Towards a Microfluidic Platform for Cell Handling
H. Höfemann1, T. Borchardt2, C. Cupelli2, R. Gronmaier1, C. Müller 3, S. Haeberle1, R. Zengerle1,2
1
HSG-IMIT, Institute for Micromachining and Information Technology, Villingen-Schwenningen, Germany
Laboratory for MEMS Applications, 3Laboratory for Process Technology, Department of Microsystems Engineering (IMTEK), University of Freiburg, Germany henning.hoefemann@hsg-imit.de
2
Abstract
This paper describes our capabilities in design, fabrication and testing of microfluidic chips for cellhandling and separation. A rapid prototyping process, based on soft embossing in thermoplastic substrates enables the fast and cost-efficient fabrication of up to several tens of identical chips as pre-stage to later micro injection molding. As an application example, we present a passive microfluidic chip for the separation of leukocytes (white blood cells, WBC) from erythrocytes (red blood cells, RBC) out of a suspension, based on the hydrodynamics within the laminar flow regime. The achieved separation efficiency is about 87 %.
(separating) or cell accumulation are required for a complete microfluidic cell handling platform. Since most of these processes are based on continuous flows (to realize a certain throughput), we consider the pressuredriven platform as one of the most promising ones for cell handling and actuation. Working towards such a platform, we focus on both, the required fabrication capabilities, as well as the theoretical understanding, fluidic design and testing of microfluidic chips. Within this paper, we describe a rapid prototyping technology for microfluidic chips in thermoplastics (“soft embossing”), as well as an example for a cell handling chip, namely a passive cell separation device.
1 Introduction
Cell handling in microfluidic channels offers novel prospects compared to classical systems, e.g. single-cell analysis [1], dielectrophoretic cell manipulation [2], or identification and separation of different cell populations regarding type [3], optical properties [4] or size [5-7]. Consequently, increasing attention has been drawn to these microfluidic technologies within the past years, especially in the field of cell-based diagnostics and regenerative medicine. Depending on the application, several cell handling operations have to be implemented in one microfluidic chip. In order to avoid starting always from the scratch, already developed basic operations, so called “unit operations”, should be used and combined whenever possible. We call this the “microfluidic platform approach” similar to the platform concepts in other technological areas (e.g. ASIC’s in microelectronics). A discussion of already established platforms as well as implemented applications can be found in a recent review paper [8]. Some of these microfluidic platforms could be also interesting for cell-handling applications. However, additional unit operations like cell alignment (flow focusing), deflection of individual cells
2 Prototyping of Microfluidic Chips
The fabrication of microchannels in PDMS (polydimethylsiloxane) using the so called “soft lithography” process [9] is very common in microfluidics research. The most convincing arguments for this well established process are its simplicity and low costs. However, the time consuming nature of the manual casting and curing process does not allow chip manufacturing on an industrial level. Therefore, a high-volume technology like injection molding is inevitable for the cost-efficient production of high quantities of microfluidic chips. In order to use the identical properties of a durable, thermoplastic material within the development phase of a microfluidic chip, however, an alternative process to soft lithography is required. This technology should be fast and cheap to realize different designs within a short time, but also allow a seamless transfer to injection molding. The “soft embossing” process as described below, enables the fast fabrication of microfluidic chips in thermoplastics, based on a replication process [10]. The soft embossing process is based on the replication of a microstructured master via a PDMS stamp into a polymer substrate (see Figure 1). The primary master is manufactured using standard micromachining processes
like lithography of SU-8 (1A) or DRIE (deep reactive ion etching) of a silicon wafer (1B). In the following step, the master is cast by a special PDMS type which serves as replication stamp after curing (2). The used PDMS is Elastosil RT 607 (Wacker Chemie, Germany) because of its good cavity filling properties due to a low viscosity in the liquid state and a relatively high hardness after curing. To prevent backside unevenness of the PDMS stamp, a flat metal plate is applied during the curing process. The flexible PDMS mold can then be easily peeled off from the master. The final chip is embossed in a thermoplastic substrate (e.g. COC) using a hot embossing machine (3). The PDMS stamp on top of the substrate is enclosed in a metal frame between two highly flattened and parallel aligned metal plates to warrant a tight fit and to prevent a deflection of the elastic mold during embossing. The whole assembly is evacuated to avoid trapping of air bubbles and heated up to 175 °C beyond the glass transition temperature of the substrate (Tg = 130°C for COC, TOPAS 5013). By applying a load of F = 3 kN for 5 min the polymer fills the microcavities of the PDMS mold. After cooling down to room temperature the PDMS stamp can be peeled off the structured chip (see Figure 2 for an example chip) and reused for the next embossing cycle. The mean life time before abrasion effects become noticeable is about 100 cycles [10].
1A SU-8
SU-8 Si Si
Figure 2. Microfluidic COC Chip with 100 µm wide channels, fabricated by Soft Embossing.
1B DRIE
The soft embossed microfluidic chips are afterwards sealed by thermal diffusion bonding or self-adhesive foils. Also different long-term stable coating technologies for global hydrophilic or locally refined hydrophobic coatings have been developed and can be applied if required for the application [10]. An example of a soft-embossed chip in COC is given in Figure 2. Through-holes and threads for standard connectors are machined after embossing the microstructures.
3 Separation of Cells
2 PDMS Si
3 PDMS COC
Figure 1. Soft embossing process flow: A structured master is fabricated by means of SU-8 lithography (1A) or deep reactive ion etching (DRIE, 1B). PDMS is cast upon the master and cured (2). The microstructures of the master are replicated in a thermoplastic substrate (e.g. COC) via the PDMS stamp by hot embossing (3).
As an application example, we present a 4-port microfluidic chip for the separation of blood cells. Namely, leukocytes are separated from erythrocytes out of a diluted suspension [11]. Leukocytes or white blood cells are spherical cells with an average diameter of 7 20 µm whereas erythrocytes or red blood cells are shaped as 2 µm thick biconcave discs with a diameter of 7 µm. The number of erythrocytes to be found in whole blood is on the scale of 106 cells per microliter, leukocytes occur at 104 cells per microliter. So a diluted suspension with an increased leukocyte percentage had to be used for the experiments. The microfluidic chips have been fabricated in PDMS and were afterwards bonded onto a microscope slide to comply with the optical inspection on a standard microscope during the experiments. A passive, hydrodynamic focusing concept has been implemented which is illustrated in Figure 3. It is based on different flow rates at the inlet side. By increasing the flow through the “Inlet NaCl”-port, the cells are pushed against the lower channel wall. The ratio of the outlet flow rates depends on the ratio of the hydrodynamic resistances of the outlet channels, respectively.
Outlet Leukocytes Inlet NaCl
Inlet Blood
Erythrocytes Leukocytes Line of Separartion
Outlet Erythrocytes
Figure 4. Orientation of Erythrocytes with their flat side parallel to the lower channel wall due to hydrodynamic focusing. The channel width is about 30 µm.
Figure 3. Separation principle: Erythrocytes and leukocytes are aligned at the lower channel wall by hydrodynamic focusing and follow their “stream lines” into different outlets according to the ratio of the outlet flow rates.
Because of the difference in diameter between the two cell types the center of mass of the smaller erythrocytes is located closer to the wall compared to the bigger leukocytes. This effect is additionally amplified by the disc-like shape of the red blood cells which leads to an orientation with the flat side of the erythrocytes parallel to the channel wall as depicted in Figure 4. After focusing the cells on the lower channel wall, the flow is split into two different outlets (“Outlet Leukocytes” and “Outlet Erythrocytes”) at a second channel branching. Depending on the flow rate ratio of the two corresponding outlet flows, a “line of separation” between the two sub-streams forms out, deciding whether the liquid leaves the channel via the perpendicular channel to the top, or through the channel to the right.
Due to the laminar flow conditions, any cell that is located above the line of separation (with its center of mass) will leave the channel via the upper outlet. Consequently, any cell which is located below will continue towards the right outlet channel, respectively. As described before, the bigger leukocytes are located further away from the wall and can thus be separated from the erythrocytes if appropriate channel dimensions and flow rates are chosen. The flow rate ratio of sample (blood) to buffer (NaCl) used for the following results is 1:16. A lower ratio decreases the separation efficiency and a higher ratio lines out the sample flow. The liquid flow fraction leaving through the leukocyte outlet is about 1.5 %. These conditions lead to a separation efficiency of about 87 %. A series of microscope images in Figure 5 shows the successful separation of leukocytes to the upper outlet channel (1, 2), while the erythrocytes follow the main stream to the right outlet (3, 4).
1
3
2
4
Figure 5. Separation experiment. Two leukocytes (marked with a white circle in picture 1 and 2) enter the focusing area from the left side and leave through the upper outlet channel. In contrast, the erythrocytes (marked with red circles in picture 3 and 4) leave through the right outlet channel. The channel width is about 30 µm.
4 Conclusion & Outlook
We presented a rapid prototyping technology for the fabrication of microfluidic chips based on a combination of micromachining (SU-8 or DRIE) and soft embossing guaranteeing a high geometrical precision and surface quality. Thus, the chain from first designs and calculations towards prototyping and pre-mass production is closed and design iterations can be realized within short time scales leading to a faster development process. The presented application for sorting of blood cells allows the separation of cells or particles with diameters between 2 and 5 µm from bigger ones by adjustment of flow rates and flow rate ratios. This continuous passive cell separation technique is an important unit operation for microfluidic cell handling. It will be further optimized and combined with additional unit operations to expand the capabilities of our pressure driven cell handling platform for future applications.
[4] X. J. Liang, A. Q. Liu, C. S. Lim, T. C. Ayi, and P. H. Yap, "Determining refractive index of single living cell using an integrated microchip," Sensors and Actuators A, vol. 133, no. 2, pp. 349-354, 2006. [5] M. Yamada, J. Kobayashi, M. Yamato, M. Seki, and T. Okano, "Millisecond treatment of cells using microfluidic devices via two-step carrier-medium exchange," Lab Chip., vol. 8, no. 5, pp. 772-778, May2008. [6] M. Yamada and M. Seki, "Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics," Lab Chip., vol. 5, no. 11, pp. 1233-1239, Nov.2005. [7] J. Takagi, M. Yamada, M. Yasuda, and M. Seki, "Continuous particle separation in a microchannel having asymmetrically arranged multiple branches," Lab Chip., vol. 5, no. 7, pp. 778-784, July2005. [8] S. Haeberle and R. Zengerle, "Microfluidic platforms for lab-on-a-chip applications," Lab Chip., vol. 7, no. 9, pp. 1094-1110, Sept.2007. [9] J. C. McDonald and G. M. Whitesides, "Poly(dimethylsiloxane) as a Material for Fabricating Microfluidic Devices," ACCOUNTS OF CHEMICAL RESEARCH, vol. 35, pp. 491-499, 2002. [10] J. Steigert, S. Haeberle, T. Brenner, C. Müller, C. P. Steinert, P. Koltay, N. Gottschlich, H. Reinecke, J. Rühe, R. Zengerle, and J. Ducree, "Rapid prototyping of microfluidic chips in COC," J. Micromech. Microeng., vol. 17, pp. 333-341, 2007. [11] T. Borchardt, "Entwicklung eines fluidischen Mikrochips zur Zellseparation." Universität Freiburg - IMTEK, 2007.
Acknowledgement
This work has been partially supported by the Federal Ministry of Education and Research, BMBF (Nr. 16SV2361).
References
[1] J. F. Zhong, Y. Chen, J. S. Marcus, A. Scherer, S. R. Quake, C. R. Taylor, and L. P. Weiner, "A microfluidic processor for gene expression profiling of single human embryonic stem cells," Lab Chip., vol. 8, no. 1, pp. 68-74, Jan.2008. [2] S. Fiedler, S. G. Shirley, T. Schnelle, and G. Fuhr, "Dielectrophoretic sorting of particles and cells in a microsystem," Anal. Chem., vol. 70, no. 9, pp. 19091915, May1998. [3] 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, and M. Toner, "Isolation of rare circulating tumour cells in cancer patients by microchip technology," Nature, vol. 450, no. 7173, pp. 1235-1239, Dec.2007.