The presented mass-producible filtration chips have demonstrated high capture efficiency. With 100% capture efficiency on 10μm beads. For unfixed tumor cell line MCF-7, the device has 87.3±1.5% capture efficiency under a constant negative pressure of 0.1psi. Other cell lines tests showed the capture efficiency would change if use different cell lines/populations. Due to the injection-molding production, the performance of the low-cost device is robust.
The recovery rate of live MCF-7 cells spiked in diluted blood is 80~89%, of Colo205, PC3 and K562 are 70% to 80%. The filtration chip could handle 15mL diluted blood (1:15) in 1~1.5 hour. For cell viability test, PC-3 prostate cancer cell line was testes to show after 48 hours filtration, and stained on-chip with Calcein AM are viable and even proliferating, which enable further cell culture and other analysis of rare cells. The system setup for chip tubeless was show the stability of performance and high cell viability. With the aspects of low cost, tubeless, high capture efficiency and viable capture makes this device enables further cell analysis and other experiments of these value cells.
Reference
(1) P. S. Steeg Nat Med 2006, 12 (8), 895-904.
(2) S. Mocellin, U. Keilholz, C. R. Rossi, D. Nitti Trends Mol Med 2006, 12 (3), 130-139.
(3) J. Kaiser Science 2010, 327 (5969), 1072-1074.
(4) M. Cristofanilli Semin Oncol 2006, 33 (3), S9-S14.
(5) S. Mocellin, D. Hoon, A. Ambrosi, D. Nitti, C. R. Rossi Clin Cancer Res 2006, 12 (15), 4605-4613.
(6) G. Wiedswang, B. Naume Nat Clin Pract Oncol 2007, 4 (3), 154-155.
(7) S. Riethdorf, H. Wikman, K. Pantel Int J Cancer 2008, 123 (9), 1991-2006.
(8) J. S. de Bono, H. I. Scher, R. B. Montgomery Clin Cancer Res 2009, 15 (4), 1506-1506.
(9) J. den Toonder Lab Chip 2011, 11 (3), 375-377.
(10) M. Naoe, Y. Ogawa, J. Morita, K. Omori, K. Takeshita, T. Shichijyo, T. Okumura, A. Igarashi, A. Yanaihara, S. Iwamoto, T. Fukagai, A. Miyazaki, H. Yoshida Cancer 2007, 109 (7), 1439-1445.
(11) S. Riethdorf, H. Fritsche, V. Muller, T. Rau, C. Schindibeck, B. Rack, W. Janni, C.
Coith, K. Beck, F. Janicke, S. Jackson, T. Gornet, M. Cristofanilli, K. Pantel Clin Cancer Res 2007, 13 (3), 920-928.
(12) J. Kraan, S. Sleijfer, M. H. Strijbos, M. Ignatiadis, D. Peeters, J. Y. Pierga, F.
Farace, S. Riethdorf, T. Fehm, L. Zorzino, A. G. J. Tibbe, M. Maestro, R.
S. Maheswaran, D. A. Haber J Clin Oncol 2009, 27 (15),
(14) L. V. Sequist, S. Nagrath, M. Toner, D. A. Haber, T. J. Lynch J Thorac Oncol 2009, 4 (3), 281-283.
(15) S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R.
Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber, M. Toner Nature 2007, 450 (7173), 1235-U10.
(16) S. L. Stott, C. H. Hsu, D. I. Tsukrov, M. Yu, D. T. Miyamoto, B. A. Waltman, S. M.
Rothenberg, A. M. Shah, M. E. Smas, G. K. Korir, F. P. Floyd, A. J. Gilman, J. B. Lord, D. Winokur, S. Springer, D. Irimia, S. Nagrath, L. V. Sequist, R. J. Lee, K. J. Isselbacher, S. Maheswaran, D. A. Haber, M. Toner P Natl Acad Sci USA 2010, 107 (43),
Pantel Clin Cancer Res 2005, 11 (10), 3678-3685.
(19) R. Gertler, R. Rosenberg, K. Fuehrer, M. Dahm, H. Nekarda, J. R. Siewert Recent Res Cancer 2003, 162 149-155.
(20) R. Rosenberg, R. Gertler, J. Friederichs, K. Fuehrer, M. Dahm, R. Phelps, S.
Thorban, H. Nekarda, J. R. Siewert Cytometry 2002, 49 (4), 150-158.
(21) E. Andreopoulou, L. Y. Yang, K. M. Rangel, J. M. Reuben, L. Hsu, S.
Krishnamurthy, V. Valero, H. A. Fritsche, M. Cristofanilli Int J Cancer 2012, 130 (7), 1590-1597.
(22) V. Melnikova, Y. Zhang, M. Pace, M. Garza, S. Sukumaran, S. Zhao, J. Woo, D.
Davis Ejc Suppl 2010, 8 (7), 196-196.
(23) J. S. Kuo, Y. X. Zhao, P. G. Schiro, L. Y. Ng, D. S. W. Lim, J. P. Shelby, D. T. Chiu Lab Chip 2010, 10 (7), 837-842.
(24) G. Vona, A. Sabile, M. Louha, V. Sitruk, S. Romana, K. Schutze, F. Capron, D.
Franco, M. Pazzagli, M. Vekemans, B. Lacour, C. Brechot, P. Paterlini-Brechot Am J Pathol 2000, 156 (1), 57-63.
(25) V. De Giorgi, D. Massi, M. Grazzini, P. Pinzani, T. Lotti J Am Acad Dermatol 2011, 64 (2), Ab9-Ab9.
(26) I. Desitter, B. S. Guerrouahen, N. Benali-Furet, J. Wechsler, P. A. Janne, Y. A.
Kuang, M. Yanagita, L. L. Wang, J. A. Berkowitz, R. J. Distel, Y. E. Cayre Anticancer Res 2011, 31 (2), 427-441.
(27) V. Hofman, C. Bonnetaud, M. I. Ilie, P. Vielh, J. M. Vignaud, J. F. Flejou, S.
Lantuejoul, E. Piaton, N. Mourad, C. Butori, E. Selva, M. Poudenx, S. Sibon, S. Kelhef, N. Venissac, J. P. Jais, J. Mouroux, T. J. Molina, P. Hofman Clin Cancer Res 2011, 17 (4), 827-835.
(34) S. M. McFaul, B. K. Lin, H. Ma Lab Chip 2012, 12 (13), 2369-2376.
(35) C. S. Tan, A. Fan, K. N. Chen, R. Reif Appl Phys Lett 2003, 82 (16), 2649-2651.
Appendix A
Design concepts of apertures on the micro-filter
This section is talking about the reasons we designed the micro-filter. First, we chose rectangle as the shape of apertures because of the simplicity in multi-layer photolithography fabrication of the mold for injection-molding. For simpler detection, the short transverse of the rectangular aperture should be the height to ensure the target cells will not be overlapping after filtration.
In addition, various dimension and arrangement of the apertures generate different pressure fields encountered by a cell in a filtration environment. It was necessary to analyze how the design of apertures affects the cells because cell membrane has its tolerance to neighboring pressure. There were several studies about cell membrane damage associated with mechanical trauma. For example, micropipette experiments on artificial phospholipids, similar to cell membrane, demonstrated that membrane damage happened when membrane tension increased over a critical level, i.e. membrane area extension outpace 3% (Evans et al. 1979). Cell lysis occured at the tension as low as 3 mN/m (Kwok and Evans 1981). Therefore, to prevent the probability of cell rupture and lysis during filtration process, the aperture was needed to be designed for lowering the pressure on the cell membrane.
A brief analysis (Daniel T. Chiu, 2010) of hydrodynamic pressure conveyed encountered by a spherical particle by the carrier fluid in various filtration environments is in sections below. Three simplified filtration conditions experienced by a spherical particle will be discussed respectively: (1) carrier fluid flows past a spherical particle; (2)
Condition 1: Carrier fluid flows past a free spherical particle. pressure to be 0), m is the viscosity of the carrier fluid, R is the radius of the particle, and V is the relative velocity of the fluid. The maximum pressure is at θ = 0°; the minimum pressure is at θ = 180° on the particle surface; so that the maximum pressure difference (𝛥𝑃𝑚𝑎𝑥) is:
𝛥𝑃𝑚𝑎𝑥 = 3 𝜇𝑉 𝑅
In filtration processing, the local pressure distribution around the particle and the maximum pressure difference were mechanical force probable to made the cell damaged. In the equation (1) and (2), the parameter we could tune, while filtering, were V and 𝑃0. The optimization of V and 𝑃0 will be discussed in section 2.3.
Figure 10 Analysis of the pressure experienced by the spherical particle in the condition of carrier fluid flows bypass
(a) Carrier fluid flows past a free spherical particle and (b) pressure distribution on the surface (Daniel T. Chiu, 2010)
Condition 2: a spherical particle captured by a single aperture
While a spherical particle captured by a single aperture (Figure 11 (a)) in the filtration processing, the pressure that carrier fluid imparting to the spherical particle strongly depended on the covered ratio of aperture area. Figure 11 錯誤! 找不到參照來 源。 (b) illustrates the normalized spherical particle pressure versus various covered percentage of aperture area. The spherical particle pressure while the aperture totally blocked were at about 10^7 and 10^9 times as large as the spherical particle pressure while ~75% aperture covered and aperture unobstructed. Obviously, to ensure the flow
(a)
(b)
Figure 11 Analysis of the pressure experienced by the captured spherical particle by a single aperture
(a) A spherical particle captured by a single aperture; (b) spherical particle pressure versus various covered percentage of aperture area. Data points were obtained by solving the Navier–Stokes equation numerically for a 5 mm (D) × 10 mm (L) channel partially blocked by a 5 mm (D) cell, at a fluid velocity of 1 mm /s. (Daniel T. Chiu, 2010)
(a) (b)
Condition 3: A spherical particle occluding an aperture in the presence of multiple parallel apertures
In this case (Figure 12 (a)), one of the parallel apertures was blocked by a single particle. Meanwhile, the other apertures were still unobstructed and allowed the carrier fluid to flow bypass. For one unblocked parallel channel, the pressure difference between the inlet and outlet of the channel (∆𝑃𝑐ℎ𝑎𝑛𝑛𝑒𝑙) came from viscous dissipation which was related to viscosity of the carrier fluid (µ), hydrodynamic diameter of the channel (𝐷𝑐ℎ𝑎𝑛𝑛𝑒𝑙) the length of the channel (L) and the velocity of the carrier fluid (V).
The viscous dissipation of one unblocked parallel channel could be calculated with the equation:
That is, the pressure difference experienced by the captured spherical particle was equal to the pressure drop of the channel, which could be lowered to 𝑛1 times with n parallel channels.
Figure 12 Multiple parallel apertures and the shape design
(a) A spherical particle occluding an aperture in the presence of multiple parallel apertures; (b) The rectangular aperture had interstitial space for flow bypass even though the captured cells connecting to each other closely but the independent single apertures had not.
In conclusion, the aperture design was not only about the apertures with key size for separating the cells by size but also the arrangement and dimension of the apertures which could prevent the cell damage while filtering. After analyzing from the condition one to three, first of all, the aperture dimension was decided to be 100µ m (W) × 8µm (H). The width of the rectangular aperture (100µm) could prevent the aperture dead blocked. Even though there were more than one cells captured by the same aperture, there still had interstitial space for flowing bypass (Figure 12 (b)). The second, the apertures were parallel, not independent to each other. The design safeguarded the captured cells from the huge incensement once the channel occluded because rest of the channels still allowed the carrier fluid to flow bypass. Finally, there were over 2000 apertures lined on the serpentine filtration part of the chip. The quantity of apertures was not only for promoting flow rate (larger total area of aperture section) in the same giving pressure but also for reducing ∆𝑃𝑐ℎ𝑎𝑛𝑛𝑒𝑙 (see eqn. (4), larger the n).
(a) (b)
Appendix B Thermal bonding chamber system
For reaching the thermal bonding environment (normal stress and temperature), a thermal bonding system was designed. The thermal bonding system was a system built up with two aluminum blocks with caves which could form a chamber between them while they were combined (Figure 13 (a)). The bottom one with PMMA dummy (Figure 13 (b)) which could be changed to feat the shape of the spares. And the top one was attached with a slice of membrane (Figure 13 (c)), which could endure the pressure from high-pressure nitrogen. While high-pressure nitrogen applied to the top chamber, the membrane (with O-ring between two blocks to prevent leakage) transferred normal stress to the spares. For keeping the bonding temperature, the hotplate was set under the bonding chamber. With stable pressure, temperature and flat surfaces on the spares to be bonded, the spares could be assembled.
Figure 13 Thermal bonding chamber system (a) a perspective cartoon drawing of