Biomed Microdevices (2009) 11:1081–1089 DOI 10.1007/s10544-009-9325-5
An open-access microfluidic model for lung-specific functional studies at an air-liquid interface Divya D. Nalayanda & Christopher Puleo & William B. Fulton & Leilani M. Sharpe & Tza-Huei Wang & Fizan Abdullah
Published online: 30 May 2009 # Springer Science + Business Media, LLC 2009
Abstract In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays. Keywords Microfluidic cell culture . Alveolar cells . Air-liquid interface
D. D. Nalayanda : W. B. Fulton : L. M. Sharpe : F. Abdullah (*) Division of Pediatric Surgery, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Harvey 319, Baltimore, MD 21287, USA e-mail:
[email protected] D. D. Nalayanda : C. Puleo : T.-H. Wang Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA T.-H. Wang Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA
1 Introduction Pulmonary disease remains a significant cause of mortality and morbidity in the world, with COPD (chronic obstructive pulmonary disease) itself claiming lives of over 120,000 US lives in 2005 alone (CDC 2007a). According to the National Institute of Health, COPD cost the nation approximately $42.6 billion in 2007 including direct health care expenditures and indirect costs from morbidity and mortality (NIH 2008). Primary risk factors include exposure to cigarette smoke, chemical vapors and particulate matter (Celli and MacNee 2004; USEPA 2007b) leading to obstructive or restrictive respiration. The inability to reverse these conditions through current medical treatment emphasizes the urgent need for clinically relevant in vitro models which facilitate drug testing and cellular studies, a major barrier to pulmonary research (Birkness et al. 1999; Carterson et al. 2005; Chen et al. 2005). Existing models to study functional and metabolic properties of the lung are limited to animal models (Martorana et al. 2008) and transwell-based immersion cultures (Gueven et al. 1996; Birkness et al. 1999). In vitro immersion flask cultures are not physiologically relevant as alveolar cells in vivo exist in an air-exposed environment. On the other end of the spectrum, animal experiments involve simulating lung injury via mechanical overinflation or exposure to toxic chemicals. Such animal models have limited control of individual experimental variables as well as are cumbersome to harvest specific cells for endpoint analysis. Thus, the need for models where precise experimental control can be achieved at the cellular level is significant. Microfluidics is one such method that provides the means to develop a precision hybrid system to study lung cells in vitro. The use of microfluidic systems present the potential benefit of
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providing a culture system that can be built to a physiologically-relevant scale with a direct means to highthroughput investigations. In this study, we present a dynamic model of the alveolar epithelium that can be used to perform mechanistic studies of drug transport in conjunction with functional studies of the alveolar epithelium. Furthermore, the presented microfluidic cell culture system allows for investigations testing the role of the provision of constant nutrients and air exposure in the developing lung.
2 Materials and methods 2.1 Experimental design Human alveolar epithelial cells, A549, were used for this study. Monolayers of A549 grown in microdevices in two culture conditions were compared. Cells were cultured exposed to ambient air and submerged in culture medium. Control groups were monolayers cultured in traditional transwells with similar cell culture surface. Experimental time points were 0 days, 7-days, 14-days and 21-days in F12 K medium supplemented with 2 mM L-Glutamine and 10% FCS. Data was also gathered at other time points but is not presented fully as it is noncontributory. Electrochemical, physical and permeability analyses were performed to evaluate the efficacy of the presented micro-culture system to conduct studies of air-exposed alveolar cultures. The microfluidic cell culture wells aid in providing appropriate microenvironments for optimal cell growth by allowing for engineering designs that incorporate a dynamic pressure drop across the cell monolayer exposed to air on the apical surface. This is an important function as it has been shown that specific environmental factors such as exposure of the alveolar cell at its apical air interface has shown to affect the alveolar type II cell differentiation (Dobbs et al. 1997). Our previous work on culturing alveolar cells under constant media perfusion (Nalayanda et al. 2007) allowed adaptation of optimized experimental parameters to the current design with constant media replenishment on the basolateral surface (Fig. 1(b)) to present a model to study the direct effects of air on lung cells in an in vitro environment. 2.2 Microfluidic device fabrication and assembly The template of the desired pattern was developed using photolithography as detailed (Fig. 1(a)). Microfluidic network layouts were created using L-Edit v10.11 software and printed on transparencies using a high DPI printer (Intandem, Inc., Baltimore, MD). 70µm thick molds were then created on cleaned silicon wafers by spin coating SU-
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8 2100 negative photoresist (60 s at 3000 rpm, Microchem Inc.). The coated wafers were then soft-baked for 5 min at 65°C followed by 20 min at 95°C, before exposing the photoresist through the photomasks (Maskaligner EVG 620, EV Group Inc. NY). The substrates were baked post exposure for 1 min at 65°C followed by 10 min at 95°C, and developed (Microchem’s SU-8 developer). Bottom layer of the Microdevice: The template (pattern in SU-8 resist on Si wafer) surface was exposed to trimethylchorosilane vapor for 15 min, to yield a finecoat on the surface. Liquid PDMS (1:10 ratio of curing agent to prepolymer mix) was poured on the template surface, degassed and cured for 30 min at 80°C. The PDMS slab with the negative relief structure of the pattern is lifted-off the wafer and small access holes were created. This layer was then O2 plasma-sealed to a glass slide (PE II-A Plasma Asher, Technics West Inc.) by exposure to O2 plasma for 2 min at 25 W, and heated in an oven at 80°C for an hour. Top layer: Flat PDMS slabs were used for the top layer of the microfluidic culture chip. The edges of the two layers were aligned and overlapping holes were punched through both the layers. These served as the cell culture wells. Membrane integration: Porous PET membranes (GE Osmonics Labstore) with 0.4µm size pores were cut to dimensions roughly larger than the culture wells and sandwiched between the two layers of PDMS constructed as detailed in Fig. 1. Liquid PDMS (1:8 ratio of curing agent to prepolymer mix) was used to seal the layers together and slow-cured at 40°C for 4 h.
2.3 Micro cell culture experiments A549 cells, human alveolar basal epithelial cells, (passage 4–10, ATCC# CCL-185) were maintained in F-12 K Nutrient Mixture (Cellgro) with 2 mM L-Glutamine, supplemented with 1% penicillin/streptomycin (P4458, Sigma-Aldrich) and 10% FCS (A-111-L, Hyclone Labs Inc., UT). Cells were grown to 70 to 85% confluency in tissue culture flasks, before being harvested using 0.25% Trypsin EDTA (Gibco 25300-054, Invitrogen Corp.) for experiments. 2.4 Cell seeding PDMS microdevices and all microfluidic components (syringes, needles, and tubes) were UV-sterilized prior to use. In addition, all fluidic connections were sterilized with 70% ethanol and rinsed with sterile PBS.
Biomed Microdevices (2009) 11:1081–1089 Fig. 1 (a) Schematic representation of fabrication steps involved in assembling the multi-layer microfluidic cell-culture device. (i) Desired pattern as raised SU-8 photoresist structures on silicon wafer. (ii) PDMS polymer mix (curing agent and pre-polymer mix) poured on the SU-8 template and cured for 30 min at 80°C. (iii) Peeled PDMS mold with the negative relief structure. (iv) Access ports punched into the PDMS mold. (v) Culture wells created in the PDMS mold. (vi) The bottom PDMS layer is adhered to a clean glass slide via O2 plasma treatment and PET membrane integrated in between the two PDMS layers. (vii) Final cell culture device with tubes providing access to the microfluidic network of channels in the lower PDMS layer (b) Schematic depicting an isometric view of the microfluidic chip detailing the various layers. Right-top insert: Cross-sectional view across the indicated dotted line. Left-bottom insert: Digital image of a prototype
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The microfluidic channels were filled with cell culture media and allowed to equilibrate for an hour at 37°C and any trapped air bubbles were forced out by applying back pressure. Cells trypsinized from conventional cultures were seeded on 0.4µm pore size membrane embedded in the microfluidic chip at a seeding density of 0.3×104 cells/cm2 of the membrane surface area. The culture chips were maintained in a controlled 37°C and 5% CO2 environment until cells reached near confluence (3 to 4 days). In addition to the constant media replenishment, driven by gravity feeding on the basolateral surface, media on the apical side was changed daily on these days of monolayer formation. Also, during this period, the microfluidic culture chips were maintained in a humidified environment to stem evaporation induced during static or low flow culture periods. Upon having confluent cultures, a syringe pump was used to maintain fresh media at a constant flow rate of 0.35 µL/min. Media above the membrane was switched to air in half the wells while the other half served as a control for cells grown submerged in media. Multiple chips were grown concurrently, and cells were cultured for up to 3 weeks. Samples were harvested regularly for the tests to characterize the cell monolayer as it formed. Cells grown in wells with an air-liquid interface were compared to those submerged in media.
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alveolar epithelial cell monolayers (Adson et al. 1994). TEER was determined by applying an AC square wave current of +/− 20µA amplitude at 12.5 Hz, and the corresponding voltage deflection was measured using a silver/silver-chloride electrode (EVOM, World Precision Instruments, Houston) at 37°C in cell culture medium (DMEM w/10% FCS). TEER measurements were obtained at regular 2-day intervals, with each data point being a mean of three resistance measurements taken per culture well (Fig. 2). The contribution of the filter and culture medium is accounted for by subtracting the electrical resistance of the porous membranes without cells from the sample values and normalized with respect to the culture surface area. In cultures maintained at the air-liquid interface, the upper culture well with the alveolar cells was incubated with freshly added media for 10 min at 37°C, before measuring the electrical resistance. After the measurement, cell culture media on the apical surface was carefully aspirated to continue the culture at an air-interface. 2.7 Paracellular permeability of sodium fluorescein The paracellular permeability for Sodium Fluorescein was determined by adding 10µg/mL of Na-fluorescein (mol.wt.376.3, F6377, Sigma Aldrich) in Krebs-Ringer (KR) buffer to the apical side of the monolayer. The
2.5 Transwell cell seeding and maintenance Cell culture media was added to the top and bottom wells of Corning PET transwells (Sigma Aldrich, cat.CLS3460) with 0.4µm pore size, and allowed to equilibrate for an hour at 37°C in the cell culture incubator. Trypsinized A549 cells were then seeded onto the membrane surface at 0.3x104 cells/cm2. Cells were supplemented with F-12 K medium with 10% FCS and allowed to proliferate to confluence over a period of 3 to 4 days. For experiments with cells exposed to air, cell culture media in the upper well of the transwell system was removed while the media in the lower basal compartment was left for cell sustenance. Thereafter, spent medium was replaced with a fresh supply once every 3 days for culture periods up to 3 weeks. These transwell cultures were used to compare results from PET membrane-incorporated microdevices. Both the membranes are of the same material and pore size, and treated similarly up to cell seeding. 2.6 TEER measurements The degree of sealing of tight junctions was assessed from TEER (Trans Epithelial Electrical Resistance) measurements to provide an indication of the integrity of the
Fig. 2 Characterization of A549 cell viability and monolayer integrity. Change in transepithelial electrical resistance of A549 cell monolayer with respect to time. On-chip (data in squares) and transwell culture data (data in circles) correspond to A549 cells seeded at a density of 0.3×104 cells cm-2 in the microfluidic chip and Corning PET transwells respectively. Two culture conditions were studied: (i) exposed to atmospheric air (open legends ○ and □) and (ii) submerged in culture media (solid legends ● and ■). Each data point represents the mean ± SEM (n = 4)
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paracellular flux of the tracer molecule was determined by serially sampling fluid from the basal compartment every 20 min for a period of 3 h, and analyzing the tracer concentration, diluted 1:10 in KR buffer, using a UV Spectrophotometer (Beckman DU-530 Life Science UV/Vis Spec). Simultaneous TEER measurements were taken to monitor the integrity of the alveolar cell monolayer. Thus, the cumulative accumulation of Nafluorescein in the effluent from the lower compartment is plotted as a function of time. The apparent permeability coefficient (Papp) of Nafluorescein flux across the alveolar monolayer is determined using the equation (Seong-Hee Park et al. 2006): V dC Papp ¼ =dt AC 0
where A is the membrane surface area (cm2); C0 is the initial concentration of sodium fluorescein in the apical compartment (µM); V is the volume of the solution in the receiving (basal) compartment (cm3), and dC/dt is the rate of Na-fluorescein transfer across the cell monolayer (µM/s). It is the slope from the linear range of the plot for accumulating tracer concentration in the receiving chamber over time. Hydrostatic pressure in addition to passive diffusion, across the cell monolayer, contributes to the transmembrane flux of the tracer molecules. The resistance offered to the cross-flow by the cell monolayer is indicative of its integrity and tight junctions. 2.8 Surfactant droplet test The surface tension of the air-exposed cell cultures is determined using the surfactant drop test. 20µL droplets of DMP/O, a 4:1 v/v mixture of Dimethylphtalate / normal Octanol, were placed on the monolayer cell surface. The solution is stained with Crystal Violet to facilitate photoimaging of the test droplets. The surface tension of the cell apical surface, hypophase, is estimated from the ratio of the diameter of the droplet deposited on the hypophase (d) to the diameter of the droplet prior to deposition (d0), calculated from the volume deposited: (d/d0). Droplet diameters were measured from images of the droplets taken using a high resolution digital camera (Canon). This method is based on the fact that the diameter of the deposited droplet decreases with reducing surface tension and vice versa. The surface tension was determined from a calibration curve, for thin liquid substrates, which gives the relationship between a range of (d/d0) values and the surface tension of the film (Schurch et al. 1976; Im et al. 1997). A549 monolayer cultures were harvested at regular intervals of 7 days to register the change in surface tension of the cell surface (Fig. 4).
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2.9 Statistical analysis Experimental results are presented as the mean ± SEM for the number of experiments indicated. The statistical significance was determined by Student’s t test (paired).
3 Results The present study presents a pulmonary microculture system that is compared to conventional transwell culture system that used similar culture surfaces. In both systems, the cultured alveolar cells were tested for surfactant production and maintenance of monolayer integrity using TEER measurements as well as metabolite permeability studies as follows. 3.1 Bioelectrical Measurements The ability of A459 cells to proliferate and maintain a functional monolayer on 0.4µm pore PET membranes with constant media nourishment on its basolateral side, with 30 min average media turnover rates, was assessed from transepithelial resistance measurements taken at various time points during the experiment. TEER is widely used as a measure of cell monolayer integrity via tight junction formation (Zhang et al. 1997) and the capacity for active ion reabsorption through the epithelium (Kim et al. 1991). TEER was determined at 37°C in cell culture medium (DMEM with 10% FCS) at regular 2 day intervals (data not shown for all the time periods to avoid crowding of data points), and normalized with respect to the culture area. Resistance offered by a blank control (culture well with media but no cells) was taken into account prior to presenting the final values. The data presented in Fig. 2 indicates an increase in resistance to flow of current due to decreased gaps in the monolayer which can be attributed to the formation of tight junctions by the cells. Some of the variables that can affect the TEER values include cell viability, cell-cell interactions, cell culture area and material used for constructing the culture wells. The latter two variables have been taken into consideration and the presented values have been normalized with respect to the culture area and a ‘blank’ (detailed above) value subtracted for contributions from the set-up and their differences. Since the initial values for the transwell system is slightly different from the microdevice set-up, the values as a percentage increase with the respect to the initial value has been used to assess the improvement in resistance values for a given culture set-up. The air-exposed cultures in the microfluidic device indicated an increase in TEER values from 16.46 ± 1.47 Ohm-cm2 (mean
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value ± SEM) to 194.9 ± 13.5 Ohm-cm2 over a span of three and a half weeks, while the air-exposed cultures in the transwell set-up registered an increase from 26.68 ± 3.80 Ohm-cm2 to 177.2 ± 10.82 Ohm-cm2. This represents almost an 11-fold increase in the TEER values in the presence of air in a micro-culture to a 6-fold increase in the transwell culture platform. In contrast, the A549 cells cultured under media exhibited a weaker improvement in the resistance measurements. The transwell culture system recorded a smaller increase in average TEER values from 30.52 ± 3.8 Ohmcm2 to 147.0 ± 8.51 Ohm-cm2 than the microfluidic culture with values improving from 20.14 ± 5.11 Ohm-cm2 to 152.04 ± 13.6 Ohm-cm2 over three and a half weeks. The microfluidic cultures under both conditions exhibited a greater improvement in TEER values than the conventional culture system. In order to generate a functional barrier in vitro, a key prerequisite is the establishment of tight junctions between adjacent cells. Tight junctions are formed towards the apical side of lateral membranes in epithelial cells and are thought to function as a barrier between apical and basolateral plasma membrane domains thus allowing the epithelial cells to maintain polarity. These resistance measurements are also a mark of the percent viable cells in the monolayer, since dead cells would breach the monolayer integrity allowing a path of low resistance for the applied current. The TEER measurement technique was also used in combination with the other assays to validate the results attained to healthy monolayers.
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was used to determine the apparent permeability constants. In detail, the slope of the curve for accrued concentration of sodium fluorescein collected on the basal side versus time, yielded the value for the term ‘dC/dt’ in the equation for determining the apparent permeability constant (Section 2.8). This specific component of the uptake followed a linear relation in the initial half of the experiment and plateaued towards the later half. The uptake of the anionic fluorescent moiety by the cell monolayers was characterized for A549 cell monolayers at 0, 7 and 21 days of confluent culture (Fig. 3) under two culture conditions of air-exposed cells and those cultured submerged in media. Further, the microdevice cultures were compared to the transwell cultures under the two (above mentioned) culture interfaces. The permeability constants for the air-exposed cultures in the microfluidic device presented the largest reduction indicating better cell viability and monolayer integrity. In comparison, media submerged cultures expressed a meager improvement over the initial 7 days (between Day-0 and day-7). In line with the microdevice results, the air-exposed transwell cultures too exhibited a decent improvement in monolayer integrity i.e., drop in permeability rates. However, in comparison to the microdevice air-cultures, the transwell cultures displayed greater permeability values. These results reinforce the enhanced formation and maintenance of alveolar cell tight junctions in an air-exposed environment with comparable or better results for the microfluidic device than the transwell system.
3.2 Development of an air-exposed functional barrier Alveolar cells in over 3 weeks of confluent cultures exhibited an increased development of transepithelial resistance in the range of 190–200 ohm-cm2. In order to evaluate the reproducibility of the microculture system, similar results were summarized from four different independent assays with culture on two different culture surface areas of 7.065 mm2 and 28.3 mm2. The mean TEER values are presented in Fig. 2. 3.3 Permeability studies Sodium fluorescein transport studies were conducted to cement the results from TEER measurements of improved monolayer formation with exposure to air in a microchip. Briefly, sodium fluorescein was used as the tracer molecule due to its ease in quantification using a standard UV spectrophotometer. Peak absorbance was determined along with the standard curve at pH 7.4 in KR buffer. Sodium fluorescein uptake was linear with time up to an hour of incubation (data not shown) and this set of data
Fig. 3 Characterization of A549 cell monolayer integrity. Apparent permeability constant for paracellular diffusion of sodium-fluorescein at various culture periods. Microchip and transwell data correspond to A549 cells seeded at density of 0.3×104 cells.cm-2 in the microfluidic chip and Corning PET transwells respectively. Two culture conditions were studied: cells cultured under continued exposure to (i) atmospheric air and (ii) culture media, on the apical side. Data points represent the mean ± SEM (n = 2); * and ** indicate p < 0.05 (student’s t test, paired)
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3.4 Characterization of cell phenotype and function upon exposure to air Once the viability and integrity of the alveolar culture was established, the monolayer was tested for phenotype maintenance by assessing their surfactant production (Fig. 4). Some of the important functions of alveolar Type II cells in vivo include synthesis of surfactant proteins, transepithelial movement of water and ions and regeneration of alveolar epithelium following lung injury (Adamson and Bowden 1974; Adamson and Bowden 1975; Verkman et al. 2000; Mason et al. 2002). Alveolar type II cells produce surfactant proteins to counteract the tendency of lung alveoli to collapse as a result of high tension developed at the air-liquid interface during respiration. These surfactant proteins form a protective film over the alveolar epithelium lowering the surface tension in lungs. The experimental results are not lower than clinical values for surface tension in the lung, which could be due to the absence of other contributing factors such as alveolar type I cells, extra cellular matrix which is a complex milieu of collagen, laminin and proteoglycans, and most importantly, the mechanical stretch stimuli induced during respiration. However, there is a clear indication of a drastic decrease (~ 40%) in the surface tension of the hypophase in case of the air-exposed cultures in comparison to the cells cultured under media which registered a mere 16% reduction over 3 weeks of confluent culture. The air-exposed transwell cultures displayed a similar trend (data not shown) of drop in surface tension values than the media-submerged transwell systems as in the case of the microfluidic set-up (Fig. 4). The relevant on-chip data comparing the airexposed and media-submerged values in the microdevices are presented.
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The microfluidic set up presented in this study has several advantages. Firstly, it provides a number of in vivo like growth parameters such as closely-packed cells with cell volume to extracellular fluid ratio of greater than one (on the basal side), constant nutrient replenishment and waste removal. With media turn-over rates in the range of a few minutes, the constant nutrient supply and waste removal in our system helps avoid the diffusional constraints inherent with conventional cell culture systems. Secondly, cells cultured over the membrane are not disturbed by any fluid flow, since the flow of media is restricted to the basolateral surface of the cell layer. Therefore, the thin sheet of hypophase over the cells is not disturbed allowing any secreted molecules from the cell to remain in the extracellular fluid (Walker et al. 2004). The effect of these features unique to our microfluidic cell culture platform are evident by the consistently higher presence of surfactant and lowered surface tension in confluent cell cultures grown in a microfluidic environment versus traditional transwell cultures. The importance of air exposure in maintaining physiologic cell behavior was demonstrated from the surfactant measurements indicating a large reduction (40%) in surface tension credited to surfactant production by the alveolar cells in comparison to a bare 16% change in cultures under media. Furthermore, the microfluidic design lends itself to subsequent studies as the open-access platform allows for easy cell seeding and manipulation as well as various
4 Discussion We present herein a novel continuously-perfused microfluidic culture system of human alveolar epithelial cells in which confluent monolayers were successfully grown on PET membranes for more than three weeks exposed to air. Functionality of the membrane was demonstrated by tight junction formation as well as surfactant production. A major advantage of the system is that it can serve as a highthroughput platform for numerous fundamental scientific investigations. Alveolar cells have been previously cultured in a microfluidic channel (Huh et al. 2007) describing an injury model via introduction of an air bubble through the alveolar cultured microchannel. Apart from being an openaccess system, this study differs in that it focuses on long term and stable culture at the air-liquid interface instead of a brief exposure at the interface.
Fig. 4 Functional characterization of A549 monolayer in the microfluidic unit. Surfactant production by alveolar epithelial cells characterized by determining the change in surface tension of the hypophase with time for various culture conditions — submerged in culture media (■ – solid boxes) and air-exposed (□ – open boxes
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functional tests such as immunostaining and surfactant droplet tests (performed in this study) which would otherwise not be possible with a closed system. Various microfluidic cell culture models have been reported with two dimensional cell cultures (Chin et al. 2004; Rhee et al. 2005; Kim et al. 2006) or three dimensional scaffold cultures with cells suspended in a supporting matrix (Tan and Desai 2005; Koh and Pishko 2006). These models provide a number of advantages of a microfluidic system widely discussed in various publications (Beebe et al. 2002; Walker et al. 2004; De et al. 2006). However, their lack of popularity among researchers could be due the complexity of a closed-system with the requirement of inherent versatility on behalf of the researcher to manipulate the micro-volumes of fluid and cell suspensions through a complex network of microchannels thus limiting its application. The open-access model discussed in this paper overcomes this predicament and allows for cell culture techniques with ease on par with the conventional culture systems, granting a hybrid system with all the advantages of a microculture system integrated with the ease and familiarity of conventional cell culture models. The micro cell culture set-up is experimentally simple and highly adaptable for performing high throughput assays and requires no special equipment. Although the current design depicts a simple parallel two-well system, this experimental set-up is capable of incorporating a high throughput design with 6x6 wells (6 wells in series and 6 wells in parallel) for parallel processing, utilizing fabrication steps similar to those depicted in Fig. 1 with minor changes to the template design. Furthermore, while the application of the present design is limited in mechanical stimuli to the basolateral surface due to its open-fluidic model on the apical surface, other hydro and aero dynamic forces can be applied by slight improvisation to a flexible closed-system. Additionally, the ability of this platform to act as a model of the air-fluid interface of the alveolar membrane presents an opportunity to turn this technology towards pulmonary drug transport studies. The increasing use of pulmonary drug delivery systems as a promising path for administration of protein or peptide drugs (Gonda 2000) underline the need for dynamic in vitro models for performing drug transport studies. However, the major barrier to transportation of macromolecules to the pulmonary circulation is the lung alveolar epithelium (Taylor and Gaar 1970) comprising of alveolar Type II and Type I cells, with the latter comprising 95% of the alveolar surface area (Haies et al. 1981). Thus an in vitro model emulating the alveolar epithelia would be an important research apparatus in providing an estimation of likely pulmonary permeability of a new drug. The microfluidic platform presented in this paper could act as the foundation of one such in vitro model
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that could be used in performing mechanistic studies of drug transport. Our experiments involving paracellular transport of sodium fluorescein support the feasibility of this approach. The decreased paracellular permeability of the fluorescent molecules across the alveolar cultures with increased culture times, indicate formation of improved tight junctions between the cells. Also, the evident difference in permeability values attained for cells cultured under media and exposed to air indicate a cumulative effect of altered composition of the hypophase and enhanced tight junctions induced by air exposure on decreasing the transport of molecules across the alveolar cell barrier. In addition, the availability of a dynamic system with short diffusion times on the basal side similar to the blood compartment in vivo moves it closer to physiological environment. Although in vitro models offer enormous knowledge of essential but complex in vivo phenomena, they can lack the complex milieu existing physiologically. Similarly, while the presented microculture model provides multiple advantages it lacks a co-culture component at this stage. This constitutes the direction of future studies to incorporate the complex interplay of various cell-cell and cell-matrix interactions, along with the potential of exposing cells to stretch stimuli within a microchannel platform.
5 Conclusions This report successfully demonstrates a multi-layer biological system for the culture of alveolar epithelial cells at an air-liquid interface in a microfluidic environment with continuous nutrient perfusion. As an in vitro model for alveolar cell monolayers sustained at an air interface, the presented microfluidic system is an improvement as compared to existing transwell culture systems in that it provides a high-throughput cell culture arrangement requiring minimal maintenance and the ability to expose cells to varying mechanical stimuli. Cells cultured in this new platform demonstrated a higher degree of monolayer integrity and a decrease in surface tension of the hypophase as compared to cells cultured under conventional media exposure. Additionally, the presented microfluidic bioreactor presented may also find interesting applications in studying paracellular flux of metabolites and drug molecules across membranes of interest. This multilayer design is amenable to expansion to include designs for exposing alveolar cells to tensile and stretch stimuli, and opens the avenue for in vitro testing of various cell types experiencing dynamic forces. This work is also encouraging in that, with further developments in methods to mimic the alveolar membrane, microfluidics may play an
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important role in optimizing parameters to study lung development, injury, and regeneration. Acknowledgements This work was supported by the Robert Garrett Fund for the Surgical Treatment of Children. The authors would like to thank Venkata Sama and Yi Zhang for assistance with data analysis and manuscript review.
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