This protocol described a method for red tumor cell enumeration, and the clinical CTC isolation. To begin, culture the tumor cells MCF-7, MDA-MB-231, and HeLa in a cell-culture flask at one times 10 to the fifth in one-millimeter of DMEM, supplemented with 10%fetal bovine serum, and 1%penicillin-streptomycin. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide.
When the cell lines grow as adherent monolayers to 95%confluence, detach them from the culture dish with 0.25%trypsin solution for two minutes. Stain the tumor cells by adding three-microliters of Calcein AM, and place the dish in the incubator for 30 minutes. At the end of incubation, digest all the cells with trypsin.
Count the culture tumor cells in PBS with a cell-counting chamber, and dilute until 100 tumor cells per one-milliliter of PBS are obtained. Next, introduce the diluted cell suspension into the microfluidic chip using a syringe with a syringe pump at varied flow rates. Obtain the capture efficiency for the various flow rates, and determine the optimal flow rate.
Count the number of tumor cells captured on the chip, and flowing out from the outlet. Calculate the capture efficiency using the given formula. Repeat the procedure to obtain the capture efficiency for different numbers of tumor cells, from 10 to 100.
Test and validate the microfluidic chip for rare numbers of tumor cells. Inject these 10 sample suspensions into the microfluidic chips using a hollow needle made with a micropipette puller to aspirate diluted tumor cells. Detect and enumerate the number of tumor cells for each sample after their capture on the chip.
Next, stain the tumor cells with Calcein AM, and enumerate 100 tumor cells in five-microliters of PBS. Spike these cells into one-milliliter of whole, normal blood sample. Introduce these cells into the chip, and enumerate the number of tumor cells captured on the chip with green immunofluorescence.
Perform in vivo enumeration as described, and calculate the capture efficiency after capture. Enumerate one to 10 tumor cells without staining. Spike these cells into one-milliliter of whole, normal blood.
Introduce these samples into the microfluidic chip, and capture the tumor cells. Add three to five-microliters of fluorescent dye into 20 to 30-microliters of PBS, and introduce this solution onto the chip with a syringe. Enumerate the number of tumor cells captured on the chip with blue and green immunofluorescence to determine the capture efficiency.
Next, modify the surface of the chip with 100-microliters of 4%3-mercaptopropyl trimethoxysilane and ethanol at room temperature for 45 minutes for the affinity-based capture of anti-epithelial cell adhesion molecule. At the end of incubation, wash the chip three times with ethanol. Then add 100-microliters of the coupling agent GMBS, and allow it to interact for 30 minutes.
At the end of incubation, use a syringe to wash the chip three times with one-milliliter of PBS. Treat the chip with 30 to 40-microliters of 10-micrograms per milliliter neutravidin at room temperature for 30 minutes, leading to immobilization of the cells onto the GMBS, and then flush with PBS to remove excess avidin. Modify the chip with three-microliters of anti-biotinylated epithelial cell adhesion molecule antibody, and incubate overnight.
All the tumor cells were captured around the array of the wave chip without any cells missing, indicating the high-capture efficiency of the microfluidic chip. Tumor cells stained with Hoechst and Calcein AM are shown here. CTCs from a gastric cancer patient captured using small ellipse micro-filters are shown here.
Also, CTCs from a colorectal cancer patient captured using trapezoid micro-filters, and emitting both blue and green fluorescence, are shown. High-capture efficiency and viability were observed for tumor cells captured using big ellipse micro-filters. Clinical immunofluorescence analysis of colorectal CTCs captured on the microfluidic chip recognized CTCs as DAPI-positive, CK-positive, CD45-negative, and the WBCs as DAPI-positive, CK-negative, CD45-positive.
Colorectal tumor cells cultured on the big ellipse chip after capture, and colorectal tumor cells captured on the big ellipse micro-filters are shown here. This method of modifying with aptamer provides a new approach to enrich CTC isolation.
