Name: creation and maintenance of a living biobank - how we do it
Uploaded: 2021-04-10T14:00:00.000+00:00
Duration: 13 min 8 s
Description: Watch this Scientific Journal Video about Creation and Maintenance of a Living Biobank - How We Do It at JoVE.com

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><tbody><tr><td>Bacillol&amp;reg; 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 Discardit&lt;sup&gt;TM &lt;/sup&gt;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&amp;reg; 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 &amp;amp; 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&amp;rsquo;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 Discardit&lt;sup&gt;TM &lt;/sup&gt;II Syringe 10ml</td><td>BD</td><td>REF 309110</td><td>tissue preparation</td></tr><tr><td>cell strainer; yellow; 100&amp;micro;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&amp;deg;C/min</td></tr><tr><td>Dewar&amp;nbsp;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&amp;micro;l</td><td>Sarstedt</td><td>REF 70.760.002</td><td>liquid transfer</td></tr><tr><td>Filter tip 1000&amp;micro;l</td><td>Sarstedt</td><td>REF 70.762.411</td><td>liquid transfer</td></tr><tr><td>Pipette 200&amp;micro;l, yellow</td><td>Eppendorf</td><td>Cat. No.: 3121 000.082</td><td>liquid transfer</td></tr><tr><td>Pipette 1000&amp;micro;l, blue</td><td>Eppendorf</td><td>Cat. No.: 3121 000.120</td><td>liquid transfer</td></tr><tr><td>incubator&amp;nbsp; BB 6220 CU</td><td>Heraeus</td><td>Cat.-No.: 51012839</td><td>cell cultivation</td></tr><tr><td>heating plate PR&amp;Auml;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 &amp;amp; Co. KG</td><td>---</td><td>sterile working bench</td></tr><tr><td>freezer -80&amp;deg;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&amp;reg;&amp;nbsp;Ampullen SF 480&amp;nbsp;mg/5&amp;nbsp;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&amp;nbsp;holder</td><td>B.Braun Aesculap</td><td>BH1 1 OR</td><td>surgical instruments</td></tr><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&amp;amp;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&amp;reg;; steril; 10x10 cm</td><td>Lohmann&amp;amp;Rauscher</td><td>REF 18506</td><td>sterile OP tissue</td></tr><tr><td>Raucotupf cotton tipped applicators</td><td>Lohmann&amp;amp;Rauscher</td><td>REF 11969</td><td>applicator</td></tr><tr><td>Corning&amp;reg; 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&amp;reg; 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>

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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

7.3K 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. ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

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.

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and ...

Protocol for Prostatectomy Specimen Mapping and Dissection

Enhancing Prostate Tumor Biobanking Reliability with Improved Sampling Technique and Histological Characterization

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be particularly challenging in the context of prostate cancer (PC) due to the unique nature of this organ and the high heterogeneity associated with this tumor. On the other hand, histopathologically characterizing samples before their storage without causing significant tissue alterations is also an intriguing challenge. In this context, we present a new method for acquiring, mapping, characterizing, and micro-dissecting resected prostate tissue based on anatomopathological criteria.
Unlike previously published protocols, this method reduces the time required for histopathological analysis of the prostate specimen without compromising its structure, which is crucial for assessing surgical margins. Furthermore, it enables the delineation and micro-macro dissection of fresh prostate tissue samples, with a focus on histological tumor areas defined by pathological criteria such as Gleason score, precursor lesions (high-grade prostatic intraepithelial neoplasia - PIN), and inflammatory lesions (prostatitis). These samples are then stored in a Biobank for subsequent research analyses.

Author Spotlight: Advancing Prostate Cancer Research Through Improved Tissue Sampling and Biobanking

This protocol describes a method for facilitating the collection of samples from radical prostatectomy specimens. The goal is to map, characterize, and micro-macro dissect tissue samples from the specimens based on anatomopathological criteria before storing them in a Biobank.

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be ...

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Medicine

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the research laboratory. Here, we developed a protocol for acquiring high-quality liquid biopsies for molecular analyses from the aqueous humor and the vitreous from patients undergoing eye surgery. In this workflow, a Mobile Operating Room Lab Interface (MORLI) cart equipped with a computer, a barcode scanner, and lab instruments, including onboard cold storage, is used to obtain and archive human biological samples. A web-based data privacy-compliant database enables annotating each sample over its lifetime, and a cartesian coordinate system allows tracking each barcoded specimen in storage, enabling quick and accurate retrieval of samples for downstream analyses. Molecular characterization of human tissue samples not only serves as a diagnostic tool (e.g., to distinguish between infectious endophthalmitis and other non-infectious intraocular inflammation) but also represents an important component of translational research, allowing the identification of new drug targets, development of new diagnostic tools, and personalized therapeutics.

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the ...