Name: working with human tissues for translational cancer research
Uploaded: 2015-11-26T14:00:00
Description: Watch this Scientific Journal Video about Working with Human Tissues for Translational Cancer Research at JoVE.com

This work was supported by the philanthropic contributions of the John G. and Marie Stella Kenedy Memorial Foundation (Grant #0727033) and the Melanoma Research Alliance. The authors wish to thank the University of Texas MD Anderson Cancer Center Institutional Tissue Bank (ITB) for coordinating tissue distribution and collection and facilitating translational research, as well as Dr. Ignacio Wistuba, Dr. Victor Prieto, the MD Anderson Cancer Center Department of Pathology and the Melanoma Moon Shot Program. Finally, the authors wish to acknowledge all patients and families affected by melanoma.

Ethics statement: This protocol is approved by the University of Texas MD Anderson Cancer Center.
1. Specimen Procurement
<ol>
	<li>Collect research tissue samples from surplus tumor and related normal tissue10-12. Store specimens on wet ice to avoid autolysis.</li>
	<li>Send all tissues, medical devices and foreign bodies removed during a surgical procedure to the Department of Pathology for necessary examination in order to facilitate a pathologic diagnosis.</li>
	<li>Handle tissue allocated for res...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IRB guidebook. , (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MD Anderson Cancer Center Institutional Tissue Bank Standard Operating Procedures. 3, (2009).</li><li>Ruffell, 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=Leukocyte+composition+of+human+breast+cancer.">Leukocyte composition of human breast cancer.</a> Proc. of the Ntnl. Aca. of Sci. 109, 2796-2801 (2012).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical flow cytometry. , (2005).</li><li>Baker, A. 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Ultimately, the methods of processing must be dictated by the desired hypothesis and anticipated technical assays to be performed (Figure 1). The following section serves as an overview to describe potential assays that may be performed on such tissue sections. It is by no means an exhaustive overview, but is intended to help guide sample processing methods and choice of downstream assays by describing techniques and listing their strengths and weaknesses.
Flow cytometry stain...

Medical research has benefited immensely from the study of both human cell lines and animal models. Cell lines allow for better control of components in a system, as well as use of cells from the proper organism. Working with human cells, while more representative, presents a drastically simplified snapshot of mechanisms which may be responsible for health and disease, because cell lines may not recapitulate the impact of other cells, stromal components, and organs which may affect responses1. On the other hand, animal models, although not entirely accurate in reproducing human disease and genetic heterogeneity, offer further insight by allowing for input of whole animal systems in most models2,3.
Shortcomings aside, both methods have their benefits and are complementary. However, the main goal remains to study human cells and organs, in a full body, multi-organ system. Accordingly, translational research has taken great strides in facilitating the study of human organs and tissues extracted from patients, in order to better grasp the mechanisms underlying disease. Translational research offers the advantage of studying the result of treatments and diseases in the entire body and in appropriate cells, thereby providing the best of both worlds between cell and animal work4.
Translational research is also not without flaws. Harvested specimens from human patients, upon removal from the host, are immediately subjected to ischemia and other external factors from which they would be otherwise protected. This may result in stress-induced molecular and genetic changes, and cause bias in subsequent studies. Therefore, much effort has been put into extracting and processing tissues through strict methods to best preserve organ and cell integrity for downstream studies5. Importantly, future applications must be considered closely when selecting methods of processing human samples, as certain methods may be detrimental to cellular components and signaling pathways while sparing others.
For any research involving human tissues, regulatory issues must be addressed. After the Tuskegee and Belmont reports, there was a growing acceptance of the fact that biomedical research was associated with inherent risks often resulting in unavoidable ethical dilemmas6. The need for national standards was recognized and the federal government created Institutional Review Boards (IRBs) as a means to uphold the principles of the Belmont Report (Respect for persons, Beneficence, Justice).
Any institution&#39;s IRB is a committee of doctors, researchers and community members who are responsible for protecting research participants. It requires that voluntary consent be obtained from all participants of biomedical research studies, and that this consent be documented in an informed consent form. The informed consent process aims to provide adequate information to a participant, so that an informed decision may be made on whether or not to enroll in a study, or to continue participation. In this regard, the informed consent document must have good readability and should be written in language that can be easily comprehended (6th to 8th grade reading level) by the target population. The possibility of coercion or undue influence must be minimized, and the subject must be given ample time to consider participation. The participant must also be allowed to exercise their right to withdraw consent at any stage during the course of the study without penalty. Voluntary informed consent is a mandatory pre-requisite for a subject&#39;s participation in research and it is also legally effective7.
As per the regulations for the protection of human subjects 21 CFR 50.25 (<a href="http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html">http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html</a>8), study participants must be informed of the purpose of the research, procedures involved in the research, alternatives to participation, all foreseeable risks and discomforts (including not only physical injury but also possible psychological, social, or economic harm, discomfort, or inconvenience), benefits of the research to society and possibly to the individual, length of time the subject is expected to participate, person to contact for answers to questions or in the event of a research-related injury or emergency, statement indicating that participation is voluntary and that refusal to participate will not result in any consequences or any loss of benefits such as a compromise in regular clinical care that the participant is otherwise entitled to receive as a result of his/her diagnoses, and finally a statement regarding the subject&#39;s right to confidentiality and right to withdraw from the study at any time without any consequences. Tissue specimens from participants who withdraw consent during the course of the study should not be banked and must be immediately exhausted. Waiver of one or more elements of informed consent may be obtained from the IRB for some research projects that could not practically be done without an alteration to the required elements or for studies where required elements are not applicable. Great care must be taken to ensure data confidentiality. The Health Insurance Portability and Accountability Act (HIPAA) is a federal law that prevents using or disclosing &#8220;Protected Health Information&#8221; (PHI) without written consent from the participants. PHI includes but is not limited to health information transmitted or maintained in any form or medium and includes fields that could be used to identify an individual9.
Through this work, we aim to outline the protocol that must be followed during tissue processing - including procurement, and storage and emphasize the importance of following these strict guidelines so as to minimize the biases that may result due to the stresses a tissue is subjected to upon extraction. We also endeavor to provide a brief overview of the downstream assays or analyses (Figure 1) that could be performed on such specimens so that readers may make a qualified judgment on what assay(s) best suits their needs.

<table>
	<tbody>
		<tr>
			<td>Needles</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel disposable scalpels</td>
			<td>Miltex</td>
			<td>4-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Cassettes</td>
			<td>Thermo Fisher</td>
			<td>58931</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA-stabilizing solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Eppendorf tubes</td>
			<td>Phenix</td>
			<td>MAX-815</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml tubes</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>StatLab Medical Products</td>
			<td>28600-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Containers</td>
			<td>Fisher Scientific</td>
			<td>23-032-059</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature solution (OCT)</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Roche</td>
			<td>11088793001</td>
			<td></td>
		</tr>
		<tr>
			<td>70&#956;m cell strainers</td>
			<td>BD Bioscience</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead stain solution</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
	</tbody>
</table>

working with human tissues for translational cancer research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

The results displayed below depict flow cytometry gating strategy for broad immune phenotyping on a processed melanoma tumor. The day of staining, tissue sample was digested and homogenized with collagenase I and DNase I incubation for 60 min, at 37 &#176;C under 225 RPM rotation. Following digestion, cell suspension was filtered through a 70 &#181;m cell strainer to eliminate debris and cells were stained with fluorescence-labeled flow cytometry antibodies for immune cell phenotyping. Shown below (Figure 3</stro...

Watch this Scientific Journal Video about Working with Human Tissues for Translational Cancer Research at JoVE.com

Working with Human Tissues for Translational Cancer Research

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

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