Circulating Tumor Cells entrapment using microfluidics

• High-throughput circulating tumor cells entrapment
• Accurate circulating tumor cells enumeration
• Early prostate cancer diagnosis
• New chip generation opportunities for other cell types (channel sizes, channels number, multiple chips connected…)

Materials

Set-up

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Methods

The CTC-HTECH chip is composed of one polydimethylsiloxane block (PDMS, polymer) obtained by soft-lithography and bonded to a glass slide by plasma treatment. The PDMS block contains a network of 6 straight parallel canals linked by a series of 40 constriction channels. Showed in the subsection “Materials”, constriction channels are also displayed on Figure 2, with an example of 2 parallel channels (red and blue).

Each constriction channel is composed of 2 trapping chambers (Figure 2d). As cancer cells  have a similar size to trapping chambers, they are deformed in the constriction channels and recover their shape within the cavities of the trapping chambers. The single Circulating Tumor Cells are captured in those trapping chambers, while the rest of the cells pass through the microchannels with the blood flow. After all of the blood sample has passed, chambers containing CTC are scanned (GFP fluorescence), allowing to determine the total number of CTCs. This unique feature is not available in other size-based CTC microfluidic chips (cf. Review CTCs).

A constant pressure of 500 mbar was applied to the blood sample reservoir using the OB1 Mk3 Pressure Controller. The resulting flow rate of the blood sample was ~ 2.4 mL/hour. By varying the supplied pressure, the OB1 pump maintains a constant liquid pressure in the system. This results in a variation of the flow rate as the cells become trapped in the microchannels but avoids excessive pressure that imposes additional mechanical stress and thus damages the cells (as syringe pumps do). Indeed, the syringe pump produces constant flow rate mode, guaranteeing the efficiency of most microfluidic devices with a limited number of microchannels. However, by increasing the numbers of microchannels in the CTC-HTECH chip, the pressure is redistributed, causing mechanical drag forces on the trapped cells, altering cytoskeleton strength, membrane stiffness, and biomechanical properties.

Figure 3a shows the CTC-HTECH device composed of the 6 rows ①-⑥ previously mentioned, where row ① is closest to the blood inlet port. The outlets 1-6 were defined as waste collection sites from rows ①-⑥, respectively. Only one outlet was opened during each trial. For example, when outlet 1 is opened, outlets 2 to 6 are closed, the Circulating Tumor Cells are trapped only in the trapping chambers of the row ①, while outlet 1 collects other blood cells (Figure 3a). When outlet 2 is opened, the CTCs are trapped in rows ① and ②, and outlet 2 collects other blood cells.

Figure 3 : (a) Illustration of the configuration of the device (not to scale) with the inlet connected to Elveflow’s OB1 Mk3 Pressure Controller ; each row and each outlet was assigned and labeled individually
(b-g) The GFP+ LNCaP-C4-2 prostate cancer cells
(b) Image of inlet with a GFP+ cell starting to enter row 1
(c) GFP+ cell trapped in row 1 after the blood flow ceased
(d) Image of the waste collection at outlet 1
(e) GFP+ cell deforming and passing through row 2
(f) Two GFP+ cells in row 3 with one cell still passing, and one cell exiting this row
(g) GFP+ cell trapped in the trapping chamber of row 4
(h) Overall capture efficiency of each outlet. The data presented here is from 3 or 4 runs on CTC-HTECH device

The main conclusion of the previous figure is that when outlet 1 was opened and only row 1 was used for capture, the trapping efficiency was 46.3% (Figure 3h green bar) ; when outlet 6 was open and rows 1–6 were used for capture, the trapping efficiency was 97.9% (Figure 3h red bar). Five or six rows of micro constriction channels are needed in order to reach a capture ratio >95%.

The authors suggested that as the number of rows increases, the overall fluidic resistance also increases and, therefore, the trapping efficiency in each row varies. Thus, when only outlet 1 was used, the low fluid resistance led to higher flow rate which was too fast for all cancer cells to be captured. Therefore, using only a couple of rows will significantly decrease the number of trapped CTCs.

In this publication, Xiang Ren et al. presented a new size-based CTC entrapment chip increasing the capture efficiency . This low-cost system allows a trapping efficiency of 96%. This is significant as the number of Circulating Tumor Cells has been shown to be diagnostic or prognostic marker for tumor. The microchip run time was ~30 min for a 1.2 mL blood sample. Compared to magnetic particles with antibody methods, such as FDA approved CellSearch system, the CTC-HTECH reduced the lengthy sample pre-processing and long analysis time of ~4-6 hours.

The efficiency of this chip is guaranteed by the used of the OB1 Mk3 Pressure Controller. It can be used for CTC enrichment and entrapment for clinical diagnosis using liquid biopsies. As a customizable device, this chip can be extended following the same design for better capture efficiency, changing channel sizes, channels number, multiple chips connection…

References