell culture. Primary cancer cell lines, derived from a metastasis of colon cancer case HROC313, passage 21 (A) and pancreatic cancer case HROP88, passage 5 (B). PDX-derived cancer cell lines of colon PDX HROC285 T0 M2 (D) and pancreatic PDX HROP10 T5 M2, passage 4 (E). Mixed culture of fibroblasts and cancer cells from pancreatic cancer HROP75, passage 8 (C) and fibroblastic overgrowth (F). <a href="https://www.jove.com/files/ftp_upload/62065/62065fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Considering changes in PDX generation protocol, mouse strains used and also experimenters over several years, as well as large differences in the amount of tumor tissue available for engraftment, it is not trivial to give the overall success rate of PDX generation. In a very recent series of PDX generation experiments performed by two researchers (S.M. and F.B.), primary outgrowth rates of 63% for colorectal PDX (an exemplary histology can be depicted from Figure 3A) and 48% for pancreatic PDX (Figure 3B) were observed. The outgrowth of murine or human lymphomas at the implantation site is relatively rare, but can mimic successful PDX outgrowth (Figure 3C). Apart from histopathological examination, concordance between PDX models and their donor patients was regularly confirmed by short tandem repeat (STR) analysis (Figure 3D). To the present day the biobank comprises &#62;50 primary and &#62;50 secondary colorectal, 3 primary and 6 secondary pancreatic cancer cell lines as well as &#62;150 colorectal and 19 pancreatic PDX models.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62065/62065fig03.jpg" /> 
Figure 3: Representative histological comparison of colorectal (A) and pancreatic PDX (B). Human lymphoma at the implantation site mimicking PDX outgrowth (C). Genetic identity testing of a PDX model (HROC430 T1 M2) to the original patient tumor tissue (HROC430Tu) by short tandem repeat (STR) analysis. Comparison of the nine STR loci, vWA, THO1, TPOX, CSF1 PO (FAM dye) and D5S818, D13S317, D7S820, D16S539 (HEX dye) using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis confirmed genetic concordance of the PDX and donor tumor (D). <a href="https://www.jove.com/files/ftp_upload/62065/62065fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Creation and Maintenance of a Living Biobank - How We Do It at JoVE.com

Creation and Maintenance of a Living Biobank - How We Do It

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

creation-and-maintenance-of-a-living-biobank-how-we-do-it

Research

JoVE Journal

Cancer Research

7.2K Views.  University Medical Center Rostock, University of Rostock. In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

Video: Creation and Maintenance of a Living Biobank - How We Do It

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients' treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.

Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples.