HHS Public Access Author manuscript Author Manuscript
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21. Published in final edited form as: Micro Total Anal Syst. 2015 October ; 2015: 413–415.
High-Throughput Microfluidic Device for Circulating Tumor Cell Isolation from Whole Blood Daniel K. Yang1, Serena Leong2, and Lydia L. Sohn1,* 1Dept.
of Mechanical Engineering, University of California, Berkeley, CA 94720 USA
2Dept.
of Bioengineering, University of California, Berkeley, CA 94720 USA
Author Manuscript
Abstract Circulating tumor cells (CTCs) are promising markers to determine cancer patient prognosis and track disease response to therapy. We present a multi-stage microfluidic device we have developed that utilizes inertial and Dean drag forces for isolating CTCs from whole blood. We demonstrate a 94.2% ± 2.1% recovery of cancer cells with our device when screening whole blood spiked with MCF-7 GFP cells.
Keywords CTC; Inertial microfluidics; Size-based separation
Author Manuscript
INTRODUCTION
Author Manuscript
Circulating tumor cells (CTCs) are cells that are shed from a primary tumor and have entered the vascular system. Enumerating and analyzing these cells can potentially determine patient prognosis, monitor disease progression, and lead to effective targeted therapies. The rarity of CTCs in cancer patients’ blood, however, is a great challenge, as only 1–10 CTCs are present in a 7.5mL sample of patient blood [1]. Current techniques for isolating and identifying CTCs rely either on the physical properties of these cells (e.g. size, charge, deformability, etc. [2–5]) or on the expression of particular markers, such as EpCAM. Since CTCs are larger than red blood cells (RBCs, ~6 μm), isolation based on cell size, i.e. membrane filters [6, 7], has been a large focus. While filtration is somewhat effective, cells can be subject to damaging high-shear stress, especially when the filters become increasingly clogged [1, 4]. Here, we describe a novel platform we have developed to isolate CTCs with high recovery from peripheral blood. Our platform is an inertial-based microfluidic device that has highthroughput, and is completely passive and label-free. We achieve a recovery efficiency of 94.2% ± 2.1% in benchmarking experiments, i.e. processing whole blood spiked with MCF-7 GFP cells at 130 μL/min.
*
CONTACT: Lydia L. Sohn; phone: +1-510-642-5434; [email protected].
Yang et al.
Page 2
Author Manuscript
THEORY We have developed an inertial microfluidic device, which employs inertial forces to manipulate cell streamlines with respect to cell diameter. A schematic of our CTC recovery platform is shown in Figure 1A. The platform consists of two stages, which when combined, isolates larger (> 15 μm) cells from whole blood. During separation, cells occupy unique equilibrium positions that are dependent on maximum fluid flowrate and cell properties, such as size. In more detail, our device consists of a combination of symmetric and asymmetric contracting and expanding microchannel arrays (sCEA and aCEA, respectively) to isolate larger (>15 μm) targeted cells from blood (Figure 1A). Stage One, which consists of the sCEA, focuses all cells to the outer wall via a balance of shear gradient
Author Manuscript
( ) and wall inertial lift forces ( ) where, fL, a,W, ρ, and Um are the dimensionless lift coefficient, cell diameter, channel width, fluid density, and maximum velocity, respectively (Figure 1A) [8]. At the end of Stage One, the cells are confined to a compact and distinct streamline induced by a net inertial lift towards the channel walls. Stage Two, which consists of the aCEA, exposes the compact and inertial focused streamline of cells to a Dean drag force (FD = 3πμUDeana) within the contraction regions, where UDean and μ are the transverse velocity and fluid viscosity, respectively (Figure 1A) [9]. The dominance of the Dean drag force is inversely proportional to cell diameter. Thus, in Stage two, large cells remain in the equilibrium streamline established, and smaller cells are pulled into a different equilibrium position [10].
METHODS Author Manuscript
Device Fabrication and Operation Our complete multi-stage device consists of a single sCEA microchannel for Stage One and four independent aCEA microchannels operating in parallel for Stage Two. In-plane filters at the entrance of the sCEA are included to remove the cellular debris that could offset the flow in the device. The device is fabricated using standard soft lithography techniques. Briefly, we create a negative master of our device via standard photolithography. A PDMS mold is then cast, inlet and outlet holes are cored, and finally treated with oxygen plasma with a Harrick Plasma System (PC-001) for 30 seconds and immediately bonded to a glass slide.
Author Manuscript
For the benchmarking experiments performed, we processed 1 mL of whole blood spiked with MCF-7 GFP cells at a ratio of 10,000 white blood cells (WBCs) to 1 MCF-7 GFP cell. We used a sample and buffer inlet flow rate of 130 and 200 μL/min, respectively. In a single run, we acquired a total of five sets of data: one set corresponding to the initial waste (i.e. red blood cells and platelets) obtained after the sample passes through Stage One (the sCEA) and four separate data sets corresponding to the waste and collection outlets from each aCEA within Stage Two. We collected all outlets separately and performed flow cytometry (Guava EasyCyte) to determine the number of MCF-7 GFP cells in each. We defined the recovery efficiency of our device as the percentage of the sum of GFP cells collected in comparison to the total number of GFP cells found in all outlets.
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 3
Blood Samples and Cell Culture
Author Manuscript
MCF-7 GFP breast-cancer cells were cultured in growth medium containing DMEM supplemented by 10% FBS, 1% Penstrep, and 1% NEAA and maintained at 37°C in 5% CO2. For our spiking experiments, cells were grown in monolayer to 80% confluence and detached with 0.25% trypsin for 3 minutes. The cell suspension was washed once with DMEM media and then single cells were spiked into healthy human donor blood obtained with consent under a University of California, Berkeley IRB-approved protocol.
RESULTS AND DISCUSSION
Author Manuscript
Our CTC isolation platform, operating at 130 μL/min, demonstrated an overall recovery efficiency of 94.2 ± 2.1% (Figure 1B). A breakdown of MCF-7 GFP cell counts with respect to each outlet is shown in Table 1. Individually, each aCEA microfluidic channel (sets 2 through 5) achieved recovery efficiencies > 92%. A P value of 0.000288 was obtain between Collection and Waste by performing a Student T-test.
CONCLUSION Our platform is advantageous over existing technologies in its efficiency to recover cancer cells from whole blood and its ease of use. The device successfully recovered MCF-7 cells spiked into healthy human blood with an efficiency of 94.2% ± 2.1% at 130 μl/min. Future benchmarking will involve processing blood spiked with cancer cells at clinically relevant ratios. Ultimately, the simplicity of our platform will allow for direct integration with downstream single cell analysis platforms for the characterization of recovered cells.
Author Manuscript
Acknowledgments This work is supported by NIH 1R21CA182375-01A1 and the Bakar Fellows Program.
References
Author Manuscript
1. Hong B, et al. Detecting circulating tumor cells: current challenges and new trends. Theranostics. 2013; 3:377–394. [PubMed: 23781285] 2. Yu M, et al. Circulating tumor cells: approaches to isolation and characterization. The Journal of cell biology. 2011; 192:373–382. [PubMed: 21300848] 3. Phillips KG, et al. Optical quantification of cellular mass, volume, and density of circulating tumor cells identified in an ovarian cancer patient. Frontiers in oncology. 2012; 2:72. [PubMed: 22826822] 4. Park JM, et al. Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood. Analytical chemistry. 2012; 84:7400–7407. [PubMed: 22881997] 5. Autebert J, et al. Microfluidic: an innovative tool for efficient cell sorting. Methods. 2012; 57:297– 307. [PubMed: 22796377] 6. Vona G, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulatingtumor cells. The American journal of pathology. 2000; 156:57–63. [PubMed: 10623654] 7. Harouaka R, et al. Circulating tumor cells: advances in isolation and analysis, and challenges for clinical applications. Pharmacology & therapeutics. 2014; 141:209–221. [PubMed: 24134902] 8. Amini H, et al. Inertial microfluidic physics. Lab on a chip. 2014; 14:2739–2761. [PubMed: 24914632]
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 4
Author Manuscript
9. Lee MG, et al. Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress. Analytical chemistry. 2013; 85:6213–6218. [PubMed: 23724953] 10. Yang D, et al. High-throughput microfluidic device for rare cell isolation. Bio-MEMS and Medical Microdevices II. 2015; 9518:95180E.
Author Manuscript Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 5
Author Manuscript Author Manuscript
Fig. 1.
Schematic, isometric view, of our CTC isolation platform. A) Stage One consists of a symmetric CEA (sCEA) microfluidic channel in which cells are focused to the outer walls of the microchannel through a balance of wall lift and shear gradient lift forces. Stage Two consists of an asymmetric CEA (aCEA) microfluidic channel in which inertial focused streamlines are exposed to Dean drag forces where small particles migrate to different equilibrium positions. B) Overall percentage of MCF-7 GFP cells found in all collection and waste outlets. Data was obtain from processing 1 mL of whole blood spiked with MCF-7 GFP cells at a ratio of 10,000 WBCs: 1. Error bars correspond to standard deviation.
Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 6
Table 1
Author Manuscript
Number of MCF-7 GFP cells per outlet and recovery efficiency. The data is derived from healthy human donor blood spiked with MCF-7 GFP cells at a ratio of 10,000 WBCs: 1 MCF-7 GFP cells. The process flow rate employed was 130 μL/min. Set
Collection (MCF-7 GFP cells)
Waste (MCF-7 GFP cells)
Efficiency
1
–
0
–
2
62
2
96.9%
3
55
3
94.8%
4
51
4
92.7%
5
60
5
92.3%
Total
228
14
94.2%
Author Manuscript Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21. Published in final edited form as: Micro Total Anal Syst. 2015 October ; 2015: 413–415.
High-Throughput Microfluidic Device for Circulating Tumor Cell Isolation from Whole Blood Daniel K. Yang1, Serena Leong2, and Lydia L. Sohn1,* 1Dept.
of Mechanical Engineering, University of California, Berkeley, CA 94720 USA
2Dept.
of Bioengineering, University of California, Berkeley, CA 94720 USA
Author Manuscript
Abstract Circulating tumor cells (CTCs) are promising markers to determine cancer patient prognosis and track disease response to therapy. We present a multi-stage microfluidic device we have developed that utilizes inertial and Dean drag forces for isolating CTCs from whole blood. We demonstrate a 94.2% ± 2.1% recovery of cancer cells with our device when screening whole blood spiked with MCF-7 GFP cells.
Keywords CTC; Inertial microfluidics; Size-based separation
Author Manuscript
INTRODUCTION
Author Manuscript
Circulating tumor cells (CTCs) are cells that are shed from a primary tumor and have entered the vascular system. Enumerating and analyzing these cells can potentially determine patient prognosis, monitor disease progression, and lead to effective targeted therapies. The rarity of CTCs in cancer patients’ blood, however, is a great challenge, as only 1–10 CTCs are present in a 7.5mL sample of patient blood [1]. Current techniques for isolating and identifying CTCs rely either on the physical properties of these cells (e.g. size, charge, deformability, etc. [2–5]) or on the expression of particular markers, such as EpCAM. Since CTCs are larger than red blood cells (RBCs, ~6 μm), isolation based on cell size, i.e. membrane filters [6, 7], has been a large focus. While filtration is somewhat effective, cells can be subject to damaging high-shear stress, especially when the filters become increasingly clogged [1, 4]. Here, we describe a novel platform we have developed to isolate CTCs with high recovery from peripheral blood. Our platform is an inertial-based microfluidic device that has highthroughput, and is completely passive and label-free. We achieve a recovery efficiency of 94.2% ± 2.1% in benchmarking experiments, i.e. processing whole blood spiked with MCF-7 GFP cells at 130 μL/min.
*
CONTACT: Lydia L. Sohn; phone: +1-510-642-5434; [email protected].
Yang et al.
Page 2
Author Manuscript
THEORY We have developed an inertial microfluidic device, which employs inertial forces to manipulate cell streamlines with respect to cell diameter. A schematic of our CTC recovery platform is shown in Figure 1A. The platform consists of two stages, which when combined, isolates larger (> 15 μm) cells from whole blood. During separation, cells occupy unique equilibrium positions that are dependent on maximum fluid flowrate and cell properties, such as size. In more detail, our device consists of a combination of symmetric and asymmetric contracting and expanding microchannel arrays (sCEA and aCEA, respectively) to isolate larger (>15 μm) targeted cells from blood (Figure 1A). Stage One, which consists of the sCEA, focuses all cells to the outer wall via a balance of shear gradient
Author Manuscript
( ) and wall inertial lift forces ( ) where, fL, a,W, ρ, and Um are the dimensionless lift coefficient, cell diameter, channel width, fluid density, and maximum velocity, respectively (Figure 1A) [8]. At the end of Stage One, the cells are confined to a compact and distinct streamline induced by a net inertial lift towards the channel walls. Stage Two, which consists of the aCEA, exposes the compact and inertial focused streamline of cells to a Dean drag force (FD = 3πμUDeana) within the contraction regions, where UDean and μ are the transverse velocity and fluid viscosity, respectively (Figure 1A) [9]. The dominance of the Dean drag force is inversely proportional to cell diameter. Thus, in Stage two, large cells remain in the equilibrium streamline established, and smaller cells are pulled into a different equilibrium position [10].
METHODS Author Manuscript
Device Fabrication and Operation Our complete multi-stage device consists of a single sCEA microchannel for Stage One and four independent aCEA microchannels operating in parallel for Stage Two. In-plane filters at the entrance of the sCEA are included to remove the cellular debris that could offset the flow in the device. The device is fabricated using standard soft lithography techniques. Briefly, we create a negative master of our device via standard photolithography. A PDMS mold is then cast, inlet and outlet holes are cored, and finally treated with oxygen plasma with a Harrick Plasma System (PC-001) for 30 seconds and immediately bonded to a glass slide.
Author Manuscript
For the benchmarking experiments performed, we processed 1 mL of whole blood spiked with MCF-7 GFP cells at a ratio of 10,000 white blood cells (WBCs) to 1 MCF-7 GFP cell. We used a sample and buffer inlet flow rate of 130 and 200 μL/min, respectively. In a single run, we acquired a total of five sets of data: one set corresponding to the initial waste (i.e. red blood cells and platelets) obtained after the sample passes through Stage One (the sCEA) and four separate data sets corresponding to the waste and collection outlets from each aCEA within Stage Two. We collected all outlets separately and performed flow cytometry (Guava EasyCyte) to determine the number of MCF-7 GFP cells in each. We defined the recovery efficiency of our device as the percentage of the sum of GFP cells collected in comparison to the total number of GFP cells found in all outlets.
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 3
Blood Samples and Cell Culture
Author Manuscript
MCF-7 GFP breast-cancer cells were cultured in growth medium containing DMEM supplemented by 10% FBS, 1% Penstrep, and 1% NEAA and maintained at 37°C in 5% CO2. For our spiking experiments, cells were grown in monolayer to 80% confluence and detached with 0.25% trypsin for 3 minutes. The cell suspension was washed once with DMEM media and then single cells were spiked into healthy human donor blood obtained with consent under a University of California, Berkeley IRB-approved protocol.
RESULTS AND DISCUSSION
Author Manuscript
Our CTC isolation platform, operating at 130 μL/min, demonstrated an overall recovery efficiency of 94.2 ± 2.1% (Figure 1B). A breakdown of MCF-7 GFP cell counts with respect to each outlet is shown in Table 1. Individually, each aCEA microfluidic channel (sets 2 through 5) achieved recovery efficiencies > 92%. A P value of 0.000288 was obtain between Collection and Waste by performing a Student T-test.
CONCLUSION Our platform is advantageous over existing technologies in its efficiency to recover cancer cells from whole blood and its ease of use. The device successfully recovered MCF-7 cells spiked into healthy human blood with an efficiency of 94.2% ± 2.1% at 130 μl/min. Future benchmarking will involve processing blood spiked with cancer cells at clinically relevant ratios. Ultimately, the simplicity of our platform will allow for direct integration with downstream single cell analysis platforms for the characterization of recovered cells.
Author Manuscript
Acknowledgments This work is supported by NIH 1R21CA182375-01A1 and the Bakar Fellows Program.
References
Author Manuscript
1. Hong B, et al. Detecting circulating tumor cells: current challenges and new trends. Theranostics. 2013; 3:377–394. [PubMed: 23781285] 2. Yu M, et al. Circulating tumor cells: approaches to isolation and characterization. The Journal of cell biology. 2011; 192:373–382. [PubMed: 21300848] 3. Phillips KG, et al. Optical quantification of cellular mass, volume, and density of circulating tumor cells identified in an ovarian cancer patient. Frontiers in oncology. 2012; 2:72. [PubMed: 22826822] 4. Park JM, et al. Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood. Analytical chemistry. 2012; 84:7400–7407. [PubMed: 22881997] 5. Autebert J, et al. Microfluidic: an innovative tool for efficient cell sorting. Methods. 2012; 57:297– 307. [PubMed: 22796377] 6. Vona G, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulatingtumor cells. The American journal of pathology. 2000; 156:57–63. [PubMed: 10623654] 7. Harouaka R, et al. Circulating tumor cells: advances in isolation and analysis, and challenges for clinical applications. Pharmacology & therapeutics. 2014; 141:209–221. [PubMed: 24134902] 8. Amini H, et al. Inertial microfluidic physics. Lab on a chip. 2014; 14:2739–2761. [PubMed: 24914632]
Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 4
Author Manuscript
9. Lee MG, et al. Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress. Analytical chemistry. 2013; 85:6213–6218. [PubMed: 23724953] 10. Yang D, et al. High-throughput microfluidic device for rare cell isolation. Bio-MEMS and Medical Microdevices II. 2015; 9518:95180E.
Author Manuscript Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 5
Author Manuscript Author Manuscript
Fig. 1.
Schematic, isometric view, of our CTC isolation platform. A) Stage One consists of a symmetric CEA (sCEA) microfluidic channel in which cells are focused to the outer walls of the microchannel through a balance of wall lift and shear gradient lift forces. Stage Two consists of an asymmetric CEA (aCEA) microfluidic channel in which inertial focused streamlines are exposed to Dean drag forces where small particles migrate to different equilibrium positions. B) Overall percentage of MCF-7 GFP cells found in all collection and waste outlets. Data was obtain from processing 1 mL of whole blood spiked with MCF-7 GFP cells at a ratio of 10,000 WBCs: 1. Error bars correspond to standard deviation.
Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
Yang et al.
Page 6
Table 1
Author Manuscript
Number of MCF-7 GFP cells per outlet and recovery efficiency. The data is derived from healthy human donor blood spiked with MCF-7 GFP cells at a ratio of 10,000 WBCs: 1 MCF-7 GFP cells. The process flow rate employed was 130 μL/min. Set
Collection (MCF-7 GFP cells)
Waste (MCF-7 GFP cells)
Efficiency
1
–
0
–
2
62
2
96.9%
3
55
3
94.8%
4
51
4
92.7%
5
60
5
92.3%
Total
228
14
94.2%
Author Manuscript Author Manuscript Author Manuscript Micro Total Anal Syst. Author manuscript; available in PMC 2016 July 21.
