The overall goal of this procedure is to establish patient derived cell cultures from malignant melanomas and isolate putative CD 1 33 positive cancer stem cells. This is accomplished by first preparing primary melanoma single cells from a tumor tissue after depleting erythrocytes from the cell pellet. If necessary, characterize the primary cell culture and deplete the fibroblasts.
The final step is to perform magnetic cell sorting of the CD 1 33 positive and CD 1 33 negative melanoma cells. Ultimately, this optimized max procedure is an excellent method to obtain highly enriched and viable populations of CD 1 33 positive and CD 1 33 negative cells. We first used the technique to validate insco data in personalized medicine where we try to predict drug response of cancer patients and for that no commercially available cell line can be used.
The main advantage of the technique is that we have rapid access to CD 1 33 positive cells, which are putative cancer stem cells in malignant melanoma. The technique will be presented by Catherine Davis, a lab technician from my laboratory. Transfer the freshly isolated tumor tissue from the surgery to the tissue culture laboratory in sterile PBS containing 1%penicillin streptomycin.
Transfer the tumor tissue to a sterile petri dish, aspirate the excess solution. Then add 500 microliters dissociation mix and mince the tumor into small pieces of two to four millimeters using fresh sterile scalpels. Now transfer two to four grams of minced tumor tissue into a gentle max C tube containing five milliliters dissociation mix.
Rinse the Petri dish with an additional 4.5 milliliters dissociation mix and add remaining tissue fragments to the gentle max C tube. Close the tube, attach it upside down onto the sleeve of the dissociation and run the program H tumor zero one. Next, incubate the sample for 30 minutes at 37 degrees Celsius under continuous rotation on the highest run speed.
Now attach the tube upside down onto the sleeve of the dissociation and run the program H tumor zero two. Then incubate the sample for 30 minutes at 37 degrees Celsius under continuous rotation at 12 RPM. Next, run the gentle max program H tumor zero three.
Resuspend the sample and pass the cell suspension through a 70 micron cell strainer into a 50 milliliter tube. If necessary, stir the cell suspension with a sterile filter tip until the complete suspension runs through. Rinse the cell strainer with five milliliters quantum 2 6 3 medium pellet the cell suspension for five minutes at 300 G and discard the supernatant.
If the cell palates appears read due to a high amount of tices and lies, red bloods cells as detailed in the companion text protocol. Re-spin the tumor cells in quantum 2 6 3 and seed a culture routinely passage cells when they reach 80%confluence. Analyze the primary cell culture phenotypically using a microscope.
Then harvest a small amount of the primary cell culture for RNA extraction following CD NA synthesis and perform R-T-P-C-R with primers for neoplastic melanoma, stem cell and stroma cell marker genes. The most important genes to analyze are the fibroblast marker CD 90, the melanoma and melanocyte marker tear the cancer stem cell marker CD 1 33, the marker for mature dendritic cells, CD 83, and a housekeeping gene like HPRT or GAAP dh. If the combined microscopic and R-T-P-C-R analysis revealed a high fiberblast contamination of the primary melanoma culture, then proceed to deplete the fibroblasts using the anti fibroblasts, microbeads and LD columns.
Wash 80%confluence cells with prewarm PBS add appropriate amount of Accutane and incubate until cells detach from the culture surface. Harvest the cells in quantum 2 6 3 medium and transfer to a 50 milliliter tube. Enumerate the cells then centrifuge the required number of cells for five minutes at 300 G and four to eight degrees Celsius.
Aspirate supernatant completely and resus. Suspend up to one times 10 to the eighth cells in 350 microliters max buffer add 100 microliters FCR blocking reagent and 50 microliters CD 1 33. One biotin.
Mix well and incubate at four degrees Celsius for 10 minutes. Wash the cells with 10 milliliters max buffer, followed by centrifugation for five minutes at 300 G and four to eight degrees Celsius. Aspirate the supernatant completely after a second wash.
Resuspend the cell pellet in 400 microliters of max buffer. Add 100 microliters anti biotin microbeads and mix well incubate at four to eight degrees Celsius for 15 minutes. Meanwhile, to prepare the max separator for the magnetic separation, attach the quadro max to the max separator multis stand.
Insert the required number of LS columns with the column wings to the front in the magnetic field of the quadro max. Place a pres separation filter into each LS column and an appropriate collection tube. Under each column, rinse the filter and column with three milliliters max buffer and discard the flow through.
After washing the cells in max buffer as described earlier, resuspend the cells in 500 microliters max buffer and apply the cell suspension onto their prepared LS column. Wash three times with three milliliters max buffer per column. Collect the total affluent of the unlabeled cd 1 33 negative cell fraction.
Next, discard the pres separation filter. Remove the column from the separator and place it on a suitable collection tube. Apply five milliliters max buffer.
Immediately flush out the labeled CD 1 33 positive cells by firmly pushing the plunger into the column to increase the purity of the negative fraction. Apply the cell suspension onto a new equilibrated LS column and collect the effluent CD 1 33 negative fraction. This resected lymph node metastasis was obtained from a late stage melanoma patient.
After mechanical and enzymatic dissociation of the tissue, the pellet of filtered cells contained a high contamination with erythrocytes. Subsequently, depletion of the red blood cells resulted in a cell pellet of light brown color. These cells were cultured in quantum 2 6 3 medium here, an R-T-P-C-R of melanocyte and melanoma, fibroblast, dendritic and stem cell marker.
Genes is used to verify that the cells originated from tumor and not from surrounding stroma. Adult melanocytes served as normal control cells for tear and the embryonic carcinoma cell line N-C-C-I-T as positive control for the stem cell marker CD 1 33. These data reveal that some primary cells are indeed positive for tear and therefore of melanocytic origin.
The culture is also negative for CD 83, the marker of mature dendritic cells. Interestingly, this melanoma cell line expresses CD 1 33, a gene crucial for asymmetric cell division, and a known cancer stem cell marker. Shown here is a brightfield micrograph of a primary cell culture highly contaminated with fibroblasts after treatment for fibroblast depletion, the cultures represent primary melanoma cells.
The primary melanoma cells were sorted into CD 1 33 positive and CD 1 33 negative cells. These data from immunofluorescent, staining and western blot analysis indicate high sorting efficacy and purity. The approach has big implications for personalized medicine because now we can use patient derived tissues and cells to better predict the drug response of each patient individually.
This method will help us to answer key questions like, where does the tumor derive from? How can patients be treated? And it will help to better redefine system biology approaches by validating their experiments.
