PDAC Stem Cell Isolation: A Method to Isolate Cancer Stem Cells from Pancreatic Tumors
筆記録
– Pancreatic cancer stem cells or CSCs are a subset of self-renewable cancer cells and can give rise to the entire tumor. CSCs reside in a well-protected niche consisting of other cells, such as fibroblasts, immune cells, endothelial cells, and extracellular matrix components, such as cytokines and growth factors. The interactions happening between the cells in such an environment help CSCs propagate and increase tumor growth.
To study the properties of CSCs, we isolate them from the pancreatic tumors. Begin by taking the cell suspension from human pancreatic tumor tissue in a CSC growth media. Next, seed the suspension, consisting primarily of fibroblasts and CSCs, on a gelatin-coated plate and incubate it for the desired duration. Fibroblasts adhere to the gelatin surface, leaving the free-floating CSCs in the cell suspension.
Take a small quantity of cell suspension and add trypan blue. Pipette this mixture to a hemocytometer and count the number of viable cells. Trypan blue enters the dead cells and stains them blue leaving the live cells colorless. Finally, store the CSCs for further analysis.
In the following protocol, we will isolate the CSCs from pancreatic tumors.
– To isolate cancer stem cells from a pancreatic ductal adenocarcinoma in a sterile biosafety cabinet, use a sterile scalpel and forceps to mince the human tumor in a 60 x15 millimeter culture dish containing one milliliter of sterile PBS, adding another three to four milliliters of PBS as necessary, until the tissue is completely dissociated.
Transfer the tissue suspension into a sterile conical tube and add PBS to a final volume of five milliliters. Then, mechanically homogenize the sample with the dissociator, centrifuge, and digest the homogenized tissue with collagenase at 37 degrees Celsius.
After an hour, centrifuge the cells, resuspending the pellet in 10 milliliters of complete medium. Filter the cell suspension through a 40-micrometer strainer and spin down the cells again, this time resuspending the pellet in five milliliters of ACK.
After five minutes at room temperature, remove the red blood cell lysis buffer by centrifugation and resuspend the pellet in cancer stem cell medium. Then, incubate the cells on a gelatin-coated dish at 37 degrees Celsius to remove most of the quickly attaching fibroblast cells. After one hour, recover the supernatant and quantify the number of viable pancreatic cancer cells.
