Working with Human Tissues for Translational Cancer Research

Summary

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.

Abstract

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.

Introduction

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 o....

Protocol

Ethics statement: This protocol is approved by the University of Texas MD Anderson Cancer Center.

1. Specimen Procurement

1. Collect research tissue samples from surplus tumor and related normal tissue^10-12. Store specimens on wet ice to avoid autolysis.
2. 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.
3. Handle tissue allocated for research in accordance with applicable research and tissue banking policies and procedures.
   NOTE: Biospecimen collection involves the coordination....

Results

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 °C under 225 RPM rotation. Following digestion, cell suspension was filtered through a 70 µm cell strainer to eliminate debris and cells were stained with fluorescence-labeled flow cytometry antibodies for immune cell phenotyping. Shown below (Figure 3) is th.......

Discussion

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.......

Materials

List of materials used in this article

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