Langmuir 2008, 24, 5955-5960
5955
Cell Culture Chip Using Low-Shear Mass Transport Ke Liu,† Rajasekar Pitchimani,‡ Dana Dang,† Keith Bayer,† Tyler Harrington,† and Dimitri Pappas*,† Departments of Chemistry and Biochemistry, and Chemical Engineering, Texas Tech UniVersity, Lubbock, Texas 79409-1061 ReceiVed NoVember 6, 2007. ReVised Manuscript ReceiVed March 13, 2008 We have developed a flow cell that allows culturing adherent cells as well as suspended cells in a stable, homogeneous, and low-shear force environment. The device features continuous medium supply and waste exchange. In this paper, a simple and fast protocol for device design, fabrication, and assembly (sealing) based on a poly(dimethylsiloxane) (PMDS)/glass slide hybrid structure is described. The cell culture system performance was monitored, and the effective shear force inside the culture well was also determined. By manipulating the device dimensions and volumetric flow rate, shear stress was controlled during experiments. Cell adhesion, growth, proliferation, and death over long-term culture periods were observed by microscopy. The growth of both endothelial and suspension cells in this device exhibited comparable characteristics to those of traditional approaches. The low-shear culture device significantly reduced shear stress encountered in microfluidic systems, allowing both adherent and suspended cells to be grown in a simple device.
1. Introduction Cell culture is a useful method for understanding cell behaviors and investigating cellular physiological and pathological responses to different culture environments. Despite the time tested popularity of traditional culture systems, such as dishes, flasks and macroscale bioreactors, microfluidic approaches have increased in use in recent years as more laboratory-on-a-chip applications have been demonstrated. To date, the microfluidic systems have successfully proven to be compatible with traditional cell seeding and protocols, and have often been used to create a stable homogeneous microenvironment for cell culture. Potential benefits of these miniature devices include a reduction in sample/ reagent usage; portability for in situ and real-time analysis; and high sensitivity and throughput.1 More recently, the polymerbased microchips have received increasing attention, primarily because of the convenient fabrication and low cost in polymer replication processes (casting, embossing, or injection molding).2,3 Poly(dimethylsiloxane) (PDMS) is a good silicon elastomer candidate for biomedical device fabrication and cell-based system applications.4 The natural attributes of PDMS can be summarized as follows: (i) biocompatibility and nontoxicity,5–8 (ii) high oxidative and thermal stability,9,10 (iii) high gas (O2 and CO2) * To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Chemistry and Biochemistry. ‡ Department of Chemical Engineering. (1) Bordenave, L.; Bareille, R.; Lefebvre, F.; Caix, J.; Baquey, C. J. Biomater. Sci., Polym. Ed. 2003, 3, 409–416. (2) Harrison, D. J.; Manz, A.; Fan, Z.; Luedi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926–1932. (3) Harrison, D. J.; Fluri, K.; Seiler, K.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895–897. (4) Hong, J. W.; Fujii, T.; Seki, M.; Yamamoto, T.; Endo, I. Electrophoresis 2001, 22, 328–333. (5) Interrante, L. V.; Shen, Q.; Li, J. Macromolecules 2001, 34, 1545–1547. (6) Lee, J.-N.; Jiang, X.-Y.; Ryan, D.; Whitesides, G. M. Langmuir 2004, 20, 11684–11691. (7) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 224–248. (8) Marois, Y.; Sigot-Luizard, M.-F.; Guidoin, R. ASAIO J. 1999, 45, 271– 280. (9) Martin, R. S.; Gawron, A. J.; Lunte, S. M.; Henry, C. S. Anal. Chem. 2000, 72, 3196–3202. (10) Melin, J.; Johansson, H.; So¨derberg, O.; Nikolajeff, F.; Landegren, U.; Nilsson, M.; Jarvius, J. Anal. Chem. 2005, 77, 7122–7130.
and low water permeability,11–13 and (iv) optical transparency. These properties contribute to the suitability of PDMS for microfluidic cell culture systems.14 Since microscope glass slides have better optical qualities and more mechanical rigidity, they have been integrated as a substrate in PDMS/glass hybrid structures in which the cells are adhered or maintained for further analysis. In this paper, an innovative microfluidic cell culture system with minimum shear stress is described. By removing the cell culture well from the main fluid channel and relying on convective and diffusive mass transport for medium supply and waste removal, the shear stress in the culture chambers was reduced significantly. The rapid prototyping and robust sealing of a PDMS/ glass hybrid chip subsequent culture of endothelial and suspension cells have been demonstrated in a low-shear environment. The chip performance and experimental results have shown excellent reliability and reproducibility. One of the unique advantages to this low-shear approach is the regulation of the fluid environment for culturing cells. Quantitative fluorescence measurements were used to calculate the effective shear stress encountered by the cells. Using a photobleaching method, the flow rate of the side channels was characterized as a function of the main channel flow rate. Since cells undergo morphological and biological modifications due to exposure to large shear stresses, which usually exist in the microchannel,15 minimization of shear stress to a favorable level is a key issue for expanding analysis of individual cell activities, cell-matrix binding, and intracellular interactions. For microdevices, the flow should not be too weak to preclude a sufficient reagent delivery, since medium flow is essential for transport of nutrients and waste. However, the liquid flow exerts shear force on a portion of the cell surface rather than uniformly (11) Nathan, D. C.; Jeffery, R. C.; Frazier, A. B.; David, M. C.; Andre´s, J. G. Langmuir 2002, 18, 5579–5584. (12) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461–3473. (13) Park, J. H.; Park, K. D.; Bae, Y. H. Biomaterials 1999, 20, 943–953. (14) Gomez-Sjoberg, R.; Leyrat, A. A.; Pirone, D. M.; Chen, C. S.; Quake, S. R. Anal. Chem. 2007, 79, 8557–8563. (15) Song, J. W.; Gu, W.; Futai, N.; Warner, K. A.; Nor, J. E.; Takayama, S. Anal. Chem. 2005, 77, 3993–3999.
10.1021/la8003917 CCC: $40.75 2008 American Chemical Society Published on Web 05/10/2008
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on the entire cell, and thus, strong fluid flow may damage the cell. Furthermore, the nonflat cell geometry significantly distorts the velocity field and correspondingly the hydrodynamic shear stress distribution, which gives rise to the uneven drag force on the cell surface (and consequent detachment of adhesion cell from the surface).15 Shear stress in culture devices has been studied numerically.16 The flow rate and aspect ratio were both shown to play a significant role in the shear stress experienced by the cell. For aspect ratios that approach those used in this work, the shear stress is expected to vary over much of the channel surface. There is an even greater need, therefore, to minimize shear stress so that the culture conditions are kept static across the channel surface. Other methods used to diminish shear stress have been reviewed previously17 and include lowering fluid flow rates18 or using micropillars to shield cultures.19 High aspect ratio culture wells were also used to shield cells from direct fluid flow.20 In this last case, shear stress was reduced to 0.001-4 dyn/cm2 (0.001-4 g cm-1 s-2). Since most microfluidic systems for biological analysis reported in the literature are based on the culture of adhesive cells, it is of great interest to develop a microfluidic layout that is capable of both adhesion and suspension cell culture. An additional novelty of the design described in this paper is that the fabrication process is simple relative to other low-shear approaches. In addition, the mass transport in the cell culture can easily be regulated by control of the main channel flow rate. Methods described here overcome the technical barriers to regulate the shear force in microfluidic devices, and address the limitations in related hydrodynamic measurements. This microfluidic device enables the exploration of single cell adhesion, cell mechanics, tissue engineering, and other cell biological assays.
2. Experimental Section 2.1. Reagents and Materials. PDMS prepolymer and crosslinking agent were purchased from GE Silicones (GE RTV 615, Waterford, NY) or Ellesworth Adhesives (Dow Sylgard 184). The microscope glass slides (76.2 × 25.4 × 1 mm3) were purchased from VWR International. Fetal bovine serum and medium (RPMI 1640 with 2.05 mM L-glutamine) were purchased from Hyclone (Logan, UT). Trypsin EDTA and phosphate buffered saline (PBS, pH 7.4) were obtained from Mediatech, Inc. (Herndon, VA) and Gibco Invitrogen (Grand Island, NY), respectively. Propidium iodide (PI) DNA intercalating dye was used as received from Molecular Probes (Eugene, Oregon). Mouse endothelial (RCL-2583) and human T lymphocyte (HuT 78) and B lymphocyte (RPMI 8226) cell lines were obtained from the American Type Culture Collection. 2.2. Microfluidic Flow Cell Fabrication. There were two procedures for making the flow cells. For culture tests, glass slides were cleaned by immersion in nitric acid (30%) for 5 min and then rinsed by deionized (DI) water, dried under an air stream, and stored for further use. Uncured PDMS was made by mixing a 5:1 or 10:1 ratio (w/w) of prepolymer and cross-linking agent. The mixture was then stirred thoroughly and degassed under vacuum for 30 min. The viscous mixtures of elastomer were carefully decanted onto precleaned microscope slides and placed in an oven at 75 °C for 20 min. The microfluidic channel and culture well (∼0.25 cm3) molds were manually cut with scalpels and punches and carefully removed from the 5:1 PDMS layer. A layer of 10:1 PDMS served as the top (16) Zeng, Y.; Lee, T.-S.; Yu, P.; Roy, P.; Low, H.-T. J. Biomech. Eng. 2006, 128, 185–193. (17) Kim, L.; Toh, Y.-C.; Voldman, J.; Yu, H. Lab Chip 2007, 7, 681–694. (18) Kim, L.; Vahey, M. D.; Lee, H. Y.; Voldman, J. Lab Chip 2006, 6, 394–406. (19) Khadamhosseini, A.; Yeh, J.; Eng, G.; Karp, J.; Kaji, H.; Borenstein, J.; Farokhzad, O. C.; Langer, R. Lab Chip 2005, 5, 1380–1386. (20) Lee, P. J.; Hung, P. J.; Rao, V. M.; Lee, L. P. Biotechnol. Bioeng. 2006, 94, 5–14.
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Figure 1. (a) Schematic of the flow channel for cell culture experiments. (top) Dimensional scale of the fluid channels and culture wells. The height of the fluid structures is 1.2 mm, and the dimensions (in mm) are listed as follows: a ) 4, b ) 2.3, c ) 1.2 mm, d ) 0.76 mm, e ) 75, and f ) 18. (bottom) Close up schematic of the culture well, showing convective/diffusive mass transport into and out of the cell at a certain shear stress. The circular culture well experiences a lower shear stress (τcell) than the main channel. (b) Photograph of the cell culture chip (fluorescence image using fluorescein for visualization). Scale bar ) 25.4 mm.
of the hybrid glass/PDMS structure. The microfluidic chip therefore consisted of a top layer of 10:1 PDMS, a center layer (which contained the fluid channels) made of 5:1 PDMS, and a bottom layer of glass. The manually cut channel dimensions are given in Figure 1a. The reproducibility of the cut channels was 15% (relative standard deviation). The surface roughness was measured to be
