The overall goal of this technique is to effectively capture viable circulating tumor cells or CTCs from whole blood. These CTCs can be placed into culture or be used for downstream analyses which require non fixed samples. This method can help answer key questions in the field of metastasis.
It can also be used to follow disease progression, response to therapy and evaluate risk of recurrence. The advantage of this technique is the ability to perform label-free capture of viable CTCs from any solid tumor while remaining independent of biomarker expression. We first had the idea of releasing CTCs from the filter because we wanted to do downstream RNA analysis and culture of CTCs, instead of culturing them in-situ on the filter.
To begin the experiment, weigh enough PIPAAm to prepare a 10%weight per volume solution in a 15 milliliter tube, containing butanol. Vortex the tube until the solution is clear. Next, cut plastic microscope slides into roughly 12 millimeter by 12 millimeter squares with a pair of scissors.
Then, using a sharp pair of straight edge scissors, cut a slot-pore microfilter wafer into eight millimeter by eight millimeters squares and place these pieces directly onto the plastic squares. Using a polyimide film tape, secure the cut filters onto the plastic squares, only on the edge and the corner of the filter so that at least, seven millimeter by seven millimeter filter area is not covered by the tape. Place the microfilter that is secured on the plastic square onto the vacuum chuck of the spin coater.
Program the spin coater to the following recipe. Then, with a standard plastic transfer pipette, dispense enough 10%weight per volume PIPAAm solution to completely cover the microfilter surface and start the spin coater. After the coating is completed, remove the PIPAAm coated microfilter from the spin coater and leave the filter attached to the plastic square during storage.
Move on to filtration cassette assembly and rehydrate the PIPAAm coated microfilter at room temperature by placing the microfilter into a petri dish. Add 1X PBS until the microfilter is fully submerged. After the hydration step, release the filter from the plastic square by peeling or cutting the polyimide film tape from the filter.
Next, assemble the microfilter into the filtration cassette by sandwiching the microfilter between the top and bottom acrylic piece along the PDMS pieces which act as a gasket. Then, clamp the acrylic cassette with clips to secure the filter, to provide a tight seal. Warm three milliliters of McCoy's cell culture medium to 37 degrees celsius.
Add 7.5 milliliters of Hank's balanced salt solution or HBSS to the 7.5 milliliters of blood sample and aspirate this diluted blood into a 25 milliliter syringe. After identifying the top of the cassette, engage the filtration cassette with the syringe and place it onto the syringe pump. Set the syringe pump to a 75 milliliter per hour flow rate.
Place a 50 milliliter tube at the bottom to collect the flow through and start the pump. After filtration is complete, disengage the filtration cassette, taking note which side is the top that has the cells caught in the PIPAAm coated surface. Aspirate one milliliter of the warm culture medium into a new syringe.
Again, determine the top of the cassette and then re-engage the filtration cassette with the syringe containing the medium and engage the bottom end of the cassette so that the PIPAAm coated surface of the filter is facing away from the syringe. Then place the syringe back onto the syringe pump and hold the six well plate right under the filtration cassette. Set the flow rate and start the pump.
Pass all the medium through the filter and collect the flow through in the petri dish. Wait for all the medium to pass through, then stop the pump and remove the syringe and filtration cassette from the pump. Next, disengage the filtration cassette from the syringe and open it to retrieve the filter from the cassette.
Place the filter with the PIPAAm surface facing down into the same petri dish used to collect the reverse flow when releasing the cells from the pores. Add the rest of the warm medium and place the petri dish into a culture incubator, set at 37 degrees celsius. Once the filter has been retrieve from the cassette, it is critical to place it in the plate with the cells facing down and if needed, gently shake it in the media to ensure the cells are detached from the filter.
After 24 hours in culture, carefully remove the microfilter using forceps and discard the filter. Gently resuspend the erythrocytes and peripheral blood mononuclear cells or PBMCs with a 1000 micro liter pipette. It is critical to be extremely gentle when resuspending the erythrocytes and PBMCs to avoid pipetting out the CTCs.
Carefully remove the entire medium containing the resuspended erythrocytes and PBMCs while leaving behind the attached cancer cells and substitute the cells with fresh medium, pre-warmed to 37 degrees celsius. Proceed with CTC viability evaluation. Using healthy donors blood spiked with cultured cancer cells, the thermal responsive technique for release of viable circulating tumor cells or CTCs, achieved capture, release and retrieval efficiency of 94%82%and 77%respectively.
The release and retrieval efficiency of uncoated filters were significantly lower. A live dead assay was performed to evaluate the cell viability before the spike into blood and after release. The cells remained viable post release and expanded in culture rapidly.
SKBr-3 cells were retrieved from blood by the PIPAAm coated slot filter and plated on a 48 well plate. At 16 hours in a culture, tumour cells adhere to the culture plate along with a hypotonic erythrocytes and leukocytes which settled at the bottom of the plate. Post-wash non-adherent cells were removed only leaving adhering tumor cells on the plate.
The same filters can be used for fixed cell capture, both for enumerating and molecular characterizations of CTCs to track progression of cancer and better cancer management. After its development, this technique paved the way for the researchers in the field of CTCs enabling functional characterization of these cells to explore future drug targets while taking a step towards personalized medicine.
