Microfluidics-based High-throughput Circulating Tumor Cell Sorting and Single-cell Sequencing Technology

We focus on developing high performance CTC isolation and analysis methods to advance precision oncology through liquid biopsy. Current commercial CTC isolation platforms have low throughput and efficiency. They are also incompatible with downstream analysis platforms, which limits biological information recovery.

This study uses a microfluidic CTC isolation platform to greatly improve the detection rates. We also apply a rare cell single cell sequencing chip for deeper liquid biopsy analysis. To begin, pipette 25 microliters of washed and resuspended streptavidin modified magnetic beads into a tube containing one microgram of biotinylated EpCAM antibody.

Incubate the mixture at room temperature while rotating at 20 revolutions per minute for 40 minutes to prepare the immuno magnetic beads. Using a magnetic rack, separate the beads from the supernatant. After removing the supernatant, resuspend the beads in 25 microliters of isolation buffer.

Pipette 20 microliters of the magnetic bead suspension within one to two seconds, ensuring no air bubbles are introduced. Immediately place the chip vertically on a magnet and allow the beads to settle for five minutes without disturbance. Collect four milliliters of the blood sample into a syringe, ensuring air bubbles are removed.

Seal the chip's inlet and outlet with liquid to eliminate air bubbles, then insert the sample inlet and outlet tubes into the chip. Using a syringe pump, inject the sample at a flow rate of 1.5 milliliters per hour. After loading the sample, inject 60 microliters of DPBS into the HB chip at a flow rate of 0.2 milliliters per hour to wash off unbound cells.

Remove the magnet and manually inject 1.5 milliliters of 5%BSA to wash the chip and release captured tumor cells. Prepare a 10%MPTS solution in ethanol. Immediately introduce 20 microliters of the solution into the HB chip and incubate at room temperature for one hour.

Rinse the HB chip once with anhydrous ethanol, then dry it at 100 degrees Celsius for one hour. Next, prepare GMBS solution in ethanol at a concentration of 0.5 milligram per milliliter. After cooling the chip to 37 degrees Celsius, introduce the GMBS solution and incubate.

Rinse the HB chip two times with double distilled water, followed by two rinses with DPBS. Immediately add 15 micrograms per milliliter of streptavidin into the HB chip and incubate at room temperature for one hour or overnight at four degrees Celsius. After incubation, rinse the HB chip two times with DPBS saline.

Now prepare the CD45 antibody buffer by adding 0.2%BSA and 20 micrograms per milliliter biotinylated CD45 antibody to DPBS and adjust to the final volume. Inject 20 microliters of the CD45 antibody buffer into the HB chip for negative selection of white blood cells. After a one-hour incubation at room temperature, rinse the chip with DPBS.

Add a blocking solution containing 3%BSA and 0.05%Tween 20 into the HB chip. Aspirate the prepared sample into a syringe, ensuring no air bubbles are present. Then seal the chip's inlet and outlet with liquid and connect inlet and outlet tubes to the chip.

Using a syringe pump, inject the sample at a flow rate of 0.6 milliliters per hour. Collect purified tumor cells from the chip outlet for counting and single cell sequencing. Pipette 200 microliters of 0.5%F-68 solution prepared in DPBS into the chip inlet.

Perform water bath sonication while holding the chip. When bubbles are visible across the microporous region, continue sonication for 30 seconds to remove bubbles from the dual wells. Next, inject 200 microliters of tumor cell suspension into the chip containing 60, 000 dual wells.

Gently pipette the cell suspension in the chip up and down twice. Then place on a de-colorizing shaker again for five minutes to resuspend uncaptured cells and allow them to settle again. Now add 200 microliters of DPBS and 0.5%F-68 solution through inlet and aspirate from outlet.

After resuspending the barcoded bead suspension, immediately inject 200 microliters of suspension into the chip inlet and shake at 10 revolutions per minute for 20 seconds. Gently pipette bead suspension twice before placing it back on the shaker. Now withdraw barcoded bead suspension from outlet, then pipette 200 microliters of 20X Tris-EDTA and 50 millimolar dithiothreitol solution.

Withdraw liquid from outlet. For cell lysis and MRNA capture, slowly add 200 microliters of cell lysis buffer into the chip inlet. Immediately add 200 microliters of mineral oil to seal the dual wells.

Remove solution flowing from chip outlet into waste reservoir. Then place the chip horizontally and let stand at room temperature for five minutes. Slowly add 200 microliters of 6X saline sodium citrate solution into inlet.

Remove the waste liquid and aspirate the remaining solution from chip outlet. Slowly add 200 microliters of 6X saline sodium citrate to fill chip. Hold a magnet near chip surface and move it slowly from inlet to outlet to gather barcoded beads.

Quickly aspirate solution and beads into a centrifuge tube containing 6X saline sodium citrate. Wash the barcoded beads three times with 200 microliters of 6X saline sodium citrate, followed by once with reverse transcription buffer. A uniform distribution of immunomagnetic beads was observed under a magnetic field while minimal residual beads after magnetic removal confirmed successful release.

The CTC isolation chip efficiently captured tumor cells from both 1 milliliter and 10 milliliter samples. The addition of the purification chip further increased tumor cell purity. In peripheral blood samples spiked with minimal LNCaP cells, the CTC sorting system maintained high capture efficiency and purity.

The single cell barcoding chip achieved an 85.6%cell and 95.7%barcoded bead occupancy ratio, resulting in a pairing rate of 81.9%Increasing the number of loaded cells improved the micro well occupancy ratio without reducing capture efficiency. The integrated CTC isolation and single cell sequencing workflow produced highly pure tumor cells and accurately retained the original tumor cell ratios. TSNE analysis distinctly separated PC3, LNCaP, and Jurkat cells into three clusters, each characterized by unique marker expression.

The Jurkat cell cluster was identified by strong expression of TRBC1, IGLL1, CD1E, and CD3D, confirmed by pathway enrichment for T-cell activation. PC3 and LNCaP clusters were distinguished by differentially expressed genes with PC3 showing high microseminoprotein expression and LNCaP expressing NEDD4.