A microfluidic cell culture platform for real-time cellular imaging
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- Hsieh, C., Huang, S., Wu, P. et al. Biomed Microdevices (2009) 11: 903. doi:10.1007/s10544-009-9307-7
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This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 μm, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.
KeywordsMicrofluidicsMicro-bioreactorsCell cultureNanoparticleCellular imagingMEMS
Depth of field
Fetal bovine serum
Oral cancer cell
Multi-modal microscopic imaging has long been a crucial technique for the study of cell biology and molecular biology. Microscopy techniques have been applied for assessing cell population dynamics, single-cell or even sub-cellular structures and functions, and cellular response to defined treatments. Moreover, cell-based assays for evaluating efficacy (Lang et al. 2006; Partlow et al. 2008; Gupta et al. 2007), biological toxicity (Xu et al. 2008; Earl et al. 2008), therapeutic modalities and nanoparticle-based formulations (Tseng et al. 2007; Patra et al. 2007; Keter et al. 2008; Wu et al. 2008a; Wu et al. 2007a) greatly rely on a full understanding of the cellular responses to the treatment and its micro-environments. For example, porous iron-oxide nanorods have been developed as delivery nanocapsules (Wu et al. 2007a). Their drug delivery efficiency, sub-cellular targeting, and therapeutic efficacy have been confirmed from microscopic observation of the morphological alterations and the viability of the treated Hela cells. Besides, dynamic time-lapsed, long-term observation of cell-based assays plays important roles in the understanding of the cell and in molecular biology but are technically challenging, especially when multiplexed, high-throughput observation are desired. In order to get high-throughput, in-depth insights from the interaction between the drug delivery system and the test cells, an automatic cell culture platform capable of microscopic imaging and precise quantification of efficacy under an in vivo-like microenvironment is crucially in demand.
Microsystems based on microfluidic control technology have been demonstrated to be suitable for cellular micro-assays. For example, a microchip device has been demonstrated to improve the cell growth of primary hepatocytes (Powers et al. 2002). Microfluidic-based devices for long-term cell culture and imaging have been extensively studied recently (Kaji et al. 2003; Sin et al. 2004) such as a cell culture chip for on-line monitoring of cultured eukaryote cells (Michael et al. 2006) and the control of axon growth and polarization from a central nervous system using a microfluidic culture platform without modulation of neurotrophins (Taylor et al. 2005). Elastomeric microchannel systems could even support in vitro mouse embryo development (Beebe et al. 2002; Raty et al. 2004). However, most of these cell culture devices require large equipment such as incubators and syringe pumps to maintain cell culture environments. These apparatus are usually costly and bulky, thus hindering their practical applications. We recently reported an automatic cell culture chip integrating a micro temperature control module and medium transport mechanisms in a single chip (Huang et al. 2007). However, the flow rates from the micropumps may influence cell physiology due to the induced high shear stress. Thus a non-continuous medium supply system which controlled the shear stress level in the culture medium was implemented. Moreover, this advanced microfluidic culture system not only kept the culture system sterile, providing an adequate nutrient supply and waste removal during the culture period, but also maintained a stable, defined and flexible culture microenvironment for delivery of externally applied stimuli such as drugs or light (Wu et al. 2006; Sittinger et al. 1997; Wu et al. 2007b). A perfusion-based, micro, cell culture chip using a spider-web micropump has been developed to provide uniform and simultaneous culture medium delivery and replacement in 8 microchannels (Wu et al. 2008b). Although the design of the spider-web shape micropumps are suitable for simultaneous multiple pumping, the flow rate in the microchannels is fixed and thus does not allow tunable shear stress inside each channel. Hence, in order to solve this problem and to provide an adequate flow rate for continuous medium supply, a membrane-based serpentine-shape (S-shape) pneumatic micropump has been proposed (Huang et al. 2008). This is achieved by fine-tuning the fluidic resistance of injected air in the designed pneumatic microchannels. Besides, the S-shape pneumatic micropump can be integrated into a perfusion-based cell culture platform to continuously supply medium (Wu et al. 2008c). In this study, for high-magnification microscopic observation of the cultured cells and their response to drug delivery, we designed an ultra-thin window to be compatible with an objective lens with a depth of field (DOF) of as small as 200 µm, in order to achieve adequate spatial resolution. The new cell culture platform has a modified S-shape micropump with micro check valves to achieve better control of the flow rate. This enables a continuous medium supply at a lower flow rate for general cell culture and at a higher flow rate for efficient cell loading into the micro-culture area; both of which can now achieved in the same device. In addition, cross-contamination is prevented by the micro check valve design. The ring-shape pillars with gradually increasing spacing intervals encircling the cell culture area could effectively reduce the shear stress induced by the medium flow. With this integrated approach, the developed system provides a new platform capable of potential high-throughput, high-magnification observation for real-time, cell-based study and functional assays.
2.1 Chip design
The micro temperature control module is composed of multiple microheaters and a micro temperature sensor (Fig. 1(d-II)) to maintain a constant temperature suitable for the cell culture process. Detailed information about the microheaters and the micro temperature sensor can be found in our previous work (Hsieh et al. 2006).
2.3 Experimental setup
2.4 Sample preparation
In order to evaluate the function of the cell micro-culture platform and the real-time cellular imaging, an oral cancer cell line OC-2 (Huang et al. 2002) is used in this study. The culture medium (HyQ® RPMI-1640; Hyclone, USA) containing 2.05 mM L-glutamine, 10% fetal bovine serum (FBS) (Gibco® 26140-079 US origin, Invitrogen Ltd., Taiwan), 100 units/ml of penicillin and streptomycin (Gibco® 15140-122, Invitrogen Ltd., Taiwan), and 25 mM HEPES (SH30237, HyClone, US) is prepared. The micro-culture platform is sterilized using 70% of ethanol and exposed to ultraviolet light in a laminar flow hood for 30 min. OC-2 cancer cell suspension at 2 × 105 cells/ml density is pumped into the micro-culture area to seed the cells using a high pulsation frequency of 18.20 Hz (flow rate = 1,837.0 µl/hr). After adhesion of the loaded cells (4 h after seeding), the medium is continuously flowed through the cell culture area at a low pulsation frequency of 0.12 Hz to achieve the desired flow rate of 9 μl/hr while the culture environment is kept at 37°C. All reservoirs are covered by sterile circular lids during the culture process to prevent contamination.
In order to observe the influence of “nano drugs” on the cancer cells, we use quantum dots (QDs) conjugated with the anti-cancer drug epirubicin (C27H29NO11, Pfizer, USA) as a model (Olinsji et al. 1997; Wu et al. 2003; Pieper et al. 2000). The quantum dots provide fluorescent labels for imaging the nano drug complex. The epirubicin is conjugated to the QDs (Qdot® 655 ITK™, Invitrogen Ltd., Taiwan) with a carboxyl group (-COOH, 1,500 carboxyl/dot) through an amino group (-NH2, 1 amino/molecule) on the molecule by covalent crosslink. The ratio of the anti-cancer drug to conjugate with the QDs is 1,500(epirubicin):1(QDs). A series of cytotoxicity testes are conducted and verified for the QDs, pure epirubicin and the nano drugs using a dimythylthiazol diphenyl tetrazolium bromide (MTT) assay (Fraga et al. 2008) at different treatment dosages (0M, 10−11M, 10−10M, 10−9M, 10−8M for the QDs and the nano drugs and 0M, 1.5 × 10−8M, 1.5 × 10−7M, 1.5 × 10−6M, 1.5 × 10−5M for the epirubicin). The MTT assay is conducted as described previously. Briefly, OC-2 cells are seeded into a 96-well cell culture plate at a final concentration of 5,000 cells/well. After incubation of the seeded OC-2 cells for 24 h, the medium is replaced by fresh culture medium containing different concentrations of QDs and the nano drugs. All experimental conditions are repeated for five times in this study. After incubation for 48 h, 20 µl of MTT solution is added to each well. After incubation for 4 h at 37°C, 100 µl of SDS solution (10% sodium dodecyl sulfate dissolved in 0.01 N HCl) is added to each well. After overnight (18–20 h) incubation, absorbance at 560 nm is measured in an ELISA plate reader (LP 400 Pasteur Diagnostics, France). For the real-time, high-magnification observation of the cells in response to the nano drugs (conjugated QDs-epirubicin), the cell culture medium containing epirubicin conjugated QDs (concentration = 10−8M) is loaded into the reservoirs (see Fig. 1)(a) and a time-lapsed videomicroscopy of the micro-cultured cells is performed continuously for 24 h.
3 Results and discussion
3.1 Characterization of microfluidic components
3.2 Cell culture and real-time cell division imaging
3.3 High-magnification, real-time imaging of nano-drug-cancer cell interactions and cytotoxicity evaluation
This study reports a new cell micro-culture platform capable of automatic cell plating, medium supply, waste removal and real-time monitoring of cellular responses to the administered anti-cancer drugs or nanoparticles. The system is comprised of a cell culture module and a micro temperature control module. Continuous medium supply and long-term temperature stability of the cell culture area can be maintained for more than a week. Moreover, the cell culture platform featured an ultra-thin culture chamber for high-resolution cellular and sub-cellular level imaging. Together with these integrated functions, the developed micro-culture platform could become a promising tool for further high-throughput drug screening and biomedical research.
The authors would like to thank the National Science Council in Taiwan for financial support of this project.