A self-contained microfluidic cell culture system
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- Zhang, B., Kim, M., Thorsen, T. et al. Biomed Microdevices (2009) 11: 1233. doi:10.1007/s10544-009-9342-4
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Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.
In vitro mammalian cell culture is an essential and powerful tool in biological science. It has made substantial contribution to the understanding of many phenomena, such as intracellular enzyme activities, intracellular flux, and cell-cell interactions, since it originated over one century ago. In recent years, cell culture in high-density array formats, including microfluidic arrays, has attracted tremendous interest due to the potential for rapid large scale cell-based assays. Many microfluidic devices have been developed for cell culture (Balagaddé et al. 2005; Hufnagel et al. 2009; Hung and Lee 2007; Petronis et al. 2006; Stangegaard et al. 2006) and to study intracellular enzyme activities (Carlo et al. 2006), cellular responses to chemical gradients (Jeon et al. 2002; Hung et al. 2005), cellular differentiation (Tourovskaia et al. 2005), dynamic gene expression (King et al. 2007) and cell cytotoxicity screening (Wang et al. 2007). Some microfluidic devices were designed for cell culture purpose in a high-throughput manner (Hung et al. 2005; Gomez-Sjoberg et al. 2007), however, few devices can establish a uniform distribution of cells in whole microfluidic cell array.
As the research emphasis shifts from bulk cell culture, where the behavior of tens of thousands of cells are assayed in individual microwells, to research at the single cell level, cell culture viability and standarized, uniform growth conditions become increasingly important. In bulk culture formats, variations in cell seeding density, nutrient delivery and waste removal are sources of stress on cell cultures that introduce intracellular variations in subsequent assay procedures. Moreover, traditional culture methodology requires technical expertise and specific facilities to handle cells harvesting, media exchanging and cell sub-culturing procedures. In this paper, we describe the development of a continuous microfluidic cell culture platform, in which cells can be repeatedly grown to confluence, trypsinized and recovered. Integrated arrays of U-shaped sieves within round microchambers promote uniform spatial seeding of small clusters of cells (~10/sieve; ~100/chamber), while the ability to tightly regulate the media delivery and removal from the chambers promotes cell viability.
2 Materials and methods
2.1 Microfluidic device fabrication
The microfluidic device used for cell culture was fabricated from poly(dimethylsiloxane) PDMS (Sylgard 184, Dow Corning) using replicate molding and soft lithography (Zhao et al. 1997). SU-8 50 (Microchem, USA) was patterned on a silicon wafer using high-resolution design transparencies to create the distinct positive-relief casting mold for the elastomer. The mold was subsequently exposed to vapor phase chloro-trimethylsilane (Aldrich) to facilitate the release of the elastomer during molding. Preparing the elastomer, Sylgard 184 liquid silicone elastomer (mixed in a ratio of 10 part A : 1 part B) was poured on the mold (~5 mm thick), and baked at 80ºC for 2 h. After baking, the cured PDMS layer was peeled from the mold, fluid inlet and outlet ports were punched using a 23 gauge luer stub (BD Biosciences). To remove residue generated during the punching process, the microchannels and connection ports were cleaned with isopropanol, dried with nitrogen, and the exposed microchannel surface on the molded device was irreversibly bonded to a glass slide using oxygen plasma (150 mTorr, 50 W, 20 s).
2.2 Cell culture
BALB/3T3 murine embryonic fibroblast cells were acquired from Advanced Type Culture Collection (ATCC), and cell culture media and supplements were purchased from Gibco Invitrogen Corp. All cell culture work was carried out in sterile tissue culture hoods and cell culture was carried out in a 5% CO2 humidified incubator at 37°C. BALB/3T3 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% calf serum. Master stocks of the BALB/3T3 cell lines, grown in T25 tissue culture flasks (Corning), were maintained by trpsinizing them at the point of confluence (0.25% in EDTA, Sigma) and passaging them at 1:5 subculture ratio. Cells grown in the microfluidic microchambers were trypsinized at the point of confluence and passaged at a ~1:3 subculture ratio, determined by fraction of cells retained in the sieves upon subsequent flushing.
2.3 Microfluidic simulations
To monitor cell trapping tendency in each micro-structured sieve in the chamber, one-way coupled Lagrangian particle simulations were carried out using recently developed program, which we refer to as ‘Lab-Chip-Designer’ (LCD). The LCD program mainly consists of three parts; importing of pre-computed flow field data, calculating of particle motion equations, and post-processing of cell trajectories (Wang et al. 2007).
Before importing flow field data into the LCD program, computational fluid dynamics (CFD) simulations were carried out using finite volume method-based commercial CFD tool (STAR-CD version 3.15a, CD-adpaco). A SIMPLE (Semi-Implicit Method for Pressure Linked Equation) algorithm with tolerance of 1.0 × 10−5 was applied to solve the momentum and continuity equations. To emulate the solutions commonly used in microfluidic devices (i.e. buffers), an aqueous solution with a density 997.5 kg/m3 was used as the model working fluid. Additionally, a flat velocity profile was applied at the initial microchamber inlet with a uniform flow rate of 1 μL/min.
Pre-computed flow field data was used to evaluate forces acting on the spherical cell, including Stokes drag and pressure gradient forces, using a spatial interpolation method (Kim et al. 2008). An equation of motion for each individual cell was superimposed on these flow fields, taking into consideration the gravitational force, elastic spring force, and a diffusive force due to Brownian motion. The migration direction and distance for each cell was evaluated using Gaussian distribution under adaptively controlled time-step at every iteration (Zhao et al. 1997). Each cell was modeled as a 10 µm diamter sphere in aqueous solution seeded at the bottom of the channel randomly distributed across the inlet. To visualize dynamic cell distribution vs. time, data was exported to generate movie frames in Tecplot, a commercial CFD post-processing software package.
3 Results and discussion
Conventional cell culture is a labor-intensive and time-consuming work, only can be performed by highly trained cell culture people with specific facility. The present microfluidic cell culture array platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, micropumps. The self-contained microfluidic cell culture array platform will benefit many areas of cell-based research.
This work was supported by an 863 project from Chinese Ministry of Science and Technology and a Korean Research Foundation Grant (KRF-2006-D00019).