Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells.

The ability to culture cells in three dimensional extracellular matrix (3D ECM) has proven to be an important tool for laboratory biology. Here, we demonstrate a microfluidic perfusion array on a 96-well plate format capable of long term 3D ECM culture within biomimetic microchambers. The array consists of 32 independent flow units, each with a 4 µl open-top culture chamber, and 350 µl inlet and outlet wells. Perfusion is generated using gravity and surface tension forces, allowing the array to be operated without any external pumps. MCF-10A mammary epithelial cells cultured in Matrigel in the microfluidic array exhibit acinus morphology over 9 days consistent with previous literature. We further demonstrated the application of the microfluidic array for in vitro anti-cancer drug screening.

Dynamic cell culture: a microfluidic function generator for live cell microscopy.

We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc.

A microfluidic system for dynamic yeast cell imaging.

The investigation of cellular processes and gene regulatory networks within living cells requires the development of improved technology for dynamic, single cell imaging. Here, we demonstrate a microfluidic system capable of mechanical trapping of yeast cells with continuous flow and flow switching capability during time-lapse high magnification fluorescence imaging. The novel functionality of the system was validated by observing the response of pheromone-induced expression of GFP in Saccharomyces cerevisiae.

Microfluidic environment for high density hepatocyte culture.

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm(2)) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 microl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.

Microfluidic tissue model for live cell screening.

We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure.

An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture.

Primary hepatocytes represent a physiologically relevant model for drug toxicity screening. Here, we created a biologically inspired artificial liver sinusoid with a microfluidic endothelial-like barrier having mass transport properties similar to the liver acinus. This unit consisted of a cord of hepatocytes (50 x 30 x 500 microm) fed by diffusion of nutrients across the microfluidic endothelial-like barrier from a convective transport vessel (10 nL/min). This configuration sustained rat and human hepatocytes for 7 days without an extracellular matrix (ECM) coating. Experiments with the metabolism mediated liver toxicant diclofenac showed no hepatotoxicity after 4 h and an IC(50) of 334 +/- 41 microM after 24 h.

Microfluidic System for Automated Cell-based Assays.

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 µl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites.

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

Nanoliter scale microbioreactor array for quantitative cell biology.

A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring.

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array.

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.

Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.