This work was supported by funds from the Midwestern University College of Health Sciences Research Facilitation Grant, awarded to EEH, and Midwestern University Office of Research and Sponsored Programs Intramural Grant, awarded to KJL. Additional support was provided by Affiliated Laboratories and Affiliated Dermatology. We thank Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel, and Ali Zaidi for their technical assistance.

All experimentation on human tissue samples was approved by Western IRB (WIRB Protocol #20142461). The single criterion for the collection of tissues for these procedures is a biopsy-proven diagnosis of SCC. No exclusion criteria were outlined in the original IRB protocol, but samples from patients with a known blood-borne communicable disease were not used. Informed consent was obtained prior to the collection of cutaneous tissue, blood, and saliva. Midwestern Institutional Review Board approved the validation work with clinic-based biorepository samples at Midwestern University (AZ#807).
1. Cutaneous Tumor Tissue Processing
NOTE: This is to be performed by a Mohs histotechnologist.
<ol>
	<li>Processing of Fresh Squamous Cell Carcinoma (SCC) Tissue for RNA and Explant Culture
	<ol>
		<li>After the tissue has been excised by the Mohs surgeon, harvest a 10 to 50 mg (&#8805;10 mm3) tissue sample from the center of the apical side of the tissue during relaxation (which is the process of manipulating the tissue to allow it to lie flat on the slide) before additional processing. 
		&#8203;NOTE: This is only possible if the tumor is large enough to allow a 10-50 mg-size sample (&#8805;10 mm2) to be removed prior to the histological assessment, which is part of the Mohs procedure. If the tumor is not large enough to allow a minimum of 2 mm3 to be removed, do not proceed with processing for RNA (skip step 5).</li>
		<li>Transfer the viable tissue to a labeled 1.5 mL tube containing culture medium (1:1 mixture of DMEM:Ham&#39;s F-12, 25 mM HEPES, 100 IU/mL of penicillin, and 100 &#181;g/mL streptomycin) using tweezers and ensure that the tissue is completely immersed in the medium. Store this tube at 4 &#176;C before additional processing.</li>
		<li>Use the tissue for RNA isolation and expression analysis (see step 5) and/or the establishment of an explant culture (see step 6) within 24 h.</li>
		<li>Place the remainder of the tumor sample that was removed from the patient (step 1.1.1) on a cryostat tissue holder (or &#34;chuck&#34;). Embed the tissue in optimal cutting temperature (OCT) compound, freezing the tissue within the holder, to allow for the completion of Mohs histology and for the preparation of additional histological slides for experimental analysis.</li>
	</ol>
	</li>
	<li>Preparation of Histological Slides
	<ol>
		<li>Cut tissue sections 7 &#181;m thick using a cryostat and create two (or more) slides for each tumor sample. Ensure that each slide contains up to three sections of the complete tumor. 
		&#8203;NOTE: One of the two slides will be stained using H&#38;E by the Mohs histotechnologist, and the additional slide(s) may be processed with IF, IHC, or FISH analysis.</li>
		<li>Place the slides in acetone for 1-2 s to fix the tissue. Set these slides aside to dry. Do not H&#38;E stain or coverslip the slide to be used for future immunofluorescence (IF), immunohistochemistry (IHC), or fluorescence in situ hybridization (FISH) analysis, depending on the experimental protocol.</li>
	</ol>
	</li>
	<li>Processing of Post-Mohs Tissue
	<ol>
		<li>Once Mohs processing has been completed (steps 1.1-1.2), work in the cryostat chamber and pry the sample from the cryo-embedding medium and place the tissue into a labeled cryogenic tube.</li>
		<li>Place the labeled vial in liquid nitrogen before the tissue has thawed. If samples are to be used for protein and/or DNA analysis, they may be transferred to liquid nitrogen or a -80 &#176;C freezer within a few minutes to be stored for future protein expression and DNA mutation analysis (see steps 4-5). 
		&#8203;NOTE: If intact RNA is desired from the post-Mohs sample, it is essential to keep the sample frozen during the removal from the cryo-embedding medium. If extreme care is not exercised at this step, the post-Mohs sample should be reserved for protein and/or DNA analysis.</li>
	</ol>
	</li>
	<li>Processing of Fresh ANT
	<ol>
		<li>At the time of Mohs closure, obtain ANT from the Mohs surgeon.</li>
		<li>Remove an equal or greater amount of ANT as was harvested from the SCC sample (see above) from the apical side of the normal tissue and transfer it to a labeled 1.5 mL tube containing culture medium (1:1 mixture of DMEM:Ham&#39;s F-12 supplemented with 25 mM HEPES, 100 IU/mL of penicillin, and 100 &#181;g/mL streptomycin) using tweezers.</li>
		<li>Completely immerse the tissue in the same culture medium and store at 4 &#176;C until subsequent processing (see steps 4-6).</li>
	</ol>
	</li>
</ol>
2. Blood, Saliva, and Buccal Swab Processing
<ol>
	<li>Obtain approximately 10 mL of blood in EDTA collection tubes via venipuncture.</li>
	<li>Transfer the blood from the collection tubes to a sterile 15- or 50 mL centrifuge tube. Remove approximately 200 &#181;L into a 1.5 mL cryogenic tube, avoiding bubbles; store this whole-blood sample at -80 &#176;C, to be used for future genotyping and tissue matching, if necessary.
	<ol>
		<li>Spin the remaining blood at 1,000 x g for 30 min and follow standard procedures to collect the plasma fraction.</li>
		<li>Aliquot the plasma into 1 mL increments in 1.5 mL cryogenic tubes and store at -80 &#176;C for use during future liquid biopsy analyses.</li>
	</ol>
	</li>
	<li>Use a sterile swab to obtain a buccal swab sample and store the swab in a sterile tube at room temperature.</li>
	<li>If needed, obtain a saliva sample and store in a sterile tube at room temperature. 
	NOTE: These samples may be used for future genotyping and tissue matching, if necessary.</li>
</ol>
3. Recordkeeping of Samples
<ol>
	<li>Transfer information from the checklist of patient samples to the biorepository database. Include patient information and diagnosis from the patient chart.</li>
	<li>Print labels with a barcode for each sample and record each in the database to link each collected sample with patient data.</li>
	<li>Record the distribution of the de-identified samples in the biorepository database prior to transferring the samples to the research lab.</li>
</ol>
4. Protein Isolation and Western Blot Analysis
<ol>
	<li>Place each tissue sample (SCC and/or ANT) into a vial containing 500 &#181;L of cell lysis buffer containing protease inhibitors per 100 mg of tissue to lyse the tissue. 
	NOTE: We used a radioimmunoprecipitation assay (RIPA) buffer, composed of 50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1.0% NP-40, and 0.1% SDS. Other cell lysis buffers can be used.</li>
	<li>Homogenize the tissues using a tissue homogenizer for 5 min in 20-s intervals at 4 &#176;C. Spin the samples at 21,000 x g and 4 &#176;C for 20 min and collect the supernatant containing protein into a fresh tube. Re-spin the supernatant using the same conditions and collect the final supernatant into a fresh tube.</li>
	<li>Electrophorese 30-50 &#181;g of protein from each sample using a 4-20% SDS-polyacrylamide gradient gel. Stain the gel with Coomassie Blue to visualize protein bands, if desired.</li>
	<li>If Western blot analysis of expressed proteins is needed, transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane following standard protocols. After transfer, Ponceau stain the PVDF membrane to visualize protein bands, if desired.</li>
	<li>Block the PVDF membrane and probe to detect proteins of interest following standard protocols. Use primary antibodies specific to the protein(s) of interest, along with corresponding secondary antibodies specific for the primary antibodies, at concentrations recommended by the manufacturer for Western blot analysis.</li>
	<li>Expose the probed membrane using a chemiluminescent substrate and image the membrane using an imaging system.</li>
</ol>
5. RNA Isolation and Quantitative Polymerase Chain Reaction (qPCR)
<ol>
	<li>Immediately place fresh tissue samples (SCC and/or ANT) into a 1.5 mL tube containing an RNA stabilization solution and place post-Mohs tissue samples into a 1.5 mL tube containing a frozen tissue transition solution, following the manufacturer&#39;s recommendations. Ensure that the tissue is completely immersed. Store these samples at -80 &#176;C until use.</li>
	<li>Remove the RNA samples from -80 &#176;C and thaw on ice. Transfer the tissue to a new 1.5 mL tube containing 1 mL of RNA extraction buffer (phenol/guanidine thiocyanate) per 50-100 mg of tissue.</li>
	<li>Lyse the tissues using a tissue homogenizer. Perform six cycles of homogenization, followed by a period of sample cooling; each cycle consists of 20 s of homogenization at 4 &#176;C and a 40 s cooling period. After homogenization, incubate the sample for 5 min at room temperature.</li>
	<li>Add 0.2 mL of chloroform per 1 mL of RNA extraction buffer and shake the tube vigorously by hand for 15 s. Incubate for 2-3 min at room temperature.</li>
	<li>Spin the samples at 12,000 x g and 4 &#176;C for 15 min. Remove the aqueous phase of the sample and place it into a new 1.5 mL tube.</li>
	<li>Add 0.5 mL of 100% isopropanol per 1 mL of the RNA extraction buffer used in step 5.2 to the aqueous phase and incubate for 10 min at room temperature. 
	NOTE: If the RNA yields are anticipated to be low (i.e., the starting tissue volume is less than 10 mg), samples may be precipitated overnight at -80 &#176;C. Additionally, 5 &#181;g of RNase-free glycogen per 500 &#181;L of RNA extraction buffer may be added during the isopropanol precipitation step to improve RNA yields.</li>
	<li>Spin the sample at 12,000 x g at 4 &#176;C for 10 min. Remove the supernatant and wash the pellet with 1 mL of 75% ethanol.</li>
	<li>Vortex the sample briefly and then spin it at 7,500 x g and 4 &#176;C for 5 min. Discard the wash and air-dry the pellet for 5-10 min.</li>
	<li>Resuspend the RNA pellet in 20 &#181;L of RNase-free water.
	<ol>
		<li>If desired, purify each RNA sample using an RNA cleanup kit following the manufacturer&#39;s recommendations. 
		NOTE: Small RNAs will be lost from the sample if this step is performed.</li>
	</ol>
	</li>
	<li>Electrophorese a 1 &#181;g sample using a 1.2% formaldehyde-MOPS agarose gel to analyze the quality of RNA. 
	NOTE: As an alternative, analyze samples using a bioanalyzer to obtain an RIN number13.</li>
	<li>Reverse-transcribe the RNA to cDNA using a reverse transcription protocol for qPCR analysis of target mRNAs (or miRNAs) of interest.</li>
</ol>
6. Tissue Explant Cultures
<ol>
	<li>Sterilize the excised tissue prior to culture by dipping the tissue into 70% ethanol.</li>
	<li>Place the tissue on a slide or dish and add enough culture medium (1:1 mixture of DMEM:Ham&#39;s F-12 supplemented with 10% FBS, 25 mM HEPES, 100 IU/mL of penicillin, and 100 &#181;g/mL streptomycin) to cover the tissue (to prevent the tissue from drying). Carefully cut the tissue into fragments (&#60;1 mm3) using a scalpel blade.</li>
	<li>Transfer the tissue fragments to a 35-mm tissue culture dish and add approximately 20 &#181;L of fetal bovine serum (FBS) on top of each fragment.</li>
	<li>Leave the dish open in a culture hood for 20-30 min, or until almost dry.</li>
	<li>Add 1 mL of culture medium (1:1 mixture of DMEM:Ham&#39;s F-12 supplemented with 10% FBS, 25 mM HEPES, 100 IU/mL of penicillin, and 100 &#181;g/mL streptomycin) to each dish and incubate at 37 &#176;C in 5% CO2. Subculture or sub-clone the cultures if needed.</li>
</ol>

<ol><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biorepositories%3A+Building+better+biobanks%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Biorepositories: Building better biobanks.</a> Nature. 486, 141-146 (2012).</li>
<li>Ambrosone, C. B., Nesline, M. K., Davis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+a+cancer+center+data+bank+and+biorepository+for+multidisciplinary+research%5BTitle%5D)+AND+%22Cancer+Epidemiol+Biomarkers+Prev%22%5BJournal%5D)">Establishing a cancer center data bank and biorepository for multidisciplinary research.</a> Cancer Epidemiol Biomarkers Prev. 15, 1575-1577 (2006).</li>
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<li>Neumeister, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tools+to+assess+tissue+quality%5BTitle%5D)+AND+%22Clin+Biochem%22%5BJournal%5D)">Tools to assess tissue quality.</a> Clin Biochem. 47, 280-287 (2014).</li>
<li>Vaught, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+NCI+perspective+on+creating+sustainable+biospecimen+resources%5BTitle%5D)+AND+%22J+Natl+Cancer+Inst+Monogr%22%5BJournal%5D)">An NCI perspective on creating sustainable biospecimen resources.</a> J Natl Cancer Inst Monogr. 2011, 1-7 (2011).</li>
<li>Fu, T., Aasi, S. Z., Hollmig, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+high-risk+squamous+cell+carcinoma+of+the+skin%5BTitle%5D)+AND+%22Curr+Treat+Options+Oncol%22%5BJournal%5D)">Management of high-risk squamous cell carcinoma of the skin.</a> Curr Treat Options Oncol. 17, (2016).</li>
<li>Rasmussen, C., Thomas-Virnig, C., Allen-Hoffmann, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Classical+human+epidermal+keratinocyte+cell+culture%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Classical human epidermal keratinocyte cell culture.</a> Methods Mol Biol. 945, 161-175 (2013).</li>
<li>Purdie, K. J., Pourreyron, C., South, A. P. Cancer cell culture: Methods and protocols. , Humana Press. 151-159 (2011).</li>
<li>Berglund, S. R., Schwietert, C. W., Jones, A. A., Stern, R. L., Lehman, J., Goldberg, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+methodology+for+sequential+extraction+of+RNA+and+protein+from+small+human+skin+biopsies%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies.</a> J Invest Dermatol. 127, 349-353 (2007).</li>
<li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RIN%3A+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements%5BTitle%5D)+AND+%22BMC+Mol+Biol%22%5BJournal%5D)">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 1-14 (2006).</li>
<li>Cooper, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+and+glycosylation+of+MUC1+in+epidermolysis+bullosa-associated+and+sporadic+cutaneous+squamous+cell+carcinomas%5BTitle%5D)+AND+%22Br+J+Dermatol%22%5BJournal%5D)">Expression and glycosylation of MUC1 in epidermolysis bullosa-associated and sporadic cutaneous squamous cell carcinomas.</a> Br J Dermatol. 151, 540-545 (2004).</li>
<li>Riihila, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+factor+H%3A+a+biomarker+for+progression+of+cutaneous+squamous+cell+carcinoma%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Complement factor H: a biomarker for progression of cutaneous squamous cell carcinoma.</a> J Invest Dermatol. 134, 498-506 (2014).</li>
<li>Ha Lan, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+p40+isoform+of+p63+has+high+specificity+for+cutaneous+sarcomatoid+squamous+cell+carcinoma%5BTitle%5D)+AND+%22J+Cutan+Pathol%22%5BJournal%5D)">Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.</a> J Cutan Pathol. 41, 831-838 (2014).</li>
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</ol>

The authors have nothing to disclose.

To the author&#39;s knowledge, this protocol is the first of its kind that focuses on the clinical procurement of cutaneous tissue samples in both a cost-effective and fast approach. Patients undergoing Mohs microsurgery are typically scheduled during specific blocks of time, and collection is limited to these periods. Sample collection involves effort from the medical assistant involved in patient care, the Mohs histotechnologist who processes the tumor samples, and a designated laboratory staff member who handles sampl...

Cancer and biomarker research relies on a supply of quality human tissue samples, and limited supply has hindered research1,2. Many dermatologic studies are limited by the inadequate supply, variable quality, and costs associated with the use of human tissue. The cost of establishing a large, dedicated biobank has been estimated to be approximately two million dollars3, and these costs place the use of human tissue out of the reach of many researchers. Furthermore, the process of generating and storing research samples poses the risk of affecting clinical operations and delaying patient care if not carefully executed. A cost-effective, clinic-based biorepository has been established that focuses on skin cancer samples following recommended best practices and sample validation4,5,6.
This protocol has been developed in a dermatology clinic that performs a large volume of Mohs micrographic surgeries to remove squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma skin cancers. Volunteer donors can be recruited from this patient population. It is important to establish the biorepository at the site of collection to rapidly capture tissue and blood from consented patients without delaying treatment. Gathering samples from the same clinic minimizes variations in collection techniques and minimizes variations in the quality of samples, which can be problematic for downstream applications7,8.
The goal of the Mohs micrographic surgery technique is to ensure that all cancer tissue is removed while preserving as much healthy tissue as possible. The procedure involves the progressive removal of thin layers of tumor tissue. Each successive layer is histologically examined (after cryosectioning the tumor tissue and performing H&#38;E staining) by a dermatologist to determine if all cancer tissue has been removed. The excision and examination of subsequent layers of tissues is executed while the patient remains in the office. This technique is considered the best treatment option for SCC9. At this point, the wound is closed and, to improve healing and cosmetic appearance, adjacent normal tissue (ANT) is frequently excised. Thus, this surgical procedure to remove a tumor is ideally suited for collecting histologically characterized tissue for future studies.
The procurement procedure for obtaining tumor tissue, adjacent normal tissue, saliva, and blood samples has been designed to have minimal impact on normal staff duties (Figure 1). Medical assistants perform the blood draws while preparing the patient for the procedure. After completion of the Mohs procedure, the Mohs histotechnologist prepares additional histological slides of the specimen and transfers the tissue to the biorepository. Costs associated with establishing the biorepository include the purchase of cryopreservation freezers, the creation of modest clinical laboratory space, and the development of an inventory tracking program.
<img alt="Figure 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 
Figure 1: Sequence of sample collection and responsible staff. Upon patient check-in and the attainment of patient consent, the medical assistant collects a buccal swab and performs a blood draw. The dermatologist and medical assistant then excise the tumor and close the wound, during which time SCC and ANT specimens are collected, respectively. A dedicated laboratory technician processes the blood and sections the SCC and ANT specimens for tissue culture, preservation, and entry into the biorepository. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/55583fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The diversity of samples collected enables a variety of experimental approaches (Figure 2). Samples collected from the patient are buccal swabs (saliva can also be collected if needed), whole blood, and excised tissue. The buccal swabs and a sample from the whole blood are saved, without processing, for genotyping and tissue matching. Whole blood is separated into white blood cell (WBC) and plasma fractions for future analyses. After Mohs processing, the frozen tumor is placed directly into liquid nitrogen and transferred to a -80 &#176;C freezer. Fresh, viable tumor tissue and ANT samples are cultured using modifications of previous techniques10,11 and then cryopreserved. During collection, the number of each sample type is recorded on a spreadsheet prior to entry into the inventory tracking program to facilitate accurate processing (Table 1).
<img alt="Figure 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 
Figure 2: Outline of clinic-based biorepository sample collection and processing. A buccal swab and blood sample are collected from the patient and stored for downstream genotyping and tissue matching. Whole blood is further processed for white blood cell (WBC) isolation and CTC analysis, as well as for plasma collection and liquid biopsy analysis. Tissue excised during the Mohs procedure is histologically processed for diagnostic purposes, after which the histologic slides can be used experimentally for further immunohistochemical analyses. Provided that the excised tissue sample is large enough, a portion of fresh tissue is removed and sectioned for protein and RNA isolation, and for the establishment of cultured cell lines. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/55583fig1large.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>Collection Date:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Patient 1</td>
			<td>Patient 2</td>
			<td>Patient 3</td>
			<td>Patient 4</td>
			<td>Patient 5</td>
		</tr>
		<tr>
			<td>Initials and Birth Date</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Color</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Type</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Location</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saliva</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Viable</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Normal Viable</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Mohs Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Normal Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Checklist to record sample collections. Data tracked and recorded with each sample collected include patient initials, birth date, and eye color (for skin typing), as well as the location of specimen removal. The number of saliva samples, blood collection volumes, and the number of viable and preserved tissue specimens collected are also recorded as references for allocations to later uses. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/Table1.xlsx">Please click here to download this file.</a>
To validate sample collection procedures, each sample type has been tested in downstream applications. Using modifications of previous techniques12, tumor and ANT have been successfully used in protein and RNA isolation and can potentially be used for DNA isolation. Viable explants established from the tissue sections have been evaluated by microscopy, while stored histological slides have been used for immunohistochemistry and immunofluorescence.
By following the protocol described here, it is possible to extend this model to other dermatology clinics, other tumor types (such as melanoma), and other surgical specialties and practices to provide human tissue samples for multifaceted research into human cancers. Slight modifications of this protocol are likely to be necessary for other practices but, in principle, this protocol is applicable to any surgical practice that routinely discards patient samples gathered in the course of patient treatment.

<table><tbody><tr><td>BD Vacutainer Plastic Blood Collection Tubes with K&lt;sub&gt;3&lt;/sub&gt;EDTA</td><td>ThermoFisher Scientific</td><td>02-685-2B</td><td></td></tr><tr><td>Electron Microscopy Sciences Double Edge Blades</td><td>ThermoFisher Scientific</td><td>50-949-411</td><td></td></tr><tr><td>Curved Medium Point General Purpose Forceps</td><td>ThermoFisher Scientific</td><td>16-100-110</td><td></td></tr><tr><td>Premium Microcentrifuge Tubes</td><td>ThermoFisher Scientific</td><td>05-408-138</td><td></td></tr><tr><td>Lonza Walkersville KGM Keratinocyte Medium</td><td>ThermoFisher Scientific</td><td>NC9791321</td><td></td></tr><tr><td>Electron Microscopy Sciences Tissue TEK OCT Compound</td><td>ThermoFisher Scientific</td><td>50-363-579</td><td></td></tr><tr><td>Leica CM1950 Cryostat</td><td>Leica Biosystems</td><td>14047743909</td><td></td></tr><tr><td>Frosted Microscope Slides</td><td>ThermoFisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Shandon Rapid-Chrome H&amp;amp;E Frozen Section Staining Kit</td><td>ThermoFisher Scientific</td><td>99-900-01</td><td></td></tr><tr><td>Nalgene Long-Term Storage Cryogenic Tubes</td><td>ThermoFisher Scientific</td><td>03-337-7X</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Boca Scientific BuccalT-Swab&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>NC9679349</td><td></td></tr><tr><td>Cell Signaling Technology 10x RIPA Buffer</td><td>ThermoFisher Scientific</td><td>50-195-822</td><td></td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail</td><td>ThermoFisher Scientific</td><td>PI78443</td><td></td></tr><tr><td>Eppendorf 5424R Microcentrifuge</td><td>ThermoFisher Scientific</td><td>05-401-203</td><td></td></tr><tr><td>Pellet Morter Cordless Homogenizer</td><td>ThermoFisher Scientific</td><td>12-141-3</td><td></td></tr><tr><td>RNAse Free Pellet Pestle</td><td>ThermoFisher Scientific</td><td>121-141-364</td><td></td></tr><tr><td>Forma SteriCycle CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>ThermoFisher Scientific</td><td>13-998-089</td><td></td></tr><tr><td>HyClone Fetal Bovine Serum</td><td>ThermoFisher Scientific</td><td>SH30071.02</td><td></td></tr><tr><td>Gibco Advanced DMEM/F-12</td><td>ThermoFisher Scientific</td><td>12-634-028</td><td></td></tr><tr><td>Gibco Penicillin-Streptomycin</td><td>ThermoFisher Scientific</td><td>15140148</td><td></td></tr><tr><td>Gibco 1 M Hepes</td><td>ThermoFisher Scientific</td><td>15-630-130</td><td></td></tr><tr><td>Nunc Cell Culture 35 mm with Vent</td><td>ThermoFisher Scientific</td><td>1256591</td><td></td></tr><tr><td>Anti-CFH monoclonal antibody clone OX-24</td><td>Abnova</td><td>MAB12583</td><td></td></tr><tr><td>Anti-p40 (p63 delta) antibody</td><td>Abnova</td><td>ABX-144A</td><td></td></tr><tr><td>Anti MUC-1 antibody</td><td>Santa Cruz Biotech</td><td>sc-7313</td><td></td></tr><tr><td>Anti-Snail + Slug (phospho S246)</td><td>Abcam</td><td>Ab63568</td><td></td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11034</td><td></td></tr><tr><td>Alexa Fluor 568 Phallodin</td><td>Molecular Probes&amp;nbsp;</td><td>A12380</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Molecular Probes&amp;nbsp;</td><td>P36941</td><td></td></tr><tr><td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td><td>Zeiss</td><td>4300009901</td><td></td></tr><tr><td>Olympus IX51 phase contrast with DP72 camera</td><td>Olympus</td><td>IX511F3</td><td></td></tr></tbody></table>

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.

Tumor and ANT have been used successfully in protein isolation and tested by Western blotting. As shown in Figure 3, cutaneous squamous cell carcinoma markers (Muc114, FHL-115, and p4016,17) can be detected in patient samples collected and stored in our clinic-based biorepository.
<img alt="Figure 3" src="/...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

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 ...

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

6.5K Views.  Midwestern University. 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.

Video: Establishment of a Clinic-based Biorepository

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

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 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 ...

Cancer Research

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

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 Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking

Previous methods for biobanking prostate tissue, following radical prostatectomy, generally involved random sampling. In order to increase efficiency, and enable a greater range of downstream applications, a more targeted method of sampling prostate tissue was developed. Here we use both magnetic resonance imaging (MRI) and biopsy data to target specific areas of the organ for sampling. The method involves use of a previously published prostate slicing device which removes a 5 mm transverse slice from a predetermined region of the prostate, followed by the removal of 6 mm punch biopsies from predetermined areas of this slice. These samples can be stored frozen or fixed for biobanking purposes, or used fresh immediately with 70% confidence of tumor content, as compared with 10% confidence from the random sampling approach. This enables the use of all standard downstream techniques such as genomics, proteomics or histological work, but also work that requires fresh tissue such as live tissue imaging or ex vivo culture.

This method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications.

Previous methods for biobanking prostate tissue, following radical prostatectomy, generally involved random sampling. In order to increase efficiency, and ...

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 ...