Working with Human Tissues for Translational Cancer Research

The overall goal of this procedure is to maximize the quality and quantity of data extracted from tissue samples. This method can help answer key questions in the field of translational cancer research, such as which mutations are harbored, which pathways are activated, and which immune cell subtypes infiltrate human tumors. The main advantage of this technique is that a large amount of data can be generated from a single surgical resection of human tissue.

One of the most critical steps prior to specimen processing is to ensure sample quality and validate the presence of viable tumor by pathological assessment as described in the protocol text, All relevant information for the specimen or tissue, such as patient name, number, treatment, and date of surgery should be made readily accessible in an institutional tissue database. Fresh tissue specimens should always be stored on wet ice and processed as quickly as possible in order to limit changes induced by extraction of the tissue sample from its natural environment. Normal tumor matched and tumor tissue samples should be processed separately.

To avoid cross-contamination, prepare a sterile Petri culture dish, a scalpel, as well as a needle or forceps for tissue processing. In order to limit the time of sample processing, lay out all required items in advance as depicted here. Place the tissue specimen in the open Petri culture dish flat against the surface.

Inspect the sample from all angles to gain a better understanding of its structure and dimensions. Pin the sample to the bottom of the dish with the needle and proceed to bisect the tissue along its largest and best representative axis. Cut out a small piece of tissue and place it in a sterile 1.5 milliliter micro centrifuge tube containing one milliliter of RNA stabilizing solution.

Place this sample on ice for the remainder of the process and store at four degrees Celsius until needed. Lay out an open and pre-labeled biopsy cassette on the lid of the Petri culture dish. Cut out a slice of tissue no thicker than three to five millimeters and transfer it to the cassette.

Place the cassette in 10%neutral buffered formin for a minimum of 72 hours. At room temperature, cut out a piece of tissue and place it in a tissue mold. Cover the tissue section in optimal cutting temperature compound and freeze it immediately at negative 20 degrees Celsius until ready for cryosectioning.

Cut out one or many pieces of tissue and transfer to a 50 milliliter tube containing max buffer. The tissues can be used fresh or stored overnight at four degrees Celsius or flow cytometry analysis. Cut the remainder of the tissue into pieces no larger than a maximum size of 0.5 millimeters by 0.5 millimeters and transfer them to cryogenic vials.

Immediately freeze the tissues in liquid nitrogen for long-term storage. To begin this procedure, prepare digestion medium with DM EMD NS one and collagenase. A place fresh or overnight stored tumor in max buffer in the lid of a Petri dish and submerge with the digestion medium.

Pin the tumor to the bottom of the dish with a needle and cut from the outside to the center with a scalpel until the tumor has a paste like texture. Transfer the tumor mix to a 50 milliliter tube and seal the tube to avoid leaks. Transfer the tube to an incubator and agitate at 225 RPM at 37 degrees Celsius for one hour.

After one hour, filter the tumor cell suspension through a 70 micrometer cell strainer centrifuge the cell suspension at 250 GS and four degrees Celsius for five minutes. Resuspend the cell pellet with one milliliter of fax buffer. After counting the cells using a hemo cytometer resus, suspend the cells at a concentration of one times 10 to the seventh cells per milliliter.

Transfer 100 microliters or 10 to the six cells to each well of a 96 well round bottom plate. Add 200 microliters of the viability stain solution to each well and mix incubate for 30 minutes at four degrees Celsius in the dark. After 30 minutes, spin down the cells at 250 Gs and four degrees Celsius for five minutes.

Aspirate the supernatant. Add the antibody mixture at the recommended titrated concentration for surface antigens in 200 microliters of fax buffer. Incubate the cells at four degrees Celsius in the dark for 30 minutes.

Spin down the cells at 250 Gs and four degrees Celsius for five minutes and aspirate the supernatant wash with 200 microliters of fax buffer and spin down the cells again, aspirate the supernatant and resus suspend the cells in 200 microliters of fax buffer. The cells are now ready for analysis by flow cytometry. These representative results depict the flow cytometry gating strategy for broad immune phenotyping on a processed melanoma tumor specimens processed as demonstrated and stored overnight in max buffer at four degree Celsius Show extremely limited mortality.

Furthermore, processing the tissue is demonstrated. Allows multicolor broad phenotyping of immune cell subsets in melanoma such as B cells, CD four T cells, CD eight T cells, gamma delta T cells, and K cells, dendritic cells, macrophages, mast cells, basophils, neutrophils, and eosinophils Once mastered. This technique can be done in four hours if it is performed properly.