CONTINUOUS SEPARATION OF BREAST CANCER CELLS …88.8 % to over 93.3 % through the multi-stage...
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CONTINUOUS SEPARATION OF BREAST CANCER CELLS FROM BLOOD USING MULTI-STAGE MULTI-ORIFICE FLOW FRACTIONATION (MS-MOFF) H.S. Moon 1a , K. Kwon 2a , T.S Sim 1 , J.C. Park 1 , J.G. Lee 1 and H.Y. Jung 2 1 Bio Lab, Emerging Tech. R&D Center, Samsung Advanced Institute of Technology, South Korea 2 School of Mechanical Engineering, Yonsei University, South Korea ABSTRACT This research presents an multi-stage multi-orifice flow fractionation (MS-MOFF) device designed for separating breast cancer cell from blood. The structure and dimensions of the MS-MOFF were determined by the hydrodynamic principles to have consistent Reynolds numbers (Re) at each multi-orifice segment. From this device, we achieved improved separation efficiency by collecting and re-separating the non-selected target cells. The recovery of breast cancer cell increased from 88.8 % to over 93.3 % through the multi-stage multi-orifice segments. This device can be utilized to isolate rare cells from human blood such as circulating tumor cell solely by using hydrodynamic forces. KEYWORDS: Multi-stage multi-orifice flow fractionation(MS-MOFF), Cell separation, Circulating tumor cell INTRODUCTION Tumor cells originated from a primary tumor mass can circulate in the peripheral blood of cancer patients via intravasa- tion during malignant progression. Those circulating tumor cells (CTCs) are highly correlated with the “invasive behaviors” of cancer cells responsible for the majority of cancer-related deaths. The isolation or detection of CTCs can serve as a power- ful tool for cancer prognosis, diagnosis, assessment of tumor sensitivity to anticancer drugs, and personalization of anti- cancer therapy[1]. Therefore to collect and analyze CTCs, rapid and efficient separation technique is required. Several micro- fluidic devices have been reported for CTC isolation using surface marker such as EpCAM[2]. However some tumor cells express low or no EpCAM, resulting in a limited binding efficiency[3]. Microfluidic devices have been exploited for the separation of polymer beads, biological cells, and colloids from a varie- ty of samples especially for biological and medical applications. Previously we introduced a novel hydrodynamic method us- ing a multi-orifice microchannel for size-based particle separation, which is called a multi-orifice flow fractionation (MOFF)[4]. In MOFF, microparticle is moved laterally due to hydrodynamic inertial forces created by a multi-orifice struc- ture. The extent of lateral movement varies according to particle size and polymer microspheres can be concentrated at dif- ferent lateral positions in a microchannel[5]. This process enables continuous and high throughput separation of different particles without the application of external forces. To increase the recovery we developed a multi-stage multi-orifice flow fractionation (MS-MOFF) combining three multi-orifice segments(Figure 1). With this device, we improved recovery and minimize loss of purity by collecting and re-separating non-selected particles of the first separation. Using the polymer beads(7μm and 15 μm) we found that the recovery successfully increased from 73.2% to 88.7% while the purity slightly de- creased from 91.4% to 89.1%(for 15 μm)[6]. In this study, using this technique, we separated breast cancer cells(MCF-7, a model for CTC) from blood and investigated the improvement of separation efficiency. THEORY As we previously reported [4, 5], our MOFF can separate different sized particles based on the inertial lift force and mo- mentum-change-induced inertial force generated by series of orifice. The size-based particle separation could be achieved in the specific range of the channel Reynolds number (Re c ). Since the Re c is a function of microchannel dimensions and flow velocity, we can adjust for it by controlling flow rate. Figure 1(a) shows the schematic view of our device. The separation regions at the end of each stage divided into 3-branched channel. One of the branch channels positioned at middle is for col- lecting bigger particles and the others are for smaller one. Due to this flow division, without any compensation, flow rate in the second stage become smaller than the first stage, which alters the Re c . In order to maintain the Re c consistently from the first to the second stage, buffer solution was injected into inlet 2 and 3. EXPERIMENTAL The device consists of three multi-orifice segments, and consists of 3 inlets, 3 filters, 3 multi-orifice segments and 5 out- lets (Fig. 1(b)). It was fabricated by PDMS using soft-lithography and assembled with glass substrate after oxygen plasma treatment. Samples and buffer were loaded to the microchannel by syringe pumps and observed by microscope equipped with high speed CCD camera (HotShot 1280; NAC Image Technology). 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 1218 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA
Transcript of CONTINUOUS SEPARATION OF BREAST CANCER CELLS …88.8 % to over 93.3 % through the multi-stage...
CONTINUOUS SEPARATION OF BREAST CANCER CELLS FROM BLOOD USING MULTI-STAGE MULTI-ORIFICE FLOW FRACTIONATION
(MS-MOFF) H.S. Moon1a, K. Kwon2a, T.S Sim1, J.C. Park1, J.G. Lee1 and H.Y. Jung2
1 Bio Lab, Emerging Tech. R&D Center, Samsung Advanced Institute of Technology, South Korea 2 School of Mechanical Engineering, Yonsei University, South Korea
ABSTRACT This research presents an multi-stage multi-orifice flow fractionation (MS-MOFF) device designed for separating breast
cancer cell from blood. The structure and dimensions of the MS-MOFF were determined by the hydrodynamic principles to have consistent Reynolds numbers (Re) at each multi-orifice segment. From this device, we achieved improved separation efficiency by collecting and re-separating the non-selected target cells. The recovery of breast cancer cell increased from 88.8 % to over 93.3 % through the multi-stage multi-orifice segments. This device can be utilized to isolate rare cells from human blood such as circulating tumor cell solely by using hydrodynamic forces. KEYWORDS: Multi-stage multi-orifice flow fractionation(MS-MOFF), Cell separation, Circulating tumor cell
INTRODUCTION
Tumor cells originated from a primary tumor mass can circulate in the peripheral blood of cancer patients via intravasa-tion during malignant progression. Those circulating tumor cells (CTCs) are highly correlated with the “invasive behaviors” of cancer cells responsible for the majority of cancer-related deaths. The isolation or detection of CTCs can serve as a power-ful tool for cancer prognosis, diagnosis, assessment of tumor sensitivity to anticancer drugs, and personalization of anti-cancer therapy[1]. Therefore to collect and analyze CTCs, rapid and efficient separation technique is required. Several micro-fluidic devices have been reported for CTC isolation using surface marker such as EpCAM[2]. However some tumor cells express low or no EpCAM, resulting in a limited binding efficiency[3].
Microfluidic devices have been exploited for the separation of polymer beads, biological cells, and colloids from a varie-ty of samples especially for biological and medical applications. Previously we introduced a novel hydrodynamic method us-ing a multi-orifice microchannel for size-based particle separation, which is called a multi-orifice flow fractionation (MOFF)[4]. In MOFF, microparticle is moved laterally due to hydrodynamic inertial forces created by a multi-orifice struc-ture. The extent of lateral movement varies according to particle size and polymer microspheres can be concentrated at dif-ferent lateral positions in a microchannel[5]. This process enables continuous and high throughput separation of different particles without the application of external forces. To increase the recovery we developed a multi-stage multi-orifice flow fractionation (MS-MOFF) combining three multi-orifice segments(Figure 1). With this device, we improved recovery and minimize loss of purity by collecting and re-separating non-selected particles of the first separation. Using the polymer beads(7μm and 15 μm) we found that the recovery successfully increased from 73.2% to 88.7% while the purity slightly de-creased from 91.4% to 89.1%(for 15 μm)[6]. In this study, using this technique, we separated breast cancer cells(MCF-7, a model for CTC) from blood and investigated the improvement of separation efficiency.
THEORY
As we previously reported [4, 5], our MOFF can separate different sized particles based on the inertial lift force and mo-mentum-change-induced inertial force generated by series of orifice. The size-based particle separation could be achieved in the specific range of the channel Reynolds number (Rec). Since the Rec is a function of microchannel dimensions and flow velocity, we can adjust for it by controlling flow rate. Figure 1(a) shows the schematic view of our device. The separation regions at the end of each stage divided into 3-branched channel. One of the branch channels positioned at middle is for col-lecting bigger particles and the others are for smaller one. Due to this flow division, without any compensation, flow rate in the second stage become smaller than the first stage, which alters the Rec. In order to maintain the Rec consistently from the first to the second stage, buffer solution was injected into inlet 2 and 3.
EXPERIMENTAL
The device consists of three multi-orifice segments, and consists of 3 inlets, 3 filters, 3 multi-orifice segments and 5 out-lets (Fig. 1(b)). It was fabricated by PDMS using soft-lithography and assembled with glass substrate after oxygen plasma treatment. Samples and buffer were loaded to the microchannel by syringe pumps and observed by microscope equipped with high speed CCD camera (HotShot 1280; NAC Image Technology).
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 1218 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
We used MCF-7 cells (~16-24 μm diame-ter) and blood cells (~6-10 μm diameter) and separate based on their size difference. MCF-7 cells were spiked into blood samples diluted (1/100) in PBS with 1x104 cells/ mL concentra-tion and introduced to the device. BSA was al-so added with 2% (w/v) concentration to pre-vent the aggregation.
In order to determine the flow rate (Rec), RBC, WBC and MCF-7 were tested with vari-ous flow rate (Rec =60, 70, 80). To optimize the collection channel dimensions, we tested various dimensions according to Qc/Qm condi-tion (Qc: Flow rate of central collecting chan-nel in 1st stage of MOFF, Qm: Total flow rate in 1st stage of MOFF). Qc/Qm value represents the portions of central collecting channel of each segment (Figure 1(a)).
After separation, the performance was evaluated by cell counting with EpCAM and CD45 surface marker and DAPI staining that allow to discriminate epithelial cells and blood cells.
RESULTS AND DISCUSSION
As shown in Figure 2, RBC, WBC and MCF-7 showed different movement with various flow rates (Rec =60, 70, 80). When Rec was 70, at the 1st stage MOFF, the RBCs and WBCs split into two positions and MCF-7 cells focused at the middle of the channel(Inside). We could separate MCF-7cells and blood cells with best performance with this flow rate condition. At the 2nd stage MOFF, RBC and WBC directed to the side channel(Outside) same as the 1st stage and exited to Outlet II~V. On the other hand, a few MCF-7 that went through the side channel at the 1st stage (non-selected target), directed to the middle of channel(Inside) and exited to Outlet I.
Figure 3 shows the separation efficiencies of MS-MOFF when Rec is 70. To maintain the Rec in 2nd stage, the buffer solution was injected into inlet 2 and 3 with flow rate of 170, 184 and 202 �L/min depending on the Qc / Qm condition (35%, 45%, 65%).
As the central collecting channel became larger, recovery of MCF-7 in-creased while recovery of blood cells decreased. Using this device we could separate 98.9±0.4 % of MCF-7 cells and 70.3±4.7 % of blood cells when Qc/Qm was 45%. Since we re-separating the non-selected MCF-7 cells using MS-MOFF, the recovery of MCF-7 in-creased from 88.8 %(single-MOFF[7]) to over 93.3 % (when Qc/Qm = 35%).
Figure 2. Trajectory of cells through the multi-orifice microchannel according to various channelReynolds numbers (Rec).
Figure 1 (a) Schematic view of the multi-stage multi-orifice flow frac-tionation (MS-MOFF). (b)Fabricated device.
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CONCLUSION
We have demonstrated a MS-MOFF that separate cancer cell from blood with improved efficiency. This technique will be useful for clinical applications, such as separation of circulating tumor cells (CTC) or rare cells from human blood samples because of its simple experi-mental set up, high operational flow rate and capability of collection of viable cells due to its label-free manner. ACKNOWLEDGEMENTS
This work was supported by the Korea Science & Engineering Founda-tion(KOSEF) grant funded by the Korea government(MEST)(2011-0003131), Na-tional Research Foundation of Ko-rea(NRF) grant funded by the Korea gov-ernment(MEST)(No. 2011-0016731) and National R&D Program for Cancer Con-trol, Ministry of Health & Welfare, Re-public of Korea (no. 1120290). a These authors contributed equally to this work.
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107, pp. 18392-18397 (2010) [3] G. Deng, et. al., “Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly in-
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[4] J.S. Park, S.H. Song, and H.I. Jung, “Continuous focusing of microparticles using inertial lift force and vorticity via multi-orifice microfluidic channels,” Lab Chip, vol. 9, pp. 93-9489 (2009)
[5] J.S. Park, S.H. Song, and H.I. Jung, “Multiorifice flow fractionation: Continuous size-based separation of microspheres using a series of contraction/expansion microchannels,” Anal. Chem., vol. 81, pp. 8280-8288 (2009)
[6] T.S. Sim, K. Kwon, J.C. Park, J.G. Lee, and H.I. Jung, “Multistage-Multiorifice Flow Fractionation (MS-MOFF): Con-tinuous size-based separation of microspheres using multiple series of contraction/expansion microchannels,” Lab Chip, vol. 11, pp. 93-99 (2011)
[7] H.S. Moon, K. Kwon, H. Han, J. Sohn, S.I. Kim, S. Lee, and H.I. Jung, “Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation(MOFF) and dielectrophoresis(DEP),” Lab Chip, vol. 11, pp.1118-1125 (2011)
CONTACT *H.I. Jung, tel: +82-2-2123-7767; [email protected]
Figure 3. Separation efficiencies of blood cells and MCF-7 cells in the MS-MOFF depending on Qc / Qm condition. Inside fraction exits to out-let 1 and outside fraction to outlet 2~5
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