High-throughput Isolation of Circulating Tumor Cells Using Cascaded

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High-throughput Isolation of Circulating Tumor Cells Using Cascaded Inertial Focusing Microfluidic Channel Aynur Abdulla, Wenjia Liu, Azarmidokht Gholamipour-Shirazi, Jiahui Sun, and Xianting Ding Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04210 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Analytical Chemistry

High-throughput Isolation of Circulating Tumor Cells Using Cascaded Inertial Focusing Microfluidic Channel Aynur Abdulla, Wenjia Liu, Azarmidokht Gholamipour-Shirazi, Jiahui Sun, Xianting Ding* State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China Corresponding author: Xianting Ding, PhD Tel: +86-21-62932274, Fax: +86-21-62932274, E-mail: [email protected] ABSTRACT: Circulating tumor cells (CTCs) are rare cells that detach from primary or metastasis tumor and flow into the blood stream. Intact and viable tumor cells are needed for genetic characterization of CTCs, new drug development, and other research. Although separation of CTCs using spiral channel with two outlets has been reported, few literatures demonstrated simultaneous isolation of different types of CTCs from human blood using cascaded inertial focusing microfluidic channel. Herein, we introduce a cascaded microfluidic device consisting of two spiral channels and one zigzag channel designed with different fluid fields, including lift force, Dean drag force, and centrifugal force. Both red blood cells (RBCs)-lysed human blood spiked with CTCs and 1:50 diluted human whole blood spiked with CTCs were tested on the presented chip. This chip successfully separated RBCs, white blood cells (WBCs), and two different types of tumor cells (human lung cancer cells (A549) and human breast cancer cells (MCF7)) simultaneously based on their physical properties. 80.75% of A549 and 73.75% of MCF-7 were faithfully separated from human whole blood. Furthermore, CTCs gathered from outlets could propagate and remained intact. The cell viability of A549 and MCF-7 were 95% and 98%, respectively. The entire separating process for CTCs from blood cells could be finished within 20 minutes. The cascaded microfluidic device introduced in this study serves as a novel platform for simultaneous isolation of multiple types of CTCs from patient blood.

Cancer-related mortality rate has steadily increased through the past decades, and it remains as a major cause of death for humans1. A research carried out by the World Health Organization (WHO) found that at least 30% of mortality was preventable if cancer patients were diagnosed and treated before the germination of metastatic cancer2. Metastasis occurs when circulating tumor cells (CTCs) detach from primary or metastasis tumor and flow into peripheral blood streams through epithelial mesenchymal transition (EMT)3. CTCs are unneglectable markers in cancer prognostics and diagnostics. Furthermore, the number of CTCs in blood facilitates as a predictor of cancer progress. However, due to the low frequency of CTCs (1~10 CTC/mL) occurrence in the peripheral blood of cancer patients and the difficulty in its accurate enumeration and separation, CTC has not been widely used for cancer diagnostics and treatments2. The potential role of CTCs during metastasis process remains unclear. Thus, to facilitate cancer diagnosis, prognosis, treatment and the study of tumor metastasis, efficient and accurate methods for CTCs enumeration, characterization and separation are in high demand4. Many researchers have explored for efficient and reliable CTC separation systems. Technologies based on biological properties such as specific expressions of biomarkers5-7 or physical properties such as size1,8-12 and deformability13-17 of CTCs were developed to separate them from whole blood with high throughput and high purity. One of the most commonly used biological methods is the antibody-based technique18

(including the FDA-approved CellSearch© system, Veridex). CellSearch© system utilizes the anti-EpCAM (epithelial cell adhesion molecule, specific for human breast cancer cells) conjugated magnetic beads for the immunomagnetic capture and isolation of CTCs. This system was regarded as the golden standard for CTCs separation and counting. However, considering the existence of epithelial mesenchymal transition (EMT) and the varying expression levels of EpCAM on different types of tumor cells, a portion of CTCs may be lost during the isolation. Furthermore, to enable follow-up investigations on the collected CTCs, techniques that could gather intact cells without labeling are more appealing. Another category of commonly used methods are the label-free isolation of CTCs, including microfluidic filters19-21, inertial focusing22-24, Deterministic Lateral Displacement (DLD)9,25, acoustics8,26,27, optics28,29, and dielectrophoresis (DEP)30,31. These methods take advantage of the fact that CTCs are larger and stiffer than regular blood cells. However, these methods also have their limitations. Acoustics, optics and dielectrophoresis (DEP) require extra force fields and longer process time. DLD offers a solution for continuously processing cell samples with an increased flow rate to as high as 10 mL/min9. Yet, the clogging issue makes it challenging to deal with concentrated fluids with relatively high cell density. Microfluidic devices using inertial focusing methods take advantage of the hydraulic phenomenon where different sizes of particles/cells occupy different equilibrium positions in the cross section of the microfluidic channel due to the force balance between inertial lift

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force and Dean drag force, and it offers a wide range of flow rate from 5µL/min32 to 8mL/min33. Thus, microfluidic devices using inertial focusing are efficient solutions to achieve a continuous, fast, and high-throughput sized-based separation of CTCs. There has being quite a lot of papers trying to adopt microfluidics for CTC separation. However, previous literatures usually reported separation of one particular type of CTC cells, and few of them demonstrated the separation of multiple types of CTCs simultaneously from real human whole blood. On the other hand, separating multiple CTCs has its practical benefit. For instance, breast cancer patient that has metastasis proliferated to the lung could obtain both CTCs from breast cancer and CTCs from lung cancer26 in blood. Isolation of the two different types of cancer cells provides a potential opportunity for further research of metastatic mechanism in tumor progression. Herein, we provide a solution for this challenge―the separation of two differently sized CTCs from blood cells by proposing a microfluidic chip with three integrated inertial-based channels, allowing for size-based, label-free, high-throughput, and high efficient separation of CTCs in blood cells. The platform demonstrated here integrates multiplex channels onto a single chip and simultaneously separates four different types of cells, namely human lung cancer cells (A549), human breast cancer cells (MCF-7), red blood cells (RBCs), and white blood cells (WBCs) based on their size differences. The cascaded microfluidic chip, consisting of two spiral channels with different dimension and a zigzag channel, has one sample inlet for cell mixture sample at a flow rate of 2mL/min and two buffer inlets for buffer solution a flow rate of 1.2mL/min. Simulated blood, 50 times diluted blood spiked with CTCs and RBCs-lysed human blood spiked with CTCs were used to characterize the performance of the cascaded microfluidic chip. The results for RBCs-lysed blood spiked with CTCs showed that this integrated platform could faithfully separate 80.75% A549 and 73.75% MCF-7. The cell viability of A549 and MCF-7 was 95% and 98%. The whole process for separating A549 and MCF-7 from blood cells using the RBCs-lysed human blood spiked with CTCs sample, including red blood lysis process, was done within 20 minutes. Intact cells were collected from the four different outlets simultaneously. RBCs, WBCs, A549 cells and MCF-7 cells all maintained high viability when using diluted blood and RBCs depleted blood. Thus, the CTCs collected from the outlets of the cascaded microfluidic channels could be used for further genetic characterization and new drug development study34-37. A table summarizing features and performances of existing reports and comparison of the technique presented in this paper is provided as Table S1 in the supplementary information.

EXPERIMENTAL SECTION Chip design Illustration of the chip and experimental procedure is shown in

Figure 1. The inertial-based cascaded microfluidic chip in this study consists of three parts, including two different five-loop spiral channels and one zigzag channel. The setup of the experiment is available in Figure S1.

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Figure 1.Illustration of the cascaded microfluidic channel and the experimental setups. An automatic controller operated three syringe pumps. Blood sample was injected into sample inlet while buffers were into injected from two buffer inlets. Four types of cells (red blood cell, white blood cell, A549, and MCF-7) were collected from the corresponding outlets (outlet 1, 2, 3, 4) simultaneously.

The chip has one sample inlet (named as sample inlet), two buffer inlets ( named as buffer 1 and buffer 2) and four outlets (named as outlet 1-4), as shown in Figure 2A. The chip is separated into three parts (Figure 2B). Part 1 and part 2 are spiral channels, while part 3 is a zigzag channel. The cross-section of part 1 is 300×80µm and the distance between two adjacent loops is 900µm. At the end of the fifth loop of part 1, the channel splits into two branches. According to the experimental results acquired from the CCD camera (Qianlangyan, Revealer 5F04M, Hefei, China), the separation point for part 1 is set at 100µm from the inner wall. The cross section of part 2 is 600×80µm and the distance between two adjacent loops is 1 mm. The dimensions are based on a previous literature34. The width of outlet 2 (closer to the inner wall) is 156µm, while the width of outlet 1 is 444µm. Part 3 is composed of a straight section, a zigzag section and a semicircle section. The cross-section of the straight and zigzag part is 200×80µm, while the radius of the semicircle is 6mm and the width at the end of the semicircle channel is 1.5mm. The width of outlet 3 (closer to the inner wall) is 320µm and width of outlet 4 is 1180µm. Dimensions are all determined and optimized according to experimental observations and numerous simulations (see supporting information for dimension optimizations in Table S2). Operating principle of the chip As shown in Figure 2, the cascaded chip used in this study consists of two spiral channels and a zigzag channel. Spiral channel is one of the most commonly used structures for particle separation1,4,32,34-36 due to the balance between the inertial lift force ( ) and Dean drag force ( )35,37,38. has been known to scale with particle diameter ( ), the maximum fluid velocity ( ) and the lift coefficient ( ), with a relationship described as: =

(1)

Where is the density of the fluid, is the maximum veloc, is the hydraulic diameter of the ity defined as 1.5 is average flow channel defined as 2⁄ . rate , is the channel width, and is the channel height.


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Analytical Chemistry Curvature to the microscale channel36,39 develops a secondary lateral flow known as Dean Flow due to the non-uniform inertial of fluids under a relative high Reynolds number ("# ). Dean Flow results in the formation of two counter-rotating vortices positioned above and below the central plane of the channel. The magnitude of the Dean Flow is described as a dimensionless number # , and the formula for # is given by: #

$ %

& ="# & '(

near the center of the channel (Figure 2C-ii). Thus, with the increase of the "# , small particles move towards the outer wall, while larger particles move towards inner wall (Figure 2C-iii)

(2)

'(

Where,) is the viscosity of the fluid, " is the radius of the channel, "# is the Reynolds number of the channel. The Dean Flow leads to Dean Drag Force ( ), which is generated due to the viscous fluid flow and velocity difference between the center and the edge of the channel. The formula for is described as below: ~ 5.4 , 10./ 0)# 1.23

(3)

In the two spiral channels on the cascaded microfluidic chip, particles or cells are affected by inertial lift force and Dean drag force, and obtain their equilibrium positions owing to the balance of and . Larger particles or cells have equilibrium positions near the inner wall, while smaller ones have equilibrium positions closer to the outer wall. When the samples flow through part 1, larger particles or cells flow along the inner wall and flow into part 3, while smaller ones flow into part 2. Two buffer inlets are used to increase the flow rates in part 2 and part 3. Numerous experimental results indicated that spiral channel is unable to separate particles or cells with sizes of 20µm and 25µm from each other. In our pilot study, DLD chip integrated with two spiral channel was also tested to separate four kind of cells simultaneously. However, DLD chip requires a much lower operation flow rate, which is not compatible with operation flow rate inside the spiral channel. To solve this challenge, a zigzag channel instead of a DLD chip was applied in part 3. The detailed information of the channel dimensions is supplied in the supplementary information as Table S2. In part 3, when particles flow through the zigzag part, they concentrate in the middle of the channel40-42. While traveling around the semicircle, the particles are affected by both centrifugal force (F5 ) and Dean drag force ), and acquire their equilibrium positions due to the balance of centrifugal force and Dean drag force. As described in the previous work36,37,39, F5 is described by: F5

6 7 28

(4)

Where, is the density of particles/cells, is the fluid velocity, 9 is the radius of the equilibrium position of the particles/cells in the semicircle channel. In the middle of the semicircle, Dean Vortices amplify radially outward movement of the particles, and they disturb the movement near the channel. However, when "# is low, all the particles occupy equilibrium positions at the center of the channel (Figure 2C-i). As the "# increases, drag force caused by Dean Vortices is gradually stronger than centrifugal force, and the particles tend to radially move inward of the channel. As a result, small particles move closer to the inner wall while bigger particles remain their original equilibrium positions

Figure 2. (A) Image of the chip, which consists of one sample inlet, two buffer inlets, and four outlets. (B) Corresponding schematic illustration of chip dimension. Width of part 1 and part 2 are 300µm and 600µm, respectively, while the width of part 3 is 200µm with a semicircle radius of 6mm. The height of all parts is 80µm. (C) The separation principle and force analysis in zigzag channel near outlets under three different Reynolds numbers (Re) (i) "# 12.7; (ii) "# 21.1; (iii) "# 33.9.

Chip fabrication Fabrication of the chip followed standard procedure described in previous literature1. The microfluidic channel mold was generated on silicon wafer using standard photolithographic techniques36, 47. The chips were made by casting degassed PDMS in a 10:1 ratio with curing agent (Sylgard 184, DOW CORNING, USA) on the mold and baked in the oven for 45 minutes at 75℃. After baking, the PDMS was peeled from the wafer, cut into shape, cleaned by air blower, and bonded to glass after surface activation with oxygen plasma (Harrick plasma, USA). The chip was then placed in the oven for 30 minutes at 85℃for improved bonding. Polystyrene particles sample preparation To build the cascaded microfluidic system, we first characterized the performance of the three parts separately with polystyrene particles (Shanghai Biochemical Co, Ltd). To determine the sizes of the polystyrene beads used to optimize the chip, we examined the cell sizes for RBC, WBC A549 and MCF-7. According to the previous literatures, the mean sizes of RBC and WBC are around 7.34µm43 and 10~12µm44, while the cell sizes of human lung cancer cells (A549) and human breast cancer cells (MCF-7) are 10~15µm and 15~25µm, respectively25. On the other hand, our measurement through ImageJ© software indicated A549 and MCF-7 cells used in our study had sizes of 14.956±1.759µm (n=25) and 22.364±2.7µm (n=25). Cells applied in our study were consistent with the literatures. Thus, to evaluate the performance of the cascaded microfluidic chip, particles with sizes of 5µm, 8µm, 15µm, and 24µm were used to mimic the behaviors of RBC, WBC, A549 and MCF-7.


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According to numerous simulation results, particle concentrations for 5µm, 8µm, 15µm and 24µm were set to be 5× 105/mL, 5×105/mL, 2×104/mL and 4×104/mL, respectively. Particles were suspended in DI water with 0.01% v/v Tween20. To further mimic the low occurrence of CTCs in blood, we then lowered the concentrations of the big particles by fixing the 5µm and 8µm particle concentrations at both 5×105 /mL, and decreasing the 15µm and 24µm particle concentrations to 1×104 /mL, 5×103 /mL or 1×103 /mL. Cell culture and simulated blood preparation Lung cancer cell line (A549, ATCC, USA) and breast cancer cell line (MCF-7, ATCC, USA) were used in this study to evaluate the hydrodynamic behavior of CTCs in the chip. Cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% penicillin–streptomycin (Invitrogen, USA). The cell culture was kept in a humidified atmosphere at 37°C containing 5% (v/v) CO2 and harvested at 80% confluence. To prepare simulated blood, the RBC and WBC were separated from fresh blood by regular centrifugation method and suspended in Cell cryopreservation solution, then frozen in a -80℃ fridge. RBC and WBC were revived ahead of the cell experiment, suspended, counted by hemocytometer, tested for cell viability using CCK-8, and spiked with counted amount of A549 and MCF-7. To have a better visualization of CTCs, A549 cells were stained with DiI (DiIC18 (3)) while MCF-7 cells were stained with DiO (DiOC18 (3)). The concentrations of the cells were set as the same as polystyrene particles study. Cells were suspended in 0.9% saline, which was also used as buffers for all the experiments unless otherwise stated. Human whole blood preparation Human whole blood was collected from six healthy volunteers with agreements at the laboratory in the Institute for Personalized Medicine, Shanghai Jiao Tong University, China. Each volunteer donated 2 tubes of 3.5 mL blood. One tube of the blood was diluted by 50 times with 0.9% saline and spiked with 100/mL A549 cells and 100/mL MCF-7 cells stained with DiI and DiO respectively. Another tube of blood spiked with 350 DiI stained A549 cells and 350 DiO stained MCF-7 cells was lysed with red blood cells lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at a ratio of 1 mL blood to 3 mL lysis buffer at room temperature on a shaking platform for 10 minutes. Then cells were collected by centrifuging at 1000g for five minutes at room temperature, and the deposited cells were suspended with 0.9% saline making the volume 7mL. Prepared blood samples were placed on ice before injecting into the cascaded microfluidic chip.

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Chip characterization During the experiments, the cascaded microfluidic chip was fixed on a positive microscope (Nikon, Eclipse Ci-S) equipped with a high-speed CCD camera. The cascaded channel was first washed with DI water to remove dusts and to enhance the removal of air bubbles in the chip. All chips and flexible Tygon® tubes that used to connect syringes to the chips were sterilized prior to any cell experiments. During the experiments, blood samples and buffer solution were filled in 10 mL syringe and injected to the chip through syringe pumps (LongerPump). Flow rate for blood sample in part 1 was set to 2mL/min, while flow rate of the buffers in part 2 and 3 was set to 1.2mL/min. Videos were captured at the four outlets and at the end point of the first spiral channel, and then analyzed through the ImageJ® software. RESULTS AND DISCUSSION Separation efficiency of polystyrene particles in the zigzag channel Herein, we mainly described the optimization process of the zigzag channel (Figure 3A). The optimization process of the spiral channel is similar as previous literatures and available in supplementary materials as Figure S2 and Video S1. The width of the straight section is 200µm, the radius of the semicircle section is 6 mm and the height is 80µm (Figure 3B). Under low Reynolds number ("# 12.7, corresponding to the flow rate of 0.6mL/min), 15µm and 24µm particles focused near the center of the semicircle. With the increase of the Reynolds number to 21.1 (corresponding to the flow rate of 1mL/min), 15µm particles moved towards the inner wall and 24µm particles maintained their positions near the center reaching their longest distance as shown in Figure 3C and supplementary Video S2. When the Reynolds number exceeds 33.9 (corresponding to the flow rate of 1.8mL/min), 15µm particles moved towards the outer wall and 24µm particles moved to the inner wall. The distances between 15µm particles and 24µm particles under different flow rates were analyzed and shown in Figure 3D. The largest separation distance was acquired at the flow rate of 1 mL/min. At this flow rate, the separation efficiency reached 97 % (Figure 3E) as indicated by flow cytometry analysis (FACSCantoll, BD Biosciences, USA).

Fluorescent staining Fluorescent staining was performed to facilitate better distinguish between A549 and MCF-7 in outlet 3 and outlet 4. A proportion of A549 and MCF-7 were harvested and diluted in DiI and DiO to make the cell concentrations as 1x106/mL. Cells were incubated with cell membrane fluorescence dyes for 40 minutes in a humidified atmosphere at 37°C containing 5% (v/v) CO2. After the incubation, cells were centrifuged at 1000 rpm for 5 minutes. Supernatant liquid was removed and the cells were washed with 37°C DMEM for three times.


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Analytical Chemistry ed microfluidic chip. The simulation results suggested that when the three parts are assembled together, the flow rate for sample inlet, buffer 1 and buffer 2 need to be adjusted to1.8 mL/min, 0.8 mL/min and 1.2 mL/min respectively in order to maintain the same separation efficiencies as the individual parts. Thirdly, we experimentally verified the simulation results and further optimized each inlet flow rate according to the experimental separation efficiency.

Figure 3. (A) Image of the zigzag channel that consists of one inlet and two outlets. (B) Dimensional illustration of the zigzag channel. (C) Movement of the 15µm particle (circled) near outlet at 1.0mL/min flow rate. Time ranges from 144ms to 152ms after particle injection. (D) Relationship of flow rates and distance between particle equilibrium position and the channel inner wall for 15µm and 24µm particles (n=3). (E) At the flow rate of 1.0mL/min, 97.5% of the particles from the inner outlet were 15µm, while 96.4% of particles from the outer outlet were 24µm. Data are presented as mean±s.d. (n=3).

Simulation of velocity distribution in integrated cascaded chip Since the integration of the three counterparts leads to the reestablishment of the pressure distribution in each part, two buffer inlets were designed to compensate the flow rate change due to this ensemble. To facilitate the decision of the flow rates for each buffer inlet and the sample inlet on the integrated cascaded chip, we investigated the flow rate distribution across the integrated chip using COMSOL© Multiphysics 5.2 software. The goal of this simulation is to provide fundamental hints to guide our experimental setup. The procedure to optimize the design of the cascaded chip is as follows: Firstly, we individually optimized the three counterparts for our cascaded microfluidic chip purely based on our experimental observations. The first counterpart is the 300× 80µm spiral channel (Figure 4A), which separates particles larger or smaller than 12µm at the flow rate of 1.8mL/min. The second counterpart is the zigzag channel (Figure 4B), which separates 15µm and 24µm particles at the flow rate of 1mL/min. The third counterpart is the 600 × 80µm spiral channel (Figure 4C), which separates 5µm and 8µm particles at the flow rate of 2.8 mL/min. Secondly, we integrated the three individual counterparts together (Figure 4D). The geometries of channel, flow conditions, and properties of suspending liquid medium in the integrated microfluidic chip are the main factors that would influence particle separation efficiency45. Therefore, we adopted the simulation at this stage. The simulation results facilitate us to determine at which flow rate for each inlet, the integrated channel will attain the same fluid dynamics as individual counterparts. The simulation results serve as consultations for us to build up the integrated cascad-

Figure 4. Flow rate simulation conducted by COMSOL© Multiphysics 5.2. (A) Simulation of flow rate in the single narrower spiral channel, which has the same dimension as part 1. (B) Simulation of flow rate in the zigzag channel, which has the same dimension as part 3. (C) Simulation of flow rate in the wider spiral channel, which has the same dimension as part 2. (D) Simulation of the optimal flow rate in the integrated cascaded channels. The three pointed position showed the same flow rate distribution inside the integrated channel as to that in each individual counterparts.

Separation efficiency of particles in the cascaded channels According to the simulation results, we experimentally tested sample inlet flow rate from 1.8mL/min to 2.2mL/min, buffer 1 flow rate from 0.6mL/min to 1.2mL/min, and buffer 2 flow rate from 0.6mL/min to 2.2mL/min. Polystyrene particles with sizes of 5µm, 8µm,15µm, and 24µm were assigned concentrations at 5×105/mL, 5×105/mL, 2×104/mL and 4× 104/mL, respectively. As experimental results suggested, 2mL/min for sample inlet and 1.2mL/min for both buffer inlets provided the optimal particle separation efficiency (Figure 5). Samples from four outlets were collected and analyzed by flow cytometry. Figure 5A represented the proportion of each particle in the initial sample. In outlet 1, 94.78% of the particles were 5µm (Figure 5B). In outlet 2, 80.79% of the particles were 8µm and 15.92% of particles were 5µm (Figure 5C). In outlet 3, 75.04% of the collected particles were 15µm (Figure 5D). In outlet 4, 84.4% of the particles were 24µm, containing low amount of 5µm and 15µm (Figure 5E). All the four types of particles were faithfully separated from one another (Figure 5F). We then further evaluated the separation efficiency by fixing the concentrations for 5µm and 8µm particles at 5× 105/mL and decreasing the concentrations for 15µm and 24µm particles to 1x104/mL, 5x103/mL and 1x103/mL to mimic the rare presence of the CTCs in real scenarios. The flow cytome-


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try analysis indicated that at these reduced concentrations, the separation efficiencies of the four types of particles were nearly the same (Figure S3, S4, S5). These experimental evidences suggested that the cascaded chip could faithfully separate the four types of particles from each other (Table S3).

Figure 5. (A) Scatter plots from flow cytometry showing the proportion of initial particles (5µm, 8µm, 15µm, 24µm) in the inlet sample. (B) ~ (E) Scatter plots from flow cytometry showing the results of separated particles in the cascaded channel when flow rate was 2mL/min for sample inlet and 1.2mL/min for the buffer inlets. (F) Particle size distribution in each outlet. Data are presented as mean±s.d. (n=3). Separation efficiency of CTCs using human blood sample Human blood samples were prepared as described in the Method section. The experimental setup is shown in Figure S1. Flow rates for the three inlets were set the same as the previous experiments for polystyrene particles. To obtain a comprehensive evaluation of the chip performance, three set of experiments were performed. First of all, the simulated blood sample was used to evaluate the separation efficiency of the cascaded microfluidic chip. As shown in Figure S6A-i (the bifurcation of part 1), the MCF-7 cells and the A549 cells flowed close to the inner wall and flowed together into part 3. Near the outlets of part 3, A549 cells flowed towards outlet 3 while most of the MCF-7 cells flowed towards outlet 4 (Figure S6A-ii). Distribution of WBCs and RBCs were nearly the same as the 5µm and 8µm polystyrene particles in part 2 (Figure S6A-iii). Viability of cells collected from each outlet were tested by CCK-8. Samples collected from outlet 3 and outlet 4 were cultured in 96 wells for further observation of the cell viability after flowing through the chip. The results of cell viability in outlet 3 (mostly A549) and outlet 4 (mostly MCF-7) after going through the chip at 0 hour, 24 hours and 48 hours, were also tested and shown in supplementary information as Figure S7, which indicated that the few blood cells left in outlet 3 and outlet 4 had little impact on the growth of CTCs. Viability of cells in outlet 1 (mostly RBCs) and cells in outlet 2 (mostly WBCs) had low viability (Figure S8). This low viability of RBCs and WBCs after the chip separation was consistent with the viability testing results for BRC and WBC before the experiment. Note the RBCs and WBCs were frozen prior to the experiment, the low viability were likely due to the freeze-and-thaw operation. We then further evaluated the performance of the chip using diluted

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fresh human blood spiked with CTCs. When the 10x diluted blood was injected to the channel, blood cells flowed everywhere in part 1. A captured image of 10x diluted blood sample flowing through the first bifurcation is provided as Figure S9 in the supplementary information. It is noticeable in the image that blood cells covered the equilibrium positions of the cancer cells, which made it prohibitive to separate cancer cells from blood cells. In order to attain better separation efficiency, we further increased the dilution times from 10x to 50x. When the dilution factor increased to 50x, most of the blood cells flowed into part 2, while cancer cells flowed into part 3(Figure S6-B). At this dilution factor, the flowing condition of MCF-7 and A549 was the same as the simulated blood. The viability of cells collected from each outlet was also tested and shown in Figure S10-A, and our results indicated that over 90% of all the cells in 4 outlets were alive compared to the simulated blood. However, to separate cancer cells from 50x diluted blood (175mL after dilution) took 87.5 minutes. Finally, RBCs-lysed human blood sample spiked with CTCs were used to evaluate the separation efficiency of the chip. Similarly, cancer cells flowed close to the inner wall of part 1 and flowed into part 3, while most of the WBCs flowed into part 2. Most of the A549 flowed toward the outlet 3 and MCF-7 flowed to the outlet 4(Figure 6-A and supplementary Videos S3, S4, and S5). Figure 6B showed the RBCs-lysed human blood spiked with CTCs (A549 and MCF-7) and samples collected from four outlets. As shown in figure 6C and figure 6D, the fluorescent microscope images indicated that most cells in outlet 3 were A549, while most of the cells in outlet 4 were MCF-7. Cells from each outlet were collected and tested for their viability by CCK-8. As shown in Figure S10-B, about 90% of the cells in outlet 3 and outlet 4 maintained viability. Meanwhile, as Figure 6-E indicated 80.75% A549 and 73.75% MCF-7 were successfully separated from the inlet sample. The composition of cell populations shown in Figure 6-F indicated that for outlet 3, 17.4% of the cells were WBCs, 63.6% of the cells were A549 and 19.3% were MCF-7. For outlet 4, 18.3% of the cells were WBCs, 13.0% of the cells were A549 and 68.7% were MCF-7 in. For outlet 1, only trace amount of WBCs and cell fragments of unwashed RBCs were presented. For outlet 2, most of the cells were WBCs with only three or four out of 4000 events are CTCs. Both bright field images and fluorescent images of each outlet are available as Figure S11. The separation efficiency and purity were calculated using the formulas given in the supplementary information (supplementary information). Moreover, the whole cell separation process including the red blood lysis stage was finished within 20 minutes. According to the previous literature26 and our experimental results, the red blood lysis solution did not affect cell viability and cell morphology. Considering the aim of this microfluidic chip is to separate intact, viable cancer cells in a label-free, high throughput way, depletion of red blood cells is a more desired approach than blood dilution, which is more time consuming.


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Figure 6. RBCs-lysed human blood spiked with A549 and MCF-7 was applied to test the cascaded microfluidic chip. (A) High speed microscopy images captured at the bifurcations for part 1 (i), outlets 1 and 2 (ii), and outlets 3 and 4 (iii) indicate the separation of RBCs, WBCs, A549 and MCF-7 at a sample flow rate of 2 mL/min and buffer flow rate of 1.2mL/min (videos are available as supplementary Video S2, S3, S4). (B) Comparison of initial sample (RBCs depleted blood sample) and collected samples from each outlet. Fluorescent microscopy results for (C) cells collected from outlet 3 and (D) cells collected from outlet 4. A549 was stained with DiI (red fluorescence) and MCF-7 was stained with DiO (green fluorescence). Scale bar is 100µm. (E) Separation efficiency of A549 and MCF-7 using RBCslysed human blood. (F) Purity of cells in each outlet. Data are presented as mean±s.d. (n=6). The Supporting Information is available free of charge on the ACS Publications website.

CONCLUSIONS In this study, we demonstrated an effective platform for simultaneously isolating four types of human cells with varied hydraulic diameters on one chip that was assembled with three cascaded channels designed with different fluid field behaviors. The cascaded microfluidic channels presented in this paper enabled label free, fast, and continuous separation of CTCs from diluted human whole blood, lysed human blood and simulated human blood. Separation of particles and cells in this chip relied on the dimensions and flow rates, which would affect the inertial lift force, Dean drag force, and centrifugal force. CTCs separated by this chip maintained high viability. The technique introduced in this study offered opportunities for further DNA or drug development research on the isolated tumor cells. Four types of different sized particles or cells could be separated simultaneously with high throughput, high separation efficiency, and decent cell viability. Separation efficiency of the zigzag channel was 97%, while separation efficiency of the 15µm and 24µm polystyrene particles and cells in the cascaded channel was slightly lower than the single channel. The separation performance of the cascaded microfluidic chip could still be improved with further improvements.

ASSOCIATED CONTENT

Figure S1. Image of the experimental system setup; Figure S2. Separation efficiency of 5µm and 8µm particles; Figure S3~Figure S5. Scatter plots captured using flow cytometry showing the proportion of particles (5µm, 8µm, 15µm, 24µm) in each outlet using different concentrations; Figure S6. Highspeed microscopy image captured at the bifurcation of part 1 and four outlets bifurcation for simulated blood and 50x diluted blood; Figure S7~Figure S8. Cell viability bar charts; Figure S9. High-speed microscopy image captured at the bifurcation of part 1 using 10x-diluted blood; Figure S10. Cell viability bar charts using different blood samples; Table S1. Features and performances of previous reports and the current design; Table S2. Dimensions of single zigzag channel used to optimize the chip in this study; Table S3. Proportion of particles in each outlets at different concentration. (PDF) SI movie 1. 5µm and 8µm particles flowed in single spiral channel near outlets at 2.8 mL/min flow rate (AVI) SI movie 2. 15µm and 24µm particles flowed in single zigzag channel near outlets at 1 mL/min flow rate. (AVI) SI movie 3. RBCs depleted blood spiked with A549 cells and MCF-7 cells flowed in the cascaded channel. The video was captured at the first bifurcation. (AVI) SI movie 4. RBCs depleted blood spiked with A549 cells and MCF-7 cells flowed in the cascaded channel. Video of the position near outlet 3 and outlet 4 of the cascaded channel. (AVI)

Supporting Information


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SI movie 5. RBCs depleted blood spiked with A549 cells and MCF-7 cells flowed in the cascaded channel. Video of the position near outlet 1 and outlet 2 of the cascaded channel. (AVI)

AUTHOR INFORMATION Corresponding Author * [email protected]

Tel: +86-21-62932274

ACKNOWLEDGMENT This work was supported by Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01 and 17DZ2203400). This work was supported by Chinese Ministry of science and technology (Grant No. 2017ZX10203205-006-002).

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High-throughput Isolation of Circulating Tumor Cells Using Cascaded

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