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Efficient separation of tumor cells from untreated whole blood using a novel multistage hydrodynamic focusing microfluidics
Talanta 207 (2020) 120261
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Efficient separation of tumor cells from untreated whole blood using a novel multistage hydrodynamic focusing microfluidics
T
Rongke Gaoa, , Lei Chenga, Shiyi Wanga, Xiaobai Bia, Xueli Wanga, Rui Wangb, Xinyu Chene, ⁎⁎ ⁎⁎⁎ Zhengbao Zhad, Feng Wangd, Xiaofeng Xuf, Gang Zhaoc, , Liandong Yua, ⁎
a
School of Instrument Science and Opto-electronic Engineering, Hefei University of Technology, Hefei, 230009, China Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China c School of Biomedical Engineering, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, China d School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China e Department of Electrical and Computer Engineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA f Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China b
ARTICLE INFO
ABSTRACT
Keywords: Microfluidic chip Circulating tumor cells Label-free Hydrodynamic focusing Multistage microfluidics
Significant progress on circulating tumor cells (CTCs) has profound impact for noninvasive tumor profiling including early diagnosis, treatment monitoring, and metastasis recognition. Therefore, CTCs based liquid biopsy technology is taking a rapid growth in the field of precision oncology. The label-free approaches relied on microfluidic chip stand out from a crowd of methods that suffer from time consuming, extensive blood samples, lost target cells and labor-intensive operation. In this paper, a label-free separation microfluidic device was developed using multistage channel, which took full advantage of inertial lift force. Our strategy demonstrated CTCs were efficiently isolated from untreated human blood samples including antibody conjugation and erythrocyte lysis. This device was applied for isolating human brain malignant glioma cells that were spiked in human peripheral blood samples. The experimental condition was optimized and exhibited an average separation efficiency of ≥ 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is > 95%, which was better than other one-step CTCs separation methods. Furthermore, the CTCs were successfully separated from untreated clinical blood sample of cancer patient on the proposed microfluidic device. The entire experimental procedures are extremely low-cost and easy manipulation. It is believed that the proposed multistage microfluidic chip can become a promising tool for CTCs separation and early diagnosis of cancer.
1. Introduction Circulating tumor cells (CTCs) are rare cell population that shed from original or metastatic tumors and circulated in the peripheral blood. As potential biomarkers of non-invasive cancer detection, CTCs play a key role in cancer therapy since it can apply for early diagnosis of metastasis [1–4]. Owing to the extremely low concentration of CTCs in human blood, enrichment is an essential pretreatment step for effective detection of CTCs. Micromanipulation techniques based on microfluidics can isolate individual cells for further biological analysis. Compared with traditional methods, microfluidic devices have a variety of advantages, such as high throughput, efficiency and sensitivity [5–7]. Besides its significance on clinical application, CTCs
enumeration can also be used to evaluate the efficacy of therapeutic treatment of cancer [8]. Over the past few decades, the enrichment technologies of CTCs have attracted significant attention. However, owing to the rareness and heterogeneity of CTCs, tumor cells circulating in the peripheral blood were hard to determinate physically the real physiological state [9,10]. Many kinds of enrichment technologies for CTCs have been put forward and could be divided into positive and negative enrichment [11,12]. Positive enrichment utilizes physical or biological properties (such as cell gravity, shape, surface protein expression, etc.) to capture CTCs and eliminate normal blood cells. It brings higher purity of isolation efficiency, but can cause bias by their isolation condition. On the other hand, the negative enrichment captures normal blood cells and
Corresponding author. Corresponding author. ⁎⁎⁎ Corresponding author. E-mail addresses: [email protected] (R. Gao), [email protected] (G. Zhao), [email protected] (L. Yu). ⁎
⁎⁎
https://doi.org/10.1016/j.talanta.2019.120261 Received 12 June 2019; Received in revised form 7 August 2019; Accepted 14 August 2019 Available online 14 August 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic illustration of PS microspheres and cell separation in fishbone units, rectangular reservoir and inertial focusing channels. (a) Schematic of cell separation using multistage microfluidic chip. (b) Optical image of entire multistage microfluidic channel and four functional portions (marked by blue dashed boxes) for cell separation. The scale bar is 100 μm. (c) Lateral migration of RBCs (red spheres), WBCs (white spheres) and CTCs (blue spheres). The balance of shear-induced lift force (Fil) and wall-induced lift force (Fwl) caused them to distribute different areas. (d) RBCs extraction progress by two side channels. (e) Inertial focusing progress in asymmetric curvature channels. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Thereinto, the micro-posts filtration enrichment captures CTCs in a series of microscale trap structures, according to the variation in cell size and deformability. J. Che et al. combined microvortice and deformability cytometry (DC) technique for CTCs enrichment and enumeration [10]. The larger cells were trapped and released by microvortice which is generated in the rectangular reservoir. DC technique could rapidly record images and provide the biophysical information (e.g. size, deformability) of single cell. It showed up to 93.8% separation efficiency for clinical samples, which is one of the best chip-based separation devices. Ma et al. developed a microfluidic chip using oscillatory flow to force the CTCs and blood cells through an array of tapered microscale constrictions [36]. It can guide the cells in different flow paths to separate and collect them respectively. Another size-based separation approach is inertial focusing microfluidics. When cells flow in a flat and narrow linear channel, they are suffered from shear-gradient lift force and wall-effect lift force together. The cells with different sizes are subject to different forces, and the microvortices would separate them. Dielectrophoresis (DEP) is also a label-free method to separate by the distinct dielectric properties of different cells under inhomogeneous electric field. An application of this phenomenon is dielectrophoretic field-flow-fractionation (DEP-FFF) system, constructed by Moon et al., which can process 10 ml clinical samples in less than 1 h [37]. Although various cells could be separated from billions of normal blood cells by utilizing the above microfluidic channels, the separation efficiency and throughput yield still need to improve [9,38,39]. It needs to be emphasized that conventional label-free cell separation techniques using inertial forces have advantages of fast flow rates. However, endothelial cells are exposed to excessive shear stress under fast flow rates and transmit mechanical stimulus into intracellular impacts that affects cellular functions and gene expression [40,41]. To compensate these problems, we report a multistage microfluidic chip to isolate and retrieve tumor cells with blood cells as shown in Fig. 1a. Preliminary separation is achieved through the first stage, twenty fishbone shaped units, with a geometry of 45° angled symmetryrhomboid chamber. In order to attain high separation efficiency, the second stage involves a rectangular reservoir to elute non-target cells
eliminates CTCs. It can isolate heterogeneous CTCs, while has relatively low purity and high cost for vast antibodies capturing the blood cells [13–16]. Furthermore, positive enrichment could be classified into affinitybinding approaches (label-dependent) and label-free approaches [17–19]. To enrich CTCs, the devices usually employ two key properties of tumor cells which are distinguished from normal blood cells: the size and expressed protein of specific epithelial markers such as EpCAM [20,21]. Affinity-binding approaches indicated that specific antigen or biomarkers on the CTCs membrane could be targeted by their corresponding antibodies. EpCAM is a well-known and wildly studied antigen for CTCs capture, which is expressed by epithelial cancer. The CellSearch system (Veridex, Warren, NJ) is the only commercialized product and clinically validated by the US FDA for enumerating CTCs, and it uses anti-EpCAM conjugated ferrofluid nanoparticles to separate CTCs. It has shown high reproducibility and consistency. However, according to its isolation capability, only half of patients who have metastatic cancer can be diagnosed and has an obvious limitation, the inability to access cellular heterogeneity after cells are enumerated [22]. Besides, affinity-based methods also suffer from the problem of epithelial mesenchymal transition (ETM), which decreases the binding efficiency of EpCAM and its antibody. In contrast, label-free CTCs sorting technology relying on cell size is one of most reliable methods to isolate CTCs and circulating tumor-derived cells (CTDCs) [23–25]. Physical property-based CTCs separation has the potential to address the shortcomings in affinity-based separation methods [26,27]. After separation and enrichment, cell viability and gene expression keep constant, which makes molecular analysis of CTCs possible [28]. In clinical scenarios, the patients might have many different types of CTCs in their peripheral blood, and that only one or few subsets possess metastatic phenotype [29–31]. Moreover, it is expected that phenotypically diverse cancer cells could be isolated by tumor cell size. Several representative label-free approaches based on microfluidics, including micro-posts filtration, deterministic lateral displacement array and inertial lift flow, have been done in previous studies. Hence, it is the most remarkable property that differentiate CTCs from other healthy blood cells [32–35]. 2
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(i.e., WBC, RBC, etc). The third stage, served as the main separation region, is consisted of an inertial focusing microchannel. It can improve some obstacles in other microfluidic devices, such as blocking, cellular stress, and cell damage, by applying inertial forces to migrate the flow route of tumor cells for separation. For this design, low flow rate was proceeded to increase the survival rate of the cells for further investigation. And it leverages adequately hydrodynamic theory to accomplish the aim of continuous particle stream separation. The novelty of our work is twofold. First, we successfully implemented rapid separation and efficiently recovered CTCs from spiked and clinical human blood sample with this multistage microfluidic chip. It is noteworthy that glioblastoma is the most common and malignant in the human central nervous system (CNS) and the insidious trait of brain tumor metastasis is considered as peripheral blood circulation [42]. Second, the combination of preliminary separation, eluting non-target cells and inertial focusing process can realize the one-stop separation of CTCs. The pretreatment process using a RBC lysis buffer or centrifugal settling was exempted. It would require well trained staffs, heavy instruments and extra sample processing time, and may lose CTCs and damage their physiological properties. Therefore, our work can significantly contribute to further study on CTCs analysis and personalized cancer treatment.
particles, Rec is the channel Reynolds number, and h is width and height of the channel, µ is the dynamic viscosity, f is the fluid density, Um is the maximum rate of the channel flow, fc (R c , x c ) is the lift coefficient of the inertial lift force [45]. The viscous drag force is another indispensable force to particle migration. It is caused by the different rates of the microfluidics and particles. Drag force for a spherical particle can be expressed as:
Fd = 3 µd (
FD ~ f Um2 dDh2 / r
In a viscoelastic fluid, individual particle focusing could be swimmingly implemented in microchannel without external force or complicated channel structures compared with that in a Newtonian fluid [43]. The human blood sample used in our experiment conforms to viscoelastic fluids. Hence, the following force analysis all based on this conditional assumption. In viscoelastic fluid, the inertial effect and viscoelastic effect may affect the movement path of particles. The individual particles that flow in viscoelastic fluid are subjected to three hydrodynamic forces, including inertial lift force Fl , elastic force Fe, and drag force Fd [43,44]. The inertial lift force Fl , including the shear-induced lift force (Fil) and wall-induced lift force (Fwl), leads the lateral migration of particles to equilibrium positions between the microfluidic channel walls and centerline where they balance each other. The shear-induced lift force generated by the parabolic rate profile impels particles migration from the centerline toward the channel wall. Meanwhile, the wall-induced lift force produced by the asymmetric wake of the particles near the wall tends to push these particles away from the wall when particles move close to the channel wall. Elastic force can also affect particles in viscoelastic fluid, which would result in different migration behaviors of particles. In a rectangular linear channel as we used in this work, inertia effect of flow affect the streamline of particles depending on their size. The streamline of small particles is easily altered by the variation of flow rate owing to negligible inertia effect. In contrast, large particles intend to stabilize closer to the centerline of the channel because of strong inertia effect. The magnitude of inertial lift force (Fl ) can quantify as follows:
f
Rep2 fc (Rc , xc )
Rep = Rec Dh =
d2 = Dh
2wh w+h
f
Um
(1)
d2
µDh
(4)
(5)
where r represents curvature radius of curve channel. When the aqueous phase containing different-size particles were injected into microfluidic chip, the large particles tended to move toward the centerline of channel resulting from the balance of shear-induced lift force (Fil) and wall-induced lift force (Fwl), whereas small particles laterally moved to both sidewalls of fishbone expansion area [18,46,47]. In our design, the small particles appeared at both sides of fishbone areas, while the large particles were concentrated on the center streamline at an appropriate flow rate, as shown in Fig. 1c. In the following second functional unit, larger particles still stabilized nearby the center of the channel owing to inertia effect of flow and the balance of two extraction side channels. Fig. 1d displayed a number of small particles was extracted into outlet (Fig. 1b ii) to achieve pretreatment. Inertial focusing effect in curving channel can efficiently separate large and small particles [48,49]. This effect makes use of Dean vortices and the inertial lift force (Fl ) to focus and push particles toward outer side of channel and then focus large particles to a stream which they can be oriented [50,51]. Meanwhile, due to less gravity and smaller size of small particles, inertia effect of flow is negligible for small particles which dispersed between the center line and inner wall of channel (Fig. 1e). For high-concentration sample (such as whole blood), the equilibrium position of small particles would move a little toward the center of channel, and the equilibrium position of large particles would stay at the same position. Furthermore, the streams of them was extended due to more particles involved in streams. In the present study, we designed a multistage microfluidic chip including four functional portions (Fig. 1b W, X, Y, Z), one inlet (Fig. 1b i) for cell suspension and three outlets for draining CTCs and RBCs (Fig. 1b ii, iii, iv). The microfluidic channel was set up for 100, 200 and 400 μm wide and 30 μm deep. All microscopic images of microfluidic channel were directly taken from a plasma bonded PDMS chip. To improve the separation efficiency, the particle diameter to the hydraulic diameter of channel ratio is larger than 0.07 [52,53]. The first portion was functioned as first-stage separation including twenty repeated 45° angled fishbone units. In the following portion, RBCs were extracted passed through a rectangular reservoir (Fig. 1b X) to outlet ii. This reservoir contains dozens of PDMS pillars to prevent channel collapse and leakage of CTCs into side channels. The rest of cell suspension continued to move along the inertial focusing channel as the third portion Y, where CTCs with large diameter moved along the outer wall and RBCs with small size diffused along inner wall. The radiuses of inner and outer curvature turn are 310 and 530 μm, respectively. Indeed, there were two kinds of curvature turn with average radius of 390 and 280 μm. This asymmetric curvature structure had been calculated that the Dean drag (FD , eq (4)) in small turns (280 μm) was about eight
2.1. The principle of cell separation
µ2
p)
Where, f and p is the flow rates of fluid of compositions and particles, respectively. Particularly, secondary cross-sectional flow field introduced by channel curvatures, which is also known as Dean vortices, has a fixed direction that is perpendicular to the flow direction. The presence of Dean vortices promotes the drag force to accelerate gather of different-size particles in the main flow direction. When moving into channel with curvature, particles are under the influence of additional drag forces, which is scaled as FD . The magnitude of FD is given by
2. Material & methods
Fl =
f
(2) (3)
Where, the Reynolds number of particles Rep , includes length from hydraulic diameter Dh of the microfluidic channel and diameter d of 3
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times larger than it in large turns (390 μm). At the end of curving channel, WBCs and RBCs were flowed into outlet iii, while CTCs flowed into outlet iv. All gathered cell fluid was stored and incubated in 5% CO2 at 37 °C in media consisting of DMEM for further profiling.
portions. The particle distributions were counted by MATLAB (The Mathworks, Inc.) to automatically analyze the microscopic image of last portion (Fig. 2b iv). 3. Results and discussion
2.2. Device fabrication
3.1. Characterization of optimal flow rate
The fabrication of microfluidic chip utilized the rapid prototyping and UV photolithography techniques which was reported by our previous paper [54]. Briefly, the photomask was printed by 25000 dpi printer and transfer the design on a clean silicon wafer with SU-8 3035 photoresist (Microchem Corp, Westborough, US). PDMS replica were produced using Sylgard 184 Silicone Elastomer Kit. After punched hole for fluid injection and ejection, it was irreversibly bonded with 3″ × 1″ glass slide by oxygen plasma using 80 W RF power for 30 s at 500 mTorr. Then the PDMS chips were stored for microfluidic experiment.
To evaluate the performance of our device, the mixture of 8 μm and 15 μm PS microspheres was applied for optimizing flow rate to achieve best separation efficiency. The flow rates varied from 3 to 30 μl/min. Then, the movement path diffused in the case of the diameter of microspheres due to inertial lift force and drag force in four functional portions of chip. Fig. 3 showed the amount of PS microspheres which were collected from outlet iv (Fig. 1b) and counted under different flow rates. It demonstrated that 9 μl/min is the optimal flow rate to separate U87 cells and RBCs utilizing this device for further analysis. When the flow rate was lower than 9 μl/min, it was noted that inertial lift force Fl is the dominant mechanism to focus microspheres on their equilibrium positions. Separation efficiency of large microspheres was increased with the increase of flow rate. Under the optimal flow rate, the balance between the inertial lift force Fl and the Dean drag force FD facilitate the single-stream focusing of large microspheres separated from small microspheres. As the flow rate increased over 9 μl/min, the drag force from Dean flow is strengthened and can affect inertial lift equilibrium positions of microspheres. It reduced the number of large microspheres at equilibrium positions close to outer channel wall, and disperse the streamline of small microspheres. Consequently, separation efficiency decreased along with the increase of flow rate.
2.3. Sample preparation Polystyrene (PS) microspheres (D = 8 μm and 15 μm), which represents RBCs and CTCs, were firstly used to preliminarily investigate and improve the chip design. Two kinds of PS microspheres were mixed and diluted in DI water with 0.5% Tween 20 and 21% glycerin. The final concentration of 8 μm and 15 μm PS microspheres was around 1 × 106/ml and 1 × 104/ml. It should be noted that PS microspheres suspension was centrifuged and removed the supernatant before dilution. The human brain malignant glioma cell line (U87) was cultured in the essential medium, supplemented with fetal bovine serum (10% v/ v), 1% glutamine, 1% penicillin-streptomycin, incubated in 5% CO2 at 37 °C. All cell culture reagents were purchased from Invitrogen. U87 cells were spiked in a human RBCs sample and untreated whole blood, respectively. A total of three human blood samples were used in this work and came from healthy donators, who agreed to join in our related research and signed the informed consent. All blood samples were collected into ethylenediamine tetraacetic acid (EDTA) vacuum tubes and used within 1 day after collection. The human RBCs samples were obtained by three rounds of centrifugation (5000 rpm, 4 min) of 1 mL human blood sample and resuspended in 10 ml phosphate buffered saline (PBS) buffer (10 × , pH 7.4). The ratio of U87 cells and RBCs was 1:5000. An acridine orange/ethidium bromide (AO/EB) staining kit (KeyGen Biotech, China) was applied to evaluate the viability of the CTCs. 100 μl U87 cells were stained by AO/EB solution. The live cells could show green fluorescence (stained by AO) and the dead cells were red fluorescence (stained by EB), they were discerned and counted under an inverted fluorescence microscope (Ti-U, Nikon, Japan). All cells were fixed before enumeration and morphological profiling by formaldehyde (Sigma-Aldrich, Missouri). Deionized water was purified using a Milli-Q water purification system (MA, USA).
3.2. Cancer cell separation using human erythrocytes spiked with human brain malignant glioma cells To assess the feasibility of our multistage microfluidic chip for real human cells, we separated U87 cells from human RBCs. The U87 cells are ~15 μm in diameter and show globular shape. The human RBCs are 6–8 μm in diameter and show biconcave shape. The dominant forces of U87 cells and RBCs may alter between inertial lift force and Dean drag force because their diameters are different. It was applied to separate the U87 cells at the optimal flow rate. The human RBCs were extracted from whole blood by centrifugation and diluted ten times. The U87 cells were spiked into diluted human RBCs with the ratio of 1:5000. The sample was introduced into chip under various flow rates. The mixed cells passed through fishbone shaped portion, where different size of cells would be located in diverse areas for the balance of change-induced inertial force. The U87 cells were concentrated at the middle area of channel. Nevertheless, the RBCs laterally migrated to the expansion chamber and continuously moved to next unit. Because U87 cells had a tendency to obey the microfluid flow pattern with inertial effect regardless of the alteration of channel structure. However, the inertial effect on RBCs can be ignored. As fishbone shaped channels outspread consecutively, the effect of initial separation deserve further reinforce. Particularly, it prepared for subsequent pretreatment of RBCs extraction. When flow into rectangular reservoir as shown in Fig. 4b, RBCs were extracted into two side channels. Whereas U87 cells rushed through reservoir at a high speed. This step removed a mass of RBCs from fluid to perform pretreatment instead of centrifugation or filtration before chip-based separation. Then, the cells poured into inertial focusing portion and divided into two streamlines as shown in Fig. 4c. Due to lateral migration of cells under inertial forces, U87 cells stabilized nearby the outer wall of channel. Meanwhile, RBCs moved along with the inner wall of channel with continual fluid flow. Fig. 4d demonstrated the streamlines of RBCs and CTCs were guided by Y-bifurcation structure to two outlets (Fig. 4d e). U87 cell was marked by blue circle. To evaluate the performance of our device, identification and
2.4. Operation workflow The setup of microfluidic and inverted optical microscope system was shown in Fig. S1. Precision syringe pumps, 1 ml Norm-Ject Plastic disposable syringes (Henke-Sass Wolf GmbH, Germany), and Tygon microbore tube (ID = 0.02 IN, Saint-Gobain PPL Corp.) were employed to access samples into the microfluidic chip. Two precision syringe pumps were employed in this work. One was used for introducing the PS microspheres suspension or blood sample, the other pump extracted RBCs or small spheres. To obtain the optimal flow rate, samples were injected into chip with different rates from 3 to 30 μl/min. The extraction flow rate was 2 μl/min during eluting non-target cells step. Bright field images were obtained by an Eclipse Ti-U inverted research microscope with DS-Qi2 monochrome camera (Nikon, Japan). Microscopic images of cell movement in microfluidic chip were recorded and analysed by Nikon NIS-Elements imaging software. Fig. 2 displayed the photograph and the detailed microscopic images of four functional 4
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Fig. 2. (a) Photograph of the multistage microfluidic channel filled with red ink. (b) Top view of the whole chip and microscopic images for four functional portions: i) fishbone units, ii) rectangular reservoir, iii) asymmetric curvature channel, iv) Y-bifurcation structure. The scale bar is 200 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and CTCs. The separation efficiency was estimated about 97% for real cells at 9 μl/min. All collected cells have normal biological morphology and could be further cultured more than 3 days, which may be crucial for tumor profiling such as quantitative PCR, whole genome sequencing, and xenograft studies. This result is comparable with ligand capture method using antibody. However, our work proposed a novel label-free and size-based separation method which permitted recovering CTCs for further investigation. It is essential for phenotype identification and molecular analysis of CTCs to better understand cancer metastases, and can significantly contributed to personalized treatment of cancer patients. 3.3. Validation experiments of human brain malignant glioma cell separation from untreated whole blood and clinical sample To validate screening capability of this device, we proceeded an experiment by separating known number of human brain malignant glioma cells spiked into whole human blood. The viscosity of peripheral blood is mainly determined by the number of RBCs due to its character of viscous non-newtonian fluid. The great viscosity of untreated blood can accelerate formation of Dean vortices to separate different-size particles in the main low flow direction. The spiked whole blood sample included RBCs, thrombocytes, WBCs, U87 cells and so on. The human WBCs of peripheral blood are 8–12 μm in diameter and overlapped with the size distributions of U87 cells. U87 cells were fluorescently stained by AO/EB solution to enumerate them in the collection outlet (iv), as shown in Fig. 7. The green fluorescence cells were U87 cells and the WBCs were marked by with white dashed boxes. It clearly demonstrated that U87 cells were still alive through separation and only few WBCs presented in collection outlet. The fluorescent cancer cells were counted at both outlets, and the average separation efficiency was greater than 90% for three samples. The purity of collected CTCs is at least 84.96% and the viability rate of all cells is > 95%.
Fig. 3. Separation efficiency of PS microspheres under various flow rates which collected from outlet iv. The efficiencies are from three individual experiments.
enumeration of cells were taken and shown in Fig. 6. The separation efficiency was calculated as the amount of collected CTC cells over the total amount of CTC cells infused through the device. The purity describes the ability of the device to capture CTCs within a background of contaminating cells (such as WBCs). The separation efficiency and purity were defined as follows:
Separation efficiency = 100 % ×
Purity = 100 %×
CTC Cellsoutput CTC Cellsinput
(6)
CTC Cellsoutput Collected Nucleated Cells (CTCs and WBCs )output
(7)
Fig. 5 displayed the photograph and microscopic images of RBCs
Fig. 4. Microscopic images of cells movements at different positions along the channel. (a) Inlet for CTCs and RBCs. (b) CTCs went through rectangular reservoir and RBCs were extracted by two side channels. (c) The sequential images of a tumor cell moved in curving channel according to time. (d) The sequential images of a tumor cell moved in Y-bifurcation structure channel according to time. (e) Outlet for RBCs. (f) Outlet for CTCs. 5
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Fig. 5. Microscopic images and photograph of RBCs and CTCs which collected from outlet iii and iv.
contained the number of 19.20 ± 3.77 identified CTCs. Moreover, phenotypic and biomarker heterogeneity of identified CTCs need to be represented for further investigation. Obviously, the physical properties between CTCs and other blood cells of cancer patients are highly discriminative for efficient label-free isolation. It is believed that the proposed multistage label-free microfluidic device have great potential for CTCs isolation. 4. Conclusion and discussion We demonstrate a novel multistage label-free enrichment method of CTCs with hydrodynamic forces on microfluidic chip. The rarity of cancer cells in billions of patient's blood cells gives rise to the challenge of CTCs detection. To improve separation efficiency, a pretreatment for removing RBCs by centrifugation or filtration is usually taken before chip-based separation. However, it is time consumption and cumbersome. Here, we successfully developed a multistage microfluidic chip to maximize the lateral migration of cells utilizing differences in cell size and deformability. The cell separation regions consisted of a fishbone shaped channel, rectangular reservoir and inertial focusing channel. A mixture of two different diameter PS microspheres suspension was applied to explore the optimal condition for the separation. The device can separate them with high separation efficiency (> 90%) under the optimal flow rate. Furthermore, U87 cells spiked RBCs and untreated human blood samples were used for evaluating possibility of clinical application and screening capability of our device. The optimized parameters made this chip to reach an average separation efficiency of 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is > 95%. Collected CTCs were able to survive for reliable identification and visualizing heterogeneity. It became an attractive technique for cancer treatment monitoring and early diagnostic of cancer or its metastasis. In our future study, cell deformability measurements and rapid identification of CTCs subpopulations and clusters would combine with this simple microfluidic chip to provide a useful tool for investigating the possibility of cancer metastasis.
Fig. 6. Separation efficiency of RBCs and CTCs under various fluid flow rates which collected from outlet iv. The efficiencies are from three individual experiments.
Fig. 7. Composite fluorescent image of CTCs (green) and WBCs (clear) which collected from outlet iv. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Conflicts of interest There are no conflicts to declare.
To further assess the possibility of clinical application, the proposed device was performed on clinical blood sample by the optimal condition. 2 ml clinical blood sample was obtained from female patient diagnosed with metastatic ovarian cancer under informed consent, and directly injected into device without RBCs lysis, centrifugation or filtration. It was divided into three portions, and each one was 0.66 ml. After flowed through four functional portions on device, 38 CTCs were collected from three portions of clinical blood sample. It should be noted that most of collected CTCs were individual presence and only a few of CTCs were clusters. It indicated that 1 mL clinical blood sample
Acknowledgements The National Natural Science Foundation of China supported this work (No. 61601165), 51875165. We also acknowledge financial support from the Fundamental Research Funds for the Central Universities (No. JZ2019HGTB0088), the China Postdoctoral Science Foundation (Nos. 2018T110613), the Anhui Key Project of Research and Development Plan (No. 1704d0802188). 6
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Appendix A. Supplementary data
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Efficient separation of tumor cells from untreated whole blood using a novel multistage hydrodynamic focusing microfluidics
T
Rongke Gaoa, , Lei Chenga, Shiyi Wanga, Xiaobai Bia, Xueli Wanga, Rui Wangb, Xinyu Chene, ⁎⁎ ⁎⁎⁎ Zhengbao Zhad, Feng Wangd, Xiaofeng Xuf, Gang Zhaoc, , Liandong Yua, ⁎
a
School of Instrument Science and Opto-electronic Engineering, Hefei University of Technology, Hefei, 230009, China Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China c School of Biomedical Engineering, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, China d School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China e Department of Electrical and Computer Engineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA f Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China b
ARTICLE INFO
ABSTRACT
Keywords: Microfluidic chip Circulating tumor cells Label-free Hydrodynamic focusing Multistage microfluidics
Significant progress on circulating tumor cells (CTCs) has profound impact for noninvasive tumor profiling including early diagnosis, treatment monitoring, and metastasis recognition. Therefore, CTCs based liquid biopsy technology is taking a rapid growth in the field of precision oncology. The label-free approaches relied on microfluidic chip stand out from a crowd of methods that suffer from time consuming, extensive blood samples, lost target cells and labor-intensive operation. In this paper, a label-free separation microfluidic device was developed using multistage channel, which took full advantage of inertial lift force. Our strategy demonstrated CTCs were efficiently isolated from untreated human blood samples including antibody conjugation and erythrocyte lysis. This device was applied for isolating human brain malignant glioma cells that were spiked in human peripheral blood samples. The experimental condition was optimized and exhibited an average separation efficiency of ≥ 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is > 95%, which was better than other one-step CTCs separation methods. Furthermore, the CTCs were successfully separated from untreated clinical blood sample of cancer patient on the proposed microfluidic device. The entire experimental procedures are extremely low-cost and easy manipulation. It is believed that the proposed multistage microfluidic chip can become a promising tool for CTCs separation and early diagnosis of cancer.
1. Introduction Circulating tumor cells (CTCs) are rare cell population that shed from original or metastatic tumors and circulated in the peripheral blood. As potential biomarkers of non-invasive cancer detection, CTCs play a key role in cancer therapy since it can apply for early diagnosis of metastasis [1–4]. Owing to the extremely low concentration of CTCs in human blood, enrichment is an essential pretreatment step for effective detection of CTCs. Micromanipulation techniques based on microfluidics can isolate individual cells for further biological analysis. Compared with traditional methods, microfluidic devices have a variety of advantages, such as high throughput, efficiency and sensitivity [5–7]. Besides its significance on clinical application, CTCs
enumeration can also be used to evaluate the efficacy of therapeutic treatment of cancer [8]. Over the past few decades, the enrichment technologies of CTCs have attracted significant attention. However, owing to the rareness and heterogeneity of CTCs, tumor cells circulating in the peripheral blood were hard to determinate physically the real physiological state [9,10]. Many kinds of enrichment technologies for CTCs have been put forward and could be divided into positive and negative enrichment [11,12]. Positive enrichment utilizes physical or biological properties (such as cell gravity, shape, surface protein expression, etc.) to capture CTCs and eliminate normal blood cells. It brings higher purity of isolation efficiency, but can cause bias by their isolation condition. On the other hand, the negative enrichment captures normal blood cells and
Corresponding author. Corresponding author. ⁎⁎⁎ Corresponding author. E-mail addresses: [email protected] (R. Gao), [email protected] (G. Zhao), [email protected] (L. Yu). ⁎
⁎⁎
https://doi.org/10.1016/j.talanta.2019.120261 Received 12 June 2019; Received in revised form 7 August 2019; Accepted 14 August 2019 Available online 14 August 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic illustration of PS microspheres and cell separation in fishbone units, rectangular reservoir and inertial focusing channels. (a) Schematic of cell separation using multistage microfluidic chip. (b) Optical image of entire multistage microfluidic channel and four functional portions (marked by blue dashed boxes) for cell separation. The scale bar is 100 μm. (c) Lateral migration of RBCs (red spheres), WBCs (white spheres) and CTCs (blue spheres). The balance of shear-induced lift force (Fil) and wall-induced lift force (Fwl) caused them to distribute different areas. (d) RBCs extraction progress by two side channels. (e) Inertial focusing progress in asymmetric curvature channels. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Thereinto, the micro-posts filtration enrichment captures CTCs in a series of microscale trap structures, according to the variation in cell size and deformability. J. Che et al. combined microvortice and deformability cytometry (DC) technique for CTCs enrichment and enumeration [10]. The larger cells were trapped and released by microvortice which is generated in the rectangular reservoir. DC technique could rapidly record images and provide the biophysical information (e.g. size, deformability) of single cell. It showed up to 93.8% separation efficiency for clinical samples, which is one of the best chip-based separation devices. Ma et al. developed a microfluidic chip using oscillatory flow to force the CTCs and blood cells through an array of tapered microscale constrictions [36]. It can guide the cells in different flow paths to separate and collect them respectively. Another size-based separation approach is inertial focusing microfluidics. When cells flow in a flat and narrow linear channel, they are suffered from shear-gradient lift force and wall-effect lift force together. The cells with different sizes are subject to different forces, and the microvortices would separate them. Dielectrophoresis (DEP) is also a label-free method to separate by the distinct dielectric properties of different cells under inhomogeneous electric field. An application of this phenomenon is dielectrophoretic field-flow-fractionation (DEP-FFF) system, constructed by Moon et al., which can process 10 ml clinical samples in less than 1 h [37]. Although various cells could be separated from billions of normal blood cells by utilizing the above microfluidic channels, the separation efficiency and throughput yield still need to improve [9,38,39]. It needs to be emphasized that conventional label-free cell separation techniques using inertial forces have advantages of fast flow rates. However, endothelial cells are exposed to excessive shear stress under fast flow rates and transmit mechanical stimulus into intracellular impacts that affects cellular functions and gene expression [40,41]. To compensate these problems, we report a multistage microfluidic chip to isolate and retrieve tumor cells with blood cells as shown in Fig. 1a. Preliminary separation is achieved through the first stage, twenty fishbone shaped units, with a geometry of 45° angled symmetryrhomboid chamber. In order to attain high separation efficiency, the second stage involves a rectangular reservoir to elute non-target cells
eliminates CTCs. It can isolate heterogeneous CTCs, while has relatively low purity and high cost for vast antibodies capturing the blood cells [13–16]. Furthermore, positive enrichment could be classified into affinitybinding approaches (label-dependent) and label-free approaches [17–19]. To enrich CTCs, the devices usually employ two key properties of tumor cells which are distinguished from normal blood cells: the size and expressed protein of specific epithelial markers such as EpCAM [20,21]. Affinity-binding approaches indicated that specific antigen or biomarkers on the CTCs membrane could be targeted by their corresponding antibodies. EpCAM is a well-known and wildly studied antigen for CTCs capture, which is expressed by epithelial cancer. The CellSearch system (Veridex, Warren, NJ) is the only commercialized product and clinically validated by the US FDA for enumerating CTCs, and it uses anti-EpCAM conjugated ferrofluid nanoparticles to separate CTCs. It has shown high reproducibility and consistency. However, according to its isolation capability, only half of patients who have metastatic cancer can be diagnosed and has an obvious limitation, the inability to access cellular heterogeneity after cells are enumerated [22]. Besides, affinity-based methods also suffer from the problem of epithelial mesenchymal transition (ETM), which decreases the binding efficiency of EpCAM and its antibody. In contrast, label-free CTCs sorting technology relying on cell size is one of most reliable methods to isolate CTCs and circulating tumor-derived cells (CTDCs) [23–25]. Physical property-based CTCs separation has the potential to address the shortcomings in affinity-based separation methods [26,27]. After separation and enrichment, cell viability and gene expression keep constant, which makes molecular analysis of CTCs possible [28]. In clinical scenarios, the patients might have many different types of CTCs in their peripheral blood, and that only one or few subsets possess metastatic phenotype [29–31]. Moreover, it is expected that phenotypically diverse cancer cells could be isolated by tumor cell size. Several representative label-free approaches based on microfluidics, including micro-posts filtration, deterministic lateral displacement array and inertial lift flow, have been done in previous studies. Hence, it is the most remarkable property that differentiate CTCs from other healthy blood cells [32–35]. 2
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(i.e., WBC, RBC, etc). The third stage, served as the main separation region, is consisted of an inertial focusing microchannel. It can improve some obstacles in other microfluidic devices, such as blocking, cellular stress, and cell damage, by applying inertial forces to migrate the flow route of tumor cells for separation. For this design, low flow rate was proceeded to increase the survival rate of the cells for further investigation. And it leverages adequately hydrodynamic theory to accomplish the aim of continuous particle stream separation. The novelty of our work is twofold. First, we successfully implemented rapid separation and efficiently recovered CTCs from spiked and clinical human blood sample with this multistage microfluidic chip. It is noteworthy that glioblastoma is the most common and malignant in the human central nervous system (CNS) and the insidious trait of brain tumor metastasis is considered as peripheral blood circulation [42]. Second, the combination of preliminary separation, eluting non-target cells and inertial focusing process can realize the one-stop separation of CTCs. The pretreatment process using a RBC lysis buffer or centrifugal settling was exempted. It would require well trained staffs, heavy instruments and extra sample processing time, and may lose CTCs and damage their physiological properties. Therefore, our work can significantly contribute to further study on CTCs analysis and personalized cancer treatment.
particles, Rec is the channel Reynolds number, and h is width and height of the channel, µ is the dynamic viscosity, f is the fluid density, Um is the maximum rate of the channel flow, fc (R c , x c ) is the lift coefficient of the inertial lift force [45]. The viscous drag force is another indispensable force to particle migration. It is caused by the different rates of the microfluidics and particles. Drag force for a spherical particle can be expressed as:
Fd = 3 µd (
FD ~ f Um2 dDh2 / r
In a viscoelastic fluid, individual particle focusing could be swimmingly implemented in microchannel without external force or complicated channel structures compared with that in a Newtonian fluid [43]. The human blood sample used in our experiment conforms to viscoelastic fluids. Hence, the following force analysis all based on this conditional assumption. In viscoelastic fluid, the inertial effect and viscoelastic effect may affect the movement path of particles. The individual particles that flow in viscoelastic fluid are subjected to three hydrodynamic forces, including inertial lift force Fl , elastic force Fe, and drag force Fd [43,44]. The inertial lift force Fl , including the shear-induced lift force (Fil) and wall-induced lift force (Fwl), leads the lateral migration of particles to equilibrium positions between the microfluidic channel walls and centerline where they balance each other. The shear-induced lift force generated by the parabolic rate profile impels particles migration from the centerline toward the channel wall. Meanwhile, the wall-induced lift force produced by the asymmetric wake of the particles near the wall tends to push these particles away from the wall when particles move close to the channel wall. Elastic force can also affect particles in viscoelastic fluid, which would result in different migration behaviors of particles. In a rectangular linear channel as we used in this work, inertia effect of flow affect the streamline of particles depending on their size. The streamline of small particles is easily altered by the variation of flow rate owing to negligible inertia effect. In contrast, large particles intend to stabilize closer to the centerline of the channel because of strong inertia effect. The magnitude of inertial lift force (Fl ) can quantify as follows:
f
Rep2 fc (Rc , xc )
Rep = Rec Dh =
d2 = Dh
2wh w+h
f
Um
(1)
d2
µDh
(4)
(5)
where r represents curvature radius of curve channel. When the aqueous phase containing different-size particles were injected into microfluidic chip, the large particles tended to move toward the centerline of channel resulting from the balance of shear-induced lift force (Fil) and wall-induced lift force (Fwl), whereas small particles laterally moved to both sidewalls of fishbone expansion area [18,46,47]. In our design, the small particles appeared at both sides of fishbone areas, while the large particles were concentrated on the center streamline at an appropriate flow rate, as shown in Fig. 1c. In the following second functional unit, larger particles still stabilized nearby the center of the channel owing to inertia effect of flow and the balance of two extraction side channels. Fig. 1d displayed a number of small particles was extracted into outlet (Fig. 1b ii) to achieve pretreatment. Inertial focusing effect in curving channel can efficiently separate large and small particles [48,49]. This effect makes use of Dean vortices and the inertial lift force (Fl ) to focus and push particles toward outer side of channel and then focus large particles to a stream which they can be oriented [50,51]. Meanwhile, due to less gravity and smaller size of small particles, inertia effect of flow is negligible for small particles which dispersed between the center line and inner wall of channel (Fig. 1e). For high-concentration sample (such as whole blood), the equilibrium position of small particles would move a little toward the center of channel, and the equilibrium position of large particles would stay at the same position. Furthermore, the streams of them was extended due to more particles involved in streams. In the present study, we designed a multistage microfluidic chip including four functional portions (Fig. 1b W, X, Y, Z), one inlet (Fig. 1b i) for cell suspension and three outlets for draining CTCs and RBCs (Fig. 1b ii, iii, iv). The microfluidic channel was set up for 100, 200 and 400 μm wide and 30 μm deep. All microscopic images of microfluidic channel were directly taken from a plasma bonded PDMS chip. To improve the separation efficiency, the particle diameter to the hydraulic diameter of channel ratio is larger than 0.07 [52,53]. The first portion was functioned as first-stage separation including twenty repeated 45° angled fishbone units. In the following portion, RBCs were extracted passed through a rectangular reservoir (Fig. 1b X) to outlet ii. This reservoir contains dozens of PDMS pillars to prevent channel collapse and leakage of CTCs into side channels. The rest of cell suspension continued to move along the inertial focusing channel as the third portion Y, where CTCs with large diameter moved along the outer wall and RBCs with small size diffused along inner wall. The radiuses of inner and outer curvature turn are 310 and 530 μm, respectively. Indeed, there were two kinds of curvature turn with average radius of 390 and 280 μm. This asymmetric curvature structure had been calculated that the Dean drag (FD , eq (4)) in small turns (280 μm) was about eight
2.1. The principle of cell separation
µ2
p)
Where, f and p is the flow rates of fluid of compositions and particles, respectively. Particularly, secondary cross-sectional flow field introduced by channel curvatures, which is also known as Dean vortices, has a fixed direction that is perpendicular to the flow direction. The presence of Dean vortices promotes the drag force to accelerate gather of different-size particles in the main flow direction. When moving into channel with curvature, particles are under the influence of additional drag forces, which is scaled as FD . The magnitude of FD is given by
2. Material & methods
Fl =
f
(2) (3)
Where, the Reynolds number of particles Rep , includes length from hydraulic diameter Dh of the microfluidic channel and diameter d of 3
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times larger than it in large turns (390 μm). At the end of curving channel, WBCs and RBCs were flowed into outlet iii, while CTCs flowed into outlet iv. All gathered cell fluid was stored and incubated in 5% CO2 at 37 °C in media consisting of DMEM for further profiling.
portions. The particle distributions were counted by MATLAB (The Mathworks, Inc.) to automatically analyze the microscopic image of last portion (Fig. 2b iv). 3. Results and discussion
2.2. Device fabrication
3.1. Characterization of optimal flow rate
The fabrication of microfluidic chip utilized the rapid prototyping and UV photolithography techniques which was reported by our previous paper [54]. Briefly, the photomask was printed by 25000 dpi printer and transfer the design on a clean silicon wafer with SU-8 3035 photoresist (Microchem Corp, Westborough, US). PDMS replica were produced using Sylgard 184 Silicone Elastomer Kit. After punched hole for fluid injection and ejection, it was irreversibly bonded with 3″ × 1″ glass slide by oxygen plasma using 80 W RF power for 30 s at 500 mTorr. Then the PDMS chips were stored for microfluidic experiment.
To evaluate the performance of our device, the mixture of 8 μm and 15 μm PS microspheres was applied for optimizing flow rate to achieve best separation efficiency. The flow rates varied from 3 to 30 μl/min. Then, the movement path diffused in the case of the diameter of microspheres due to inertial lift force and drag force in four functional portions of chip. Fig. 3 showed the amount of PS microspheres which were collected from outlet iv (Fig. 1b) and counted under different flow rates. It demonstrated that 9 μl/min is the optimal flow rate to separate U87 cells and RBCs utilizing this device for further analysis. When the flow rate was lower than 9 μl/min, it was noted that inertial lift force Fl is the dominant mechanism to focus microspheres on their equilibrium positions. Separation efficiency of large microspheres was increased with the increase of flow rate. Under the optimal flow rate, the balance between the inertial lift force Fl and the Dean drag force FD facilitate the single-stream focusing of large microspheres separated from small microspheres. As the flow rate increased over 9 μl/min, the drag force from Dean flow is strengthened and can affect inertial lift equilibrium positions of microspheres. It reduced the number of large microspheres at equilibrium positions close to outer channel wall, and disperse the streamline of small microspheres. Consequently, separation efficiency decreased along with the increase of flow rate.
2.3. Sample preparation Polystyrene (PS) microspheres (D = 8 μm and 15 μm), which represents RBCs and CTCs, were firstly used to preliminarily investigate and improve the chip design. Two kinds of PS microspheres were mixed and diluted in DI water with 0.5% Tween 20 and 21% glycerin. The final concentration of 8 μm and 15 μm PS microspheres was around 1 × 106/ml and 1 × 104/ml. It should be noted that PS microspheres suspension was centrifuged and removed the supernatant before dilution. The human brain malignant glioma cell line (U87) was cultured in the essential medium, supplemented with fetal bovine serum (10% v/ v), 1% glutamine, 1% penicillin-streptomycin, incubated in 5% CO2 at 37 °C. All cell culture reagents were purchased from Invitrogen. U87 cells were spiked in a human RBCs sample and untreated whole blood, respectively. A total of three human blood samples were used in this work and came from healthy donators, who agreed to join in our related research and signed the informed consent. All blood samples were collected into ethylenediamine tetraacetic acid (EDTA) vacuum tubes and used within 1 day after collection. The human RBCs samples were obtained by three rounds of centrifugation (5000 rpm, 4 min) of 1 mL human blood sample and resuspended in 10 ml phosphate buffered saline (PBS) buffer (10 × , pH 7.4). The ratio of U87 cells and RBCs was 1:5000. An acridine orange/ethidium bromide (AO/EB) staining kit (KeyGen Biotech, China) was applied to evaluate the viability of the CTCs. 100 μl U87 cells were stained by AO/EB solution. The live cells could show green fluorescence (stained by AO) and the dead cells were red fluorescence (stained by EB), they were discerned and counted under an inverted fluorescence microscope (Ti-U, Nikon, Japan). All cells were fixed before enumeration and morphological profiling by formaldehyde (Sigma-Aldrich, Missouri). Deionized water was purified using a Milli-Q water purification system (MA, USA).
3.2. Cancer cell separation using human erythrocytes spiked with human brain malignant glioma cells To assess the feasibility of our multistage microfluidic chip for real human cells, we separated U87 cells from human RBCs. The U87 cells are ~15 μm in diameter and show globular shape. The human RBCs are 6–8 μm in diameter and show biconcave shape. The dominant forces of U87 cells and RBCs may alter between inertial lift force and Dean drag force because their diameters are different. It was applied to separate the U87 cells at the optimal flow rate. The human RBCs were extracted from whole blood by centrifugation and diluted ten times. The U87 cells were spiked into diluted human RBCs with the ratio of 1:5000. The sample was introduced into chip under various flow rates. The mixed cells passed through fishbone shaped portion, where different size of cells would be located in diverse areas for the balance of change-induced inertial force. The U87 cells were concentrated at the middle area of channel. Nevertheless, the RBCs laterally migrated to the expansion chamber and continuously moved to next unit. Because U87 cells had a tendency to obey the microfluid flow pattern with inertial effect regardless of the alteration of channel structure. However, the inertial effect on RBCs can be ignored. As fishbone shaped channels outspread consecutively, the effect of initial separation deserve further reinforce. Particularly, it prepared for subsequent pretreatment of RBCs extraction. When flow into rectangular reservoir as shown in Fig. 4b, RBCs were extracted into two side channels. Whereas U87 cells rushed through reservoir at a high speed. This step removed a mass of RBCs from fluid to perform pretreatment instead of centrifugation or filtration before chip-based separation. Then, the cells poured into inertial focusing portion and divided into two streamlines as shown in Fig. 4c. Due to lateral migration of cells under inertial forces, U87 cells stabilized nearby the outer wall of channel. Meanwhile, RBCs moved along with the inner wall of channel with continual fluid flow. Fig. 4d demonstrated the streamlines of RBCs and CTCs were guided by Y-bifurcation structure to two outlets (Fig. 4d e). U87 cell was marked by blue circle. To evaluate the performance of our device, identification and
2.4. Operation workflow The setup of microfluidic and inverted optical microscope system was shown in Fig. S1. Precision syringe pumps, 1 ml Norm-Ject Plastic disposable syringes (Henke-Sass Wolf GmbH, Germany), and Tygon microbore tube (ID = 0.02 IN, Saint-Gobain PPL Corp.) were employed to access samples into the microfluidic chip. Two precision syringe pumps were employed in this work. One was used for introducing the PS microspheres suspension or blood sample, the other pump extracted RBCs or small spheres. To obtain the optimal flow rate, samples were injected into chip with different rates from 3 to 30 μl/min. The extraction flow rate was 2 μl/min during eluting non-target cells step. Bright field images were obtained by an Eclipse Ti-U inverted research microscope with DS-Qi2 monochrome camera (Nikon, Japan). Microscopic images of cell movement in microfluidic chip were recorded and analysed by Nikon NIS-Elements imaging software. Fig. 2 displayed the photograph and the detailed microscopic images of four functional 4
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Fig. 2. (a) Photograph of the multistage microfluidic channel filled with red ink. (b) Top view of the whole chip and microscopic images for four functional portions: i) fishbone units, ii) rectangular reservoir, iii) asymmetric curvature channel, iv) Y-bifurcation structure. The scale bar is 200 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and CTCs. The separation efficiency was estimated about 97% for real cells at 9 μl/min. All collected cells have normal biological morphology and could be further cultured more than 3 days, which may be crucial for tumor profiling such as quantitative PCR, whole genome sequencing, and xenograft studies. This result is comparable with ligand capture method using antibody. However, our work proposed a novel label-free and size-based separation method which permitted recovering CTCs for further investigation. It is essential for phenotype identification and molecular analysis of CTCs to better understand cancer metastases, and can significantly contributed to personalized treatment of cancer patients. 3.3. Validation experiments of human brain malignant glioma cell separation from untreated whole blood and clinical sample To validate screening capability of this device, we proceeded an experiment by separating known number of human brain malignant glioma cells spiked into whole human blood. The viscosity of peripheral blood is mainly determined by the number of RBCs due to its character of viscous non-newtonian fluid. The great viscosity of untreated blood can accelerate formation of Dean vortices to separate different-size particles in the main low flow direction. The spiked whole blood sample included RBCs, thrombocytes, WBCs, U87 cells and so on. The human WBCs of peripheral blood are 8–12 μm in diameter and overlapped with the size distributions of U87 cells. U87 cells were fluorescently stained by AO/EB solution to enumerate them in the collection outlet (iv), as shown in Fig. 7. The green fluorescence cells were U87 cells and the WBCs were marked by with white dashed boxes. It clearly demonstrated that U87 cells were still alive through separation and only few WBCs presented in collection outlet. The fluorescent cancer cells were counted at both outlets, and the average separation efficiency was greater than 90% for three samples. The purity of collected CTCs is at least 84.96% and the viability rate of all cells is > 95%.
Fig. 3. Separation efficiency of PS microspheres under various flow rates which collected from outlet iv. The efficiencies are from three individual experiments.
enumeration of cells were taken and shown in Fig. 6. The separation efficiency was calculated as the amount of collected CTC cells over the total amount of CTC cells infused through the device. The purity describes the ability of the device to capture CTCs within a background of contaminating cells (such as WBCs). The separation efficiency and purity were defined as follows:
Separation efficiency = 100 % ×
Purity = 100 %×
CTC Cellsoutput CTC Cellsinput
(6)
CTC Cellsoutput Collected Nucleated Cells (CTCs and WBCs )output
(7)
Fig. 5 displayed the photograph and microscopic images of RBCs
Fig. 4. Microscopic images of cells movements at different positions along the channel. (a) Inlet for CTCs and RBCs. (b) CTCs went through rectangular reservoir and RBCs were extracted by two side channels. (c) The sequential images of a tumor cell moved in curving channel according to time. (d) The sequential images of a tumor cell moved in Y-bifurcation structure channel according to time. (e) Outlet for RBCs. (f) Outlet for CTCs. 5
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Fig. 5. Microscopic images and photograph of RBCs and CTCs which collected from outlet iii and iv.
contained the number of 19.20 ± 3.77 identified CTCs. Moreover, phenotypic and biomarker heterogeneity of identified CTCs need to be represented for further investigation. Obviously, the physical properties between CTCs and other blood cells of cancer patients are highly discriminative for efficient label-free isolation. It is believed that the proposed multistage label-free microfluidic device have great potential for CTCs isolation. 4. Conclusion and discussion We demonstrate a novel multistage label-free enrichment method of CTCs with hydrodynamic forces on microfluidic chip. The rarity of cancer cells in billions of patient's blood cells gives rise to the challenge of CTCs detection. To improve separation efficiency, a pretreatment for removing RBCs by centrifugation or filtration is usually taken before chip-based separation. However, it is time consumption and cumbersome. Here, we successfully developed a multistage microfluidic chip to maximize the lateral migration of cells utilizing differences in cell size and deformability. The cell separation regions consisted of a fishbone shaped channel, rectangular reservoir and inertial focusing channel. A mixture of two different diameter PS microspheres suspension was applied to explore the optimal condition for the separation. The device can separate them with high separation efficiency (> 90%) under the optimal flow rate. Furthermore, U87 cells spiked RBCs and untreated human blood samples were used for evaluating possibility of clinical application and screening capability of our device. The optimized parameters made this chip to reach an average separation efficiency of 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is > 95%. Collected CTCs were able to survive for reliable identification and visualizing heterogeneity. It became an attractive technique for cancer treatment monitoring and early diagnostic of cancer or its metastasis. In our future study, cell deformability measurements and rapid identification of CTCs subpopulations and clusters would combine with this simple microfluidic chip to provide a useful tool for investigating the possibility of cancer metastasis.
Fig. 6. Separation efficiency of RBCs and CTCs under various fluid flow rates which collected from outlet iv. The efficiencies are from three individual experiments.
Fig. 7. Composite fluorescent image of CTCs (green) and WBCs (clear) which collected from outlet iv. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Conflicts of interest There are no conflicts to declare.
To further assess the possibility of clinical application, the proposed device was performed on clinical blood sample by the optimal condition. 2 ml clinical blood sample was obtained from female patient diagnosed with metastatic ovarian cancer under informed consent, and directly injected into device without RBCs lysis, centrifugation or filtration. It was divided into three portions, and each one was 0.66 ml. After flowed through four functional portions on device, 38 CTCs were collected from three portions of clinical blood sample. It should be noted that most of collected CTCs were individual presence and only a few of CTCs were clusters. It indicated that 1 mL clinical blood sample
Acknowledgements The National Natural Science Foundation of China supported this work (No. 61601165), 51875165. We also acknowledge financial support from the Fundamental Research Funds for the Central Universities (No. JZ2019HGTB0088), the China Postdoctoral Science Foundation (Nos. 2018T110613), the Anhui Key Project of Research and Development Plan (No. 1704d0802188). 6
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Appendix A. Supplementary data
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