Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue
Transcription
First, weigh a tissue fragment and measure its length, breadth, and height. Place the tissue in a culture dish with a culture medium and dice it to small pieces using a scalpel. Transfer everything to a C-tube for homogenization using a Pasteur pipette.
Place the tube in a mechanical dissociator and run cycles as required. The apparatus uses mechanical forces to extract viable single cells from the tissue sample while maintaining cellular integrity, including the surface proteins. Decant the homogenate through a cell strainer fixed on a centrifuge tube.
Rehomogenize the remaining tissue and strain the homogenate through the cell strainer into the tube. Centrifuge the homogenate and remove the supernatant. Now, resuspend the cell pellet in the culture medium and transfer a small amount of the cell suspension to a tube containing trypan blue stain.
After staining, transfer the cells onto a hemocytometer slide. Count the transparent viable cells. The dead cells take up the stain, as their cell membrane is not intact. In the following protocol, we will perform non-enzymatic dissociation of fresh human tissue for quantitative and qualitative analysis of CD45+ cells.
– 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 1 milliliter of the appropriate chemically defined, serum-free, hematopoietic cell medium and use a sterile scalpel to dice the samples into small 1 to 2 square millimeter pieces. Next, transfer the dish contents into a mechanical dissociator C-tube and rinse the dish and scalpel with 2 milliliters of medium.
Add the wash to the C-tube and then run the 8.01 mechanical dissociator 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 Pasteur pipette from washing the petri dish to transfer any liquid remaining in the C-tube into the cell strainer.
Next, use a 1-milliliter micropipette to transfer the filtered liquid into a 15-milliliter conical tube and rinse the C-tube with an additional 3 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 1-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 3 more milliliters of medium so that the unhomogenized tissue drops back into the C-tube.
Rehomogenize the tissue for two more cycles as just demonstrated, and then pour the second homogenate through the cell strainer again. Rinse the C-tube with another 3 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 9 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 at room temperature. Decant the supernatant from the 15-milliliter tube into a 1.5-milliliter tube, placing it at 4 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.
Resuspended 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 trypan 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 degrees Celsius for future downstream analysis.
