A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The overall goal of this procedure is to rapidly homogenize human tissue fragments into a single cell suspension without enzymatic digestion for qualitative and quantitative analysis and the isolation of specific lymphocyte subpopulations. This is accomplished by first using a sterile scalpel to dice the tissue fragments into small pieces. Next, the tissue slurry is transferred to a mechanical dissociated tube where upon the fragments are further homogenized into a single cell suspension.

The cells are then collected by centrifugation for downstream analysis. Ultimately, flow cytometry can be used to analyze the infiltrating lymphocytes, subpopulations Asians. My laboratory developed this technique because we were looking for a way where we could image tumor infiltrating lymphocytes without dramatically affecting their native state.

We wanted something where we could rapidly isolate the cells and analyze them as they were at surgery. This technique has been used on more than 300 breast tumors and normal tissues in our laboratory, and is easy to use and can be used on a daily basis. It can be used to examine prognostic markers, response to treatment, and long-term clinical outcome.

Demonstrating this technique today will be John Nicola Loic. A technician in my laboratory Begin by weighing the tissue sample and then measuring the length, width, and height of the tissue. Then transfer the tissue fragment into a small Petri dish containing one milliliter of the appropriate, chemically defined serum free hematopoietic cell medium, and use a sterile scalpel to dice the samples into small one to two square millimeter pieces.

Next, transfer the dish contents into a mechanical dissociation C tube and rinse the dish and scalpel with two milliliters of medium. Add the wash to the C tube and then run the a 0.01 mechanical dissociation program two times in succession to gently homogenize the tissue fragments into a single cell suspension. After the second cycle, decant the homogenate through a 40 micrometer cell strainer into a 50 milliliter conical tube.

Then use the same pest your pipette from washing the Petri dish to transfer any liquid remaining in the C tube into the cell strainer. Next, use a one milliliter micro pipette to transfer the filtered liquid into a 15 milliliter conical tube and rinse the C tube with an additional three milliliters of medium. Transfer this wash through the cell strainer into the 50 milliliter tube.

Then gently move the unhomogenized tissue around the strainer with a clean one milliliter tip to squeeze the maximum amount of residual liquid trapped in the tissue into the 50 milliliter tube. Now, place the cell strainer upside down on top of the original C tube and rinse the strainer with three more milliliters of medium so that the unhomogenized tissue drops back into the C tube. Re homogenize the tissue for two more cycles as just demonstrated, and then pour this second homogenate through the cell strainer.

Again, rinse the C tube with another three milliliters of medium. Then filter the supernatant through the strainer and squeeze the residual liquid out of the tissue as just demonstrated. At this point, a volume of approximately 2.5 milliliters of single cell suspension should be present in the 15 milliliter tube, and approximately nine milliliters should be observed in the 50 milliliter tube.

To separate the tissue supernatant from the cells first centrifuge, both tubes of homogenate for 15 minutes at 600 Gs and room temperature, decant the supernatant from the 15 milliliter tube into a 1.5 milliliter tube, placing it at four degrees Celsius and discard the supernatant from the 50 milliliter tube. Then gently tap each tube on a hard surface to break up each of the cell pellets. Resuspend the loose cell pellet in the 50 milliliter tube with 500 microliters of medium, and transfer the cells to the 15 milliliter tube to resuspend the second pellet.

Then rinse the 50 milliliter tube with another 500 microliters of medium and transfer the wash to the 15 milliliter tube for maximum cell recovery. After counting the number of viable cells by trian blue exclusion pellet the cell suspension. Finally, spin down the reserved tissue supernatant.

Then carefully transfer the supernatant to at least one clean tube without touching or disturbing the pellet and store it at negative 80 degree Celsius for future downstream analysis. In these graphs, representative flow cytometry dot plots of dissociated tumor cell homogenates, followed by labeling with specific antibodies against leukocytes and epithelial cell markers are shown as demonstrated. This protocol does not significantly alter the viability of the CD 45 positive cells.

However, there is a considerable loss of viable EPAM positive cells. Despite this viable EPAM positive and CD 45 positive subsets can be segregated on the basis of their size and structure into large epithelial tumor cells, small epithelial cells, or large and small CD 45 positive cells. Representative flow cytometry dot plots of the major tumor infiltrating subsets are shown here with the lymphocytes gated on the viability of the CD 45 positive cells.

Finally, the cytokine expression or total immunoglobulins present in breast cancer supernatant can be compared with the supernatant from normal breast tissue revealing as demonstrated in this representative experiment, increases in both types of proteins associated with the tumor tissue While attendee is procedure, it's important to remember to work quickly and clean and to recover the maximum amount of material at each step of the procedure to obtain a sufficient number of cells for your endpoint analysis.