The overall goal of this procedure is to rapidly isolate xenotransplanted human tumor cells from mice to improve the downstream analysis of the desired target cells. The elimination of the contuminating mouse cells by this method allows the specific assessment of human tumor cell gene expression or in vitro direct responses. The main advantage of this technique is that the human tumor cells can be quickly and easily isolated without having to identify any surface markers on the target cells.
Begin by using forceps and a scalpel to remove the fat and necrotic areas from the tumor sample. Then mince the tumor into two to four millimeter pieces. Next, transfer the tissue fragments into a single cell suspension dissociation tube containing the digestion mix and tightly close the tube.
Place the tube upside-down onto a benchtop tissue dissociator mount and confirm that the tissue pieces are in the rotor stator area. Attach the heater to the dissociator, then click the folder symbol on the touch screen and use the up and down arrows to select the appropriate tumor dissociation mode. Next, select the mount position where the sample is located and start the dissociation.
When a single cell suspension has been obtained, quickly centrifuge the tube to collect the cells at the bottom of the tube. Filter the cells through a 70-micron mesh strainer into a 50-milliliter conical tube and rinse the strainer with 20 milliliters of medium, collecting the wash in the sample tube. Then centrifuge the cells and resuspend the human tumor mouse stromal cell pellet in five milliliters of PB buffer.
After counting, collect the cells with another centrifugation and resuspend the pellet in 80 microliters of PB buffer per one x 10 to the seventh total cells. For this step, it's important to use a microscope and try from blue exclusion rather than a cell counter to obtain an accurate count of the viable cells. Cell counters frequently exhibit problems in handling heterogeneous samples after tissue dissociation.
Next, mix 20 microliters of magnetic labeling reagent for mouse cells into the cell suspension and incubate the cells for 15 minutes in a refrigerator. While the cells are being labeled, place an LS column into a suitable magnet and rinse the column with three milliliters of PB buffer. At the end of the incubation, add 420 microliters of buffer to bring the total sample volume up to 500 microliters saving a 50 microliter aliquot at two to eight degrees Celsius for later flow cytometric or molecular analysis.
Then add the cells onto the column using an equilibrated 70-micron mesh strainer. Collect the unlabeled target cell flow through. Then as soon as the reservoir is empty, wash the column two times with one milliliter of buffer collecting the eluent in the same tube as the negative cell fraction.
To collect the magnetic bead-bound mouse cells, transfer the column into a new conical tube and plunge three milliliters of buffer through the column. Using the novel combination of anti-mouse antibodies is significantly more efficient for more comprehensive mouse cell detection in target tissues for xenotransplantation than combined CD45 and MHC class one labeling. Indeed, utilizing this novel combination of antibodies for magnetic cell sorting facilitates the isolation of human tumor cells independent of the tumor type.
In addition to the mouse cell depletion, debris is also removed during the magnetic cell separation. The mouse-free human tumor cells can then be cultured, leading to the generation of pure human tumor cell cultures. The removal of mouse cells before performing whole exome sequencing on xenographed tumor samples not only significantly increases the total amount of reads, but also considerably reduces the number of host-derived reads mapped to the human reference genome and the number of falsely predicted single nucleotide polymorphisms.
The in silico removal of mouse-derived reads by bioinformatics methods cannot fully replace the experimental procedure, however, as an unambiguous sequence-based assignment to the species of origin is not possible for all reads. As shown here for the number of predicted SNPs, the in silico procedure cannot correct for the enhanced quality and concommitant higher read coverage of the in vitro depleted samples. Once mastered, the entire procedure, including the tumor preparation, dissociation, counting, and magnetic separation can be completed in two hours if it is performed properly.
While attempting this procedure, it's important to prepare the tissue sample appropriately for dissociation, to not use aggressive enzymes, and to make sure to obtain an accurate cell count. Following this procedure, the purified human tumor cells can be used for any kind of downstream analysis, cultivation, or even subsequent magnetic or flow-based cell sorting. After its development, this technique paved the way for researchers in the field of cancer research to explore human tumor and cancer stem cells from patient and semi-direct xenograft without any bias from the mouse cells.
After watching this video, you should have a good understanding of how quick and easy it is to improve your downstream assays by working with pure populations of target cells.
