The Cell Chamber River Model to Limit Cellular Stress and Response While Delivering and Removing Stimuli

BRENDAN CASEY, LUCY HE, ALVIN KPAEYEH , JENNIFER OLSON
Biomedical Engineering, Duke University May 4, 2005

Abstract Our design group set out to create a
reaction chamber capable of quickly delivering and removing stimuli to cells. It was imperative that the chamber not induce any superfluous cellular responses. After considering multiple design ideas, the fluid flow "river" model was selected. Two prototypes were built and experiments were conducted to determine prototype capabilities. The final prototype satisfied all primary design objectives and showed promise for future usage in live cell experiments, albeit with some modifications.

withdraws the media from the cell culture while the other introduces the stimuli. One of the major drawbacks with the current experimental design, stems from the fact that direct introduction of the stimulus upon the cells may lead to extraneous calcium responses.23 Dr. Setton approached the design team to create a system, which would be able to deliver a stimulus to a cell culture in a controlled and efficient manner without any superfluous cellular responses.

Design Requirements Introduction
The foundation of tissue engineering lies within the complex communication schemes developed between individual cells, between the cell and its external environment, and within the cell itself. A prime example of this occurs during stem cell maturation. During growth, from a fetus, cells are directed or induced towards a very specific type of development. This direction occurs via chemical signaling (chemotaxis) and originates from the cellular environment (surrounding cells) and/or from the cell itself. Therefore, much of the research within tissue engineering revolves around observing cellular response to external and internal cues such as mitogens, chemokines, cytokines, and hormones. The goal of tissue engineering is the successful manipulation of these signaling pathways in order to obtain specialized cells with specific characteristics, e.g. cartilage cells.1 As a prominent researcher in orthopedic tissue engineering, our client, Dr. Lori A. Setton, is very interested in how cells respond to specific cytokine factors, e.g. such as tumor necrosis factors (TNF,). Currently, the Setton lab labels (fluorescent label) intracellular calcium and observes the elicitation calcium by the cell upon the introduction of the stimuli. This calcium elicitation is observed and quantified using an inverted confocal microscope. In order to facilitate the experimental process the lab has constructed a device consisting of a Petri dish with two attached syringes (and corresponding syringe pumps). One syringe The design team, in conjunction with Dr. Setton, agreed upon a set of specific design requirements that would need to be achieved in order to produce a successful product. These requirements include: (1) reaction control, (2) minimizing fluid shear stress, (3) existing equipment compatibility, (4) open gas exchange, and (5) auto-clavable. The team assumed that any device constructed would sustain cell life. It was imperative that the product deliver stimuli in a controlled and efficient manner, thus exposing all cells to an even concentration environment for the duration of the experiment (10-15 minutes). The termination of the experiment would depend upon stimuli removal, which should occur quickly and efficiently. Previous experiments have shown fluid shear stress to cause extraneous cell receptor response and cell detachment from the adhesion site, necessitating the need to minimize flow induced fluid shear stress for accurate experimental data. The product needs to be conducive to the current experiment setup: (1) allow for fluorescence measurements on an inverted confocal microscope and (2) make use of the Transwell™ cell inserts, thereby reducing the need to alter the Setton lab’s plating protocol. Meeting this requirement would facilitate product integration with live cell experiments. To simplify the design and ensure adequate gas exchange between the cells and environment, the product should expose the cells to open air.

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Another concern arising from the shear stress would be cell detachment. input spouts would feed into each of the input holes of the inserts. the sterilization of the showerhead and connected parts could prove to be a complicated endeavor. it is of utmost importance that the product maintains a sterile environment for the cells. would be connected to a tube aligned at 45-degree angle. immobilized over the cell insert in a closed container. and cost. The container would have user controlled drainage holes near the bottom to allow for gravity driven outflow. This could be accomplished through disposable parts and/or components that can be autoclaved. A stimuli stream would be allowed to flow down the plane and over the cells. effectively submerging them. in the hopes that the mist would be gentle enough to cause minimal shear stress on the cells. (b) Side cut-away view with holding reservoir and drain (red) shown Figure 1: The slide with recycling tube (green) and cells (yellow) shown The Funnel: The funnel idea consisted of a circular design wherein 16 inserts would line the inner wall. Each cell insert would have two holes. located near the bottom of the insert. However. e. The stimuli flow would be driven by gravity and the runoff would be collected and recycled for further use. 2(a) 2(b) Figure 2: (a) Bird’s eye view of Funnel concept. The major drawback of this design would be the complex method of stimuli delivery and removal that would most likely cause shear stress on the cells. Cells would be plated on a clear plastic material at an inclined angle. Along the outer wall of the design. away from the cells. The drainage hole. Finally. This would also cause numerous problems in terms of sterilization. fabrication tools.g. thus allowing the stimuli stream to diffuse through the membrane to the cells without exposure to the flow field. Figure (3): Showerhead concept shown with insert in closed container 2/19 . The feasibility of engineering a hydraulics system capable of generating a fine stimuli mist with minimal shear stress would be of concern. This design would incorporate a showerheadlike stimuli dispenser. the rate of stimuli funneling could be user controlled. Stimuli would be simultaneously delivered to all inserts via an infusion pump. from the direct contact with a moving flow field. A suggestion was made that a membrane "cap" could be introduced over the cells. one for stimuli inflow and the other for drainage. Ideally. due to monetary limitations. Design Alternatives The Slide: This design would attempt to exploit a nonviscous fluid’s tendency to “adhere” to surfaces over which it flows. The showerhead would generate a stimuli mist that would interact with the cells. Furthermore. Questions were also raised regarding whether the stimuli mist would settle into the container during the experiment to allow for gravity drainage or if another method of drainage should be considered. The major concern of this design would be the amount of shear stress the cells would be exposed to. All the drainage tubes would collect into a single reservoir and funnel the stimuli The Showerhead: The origins of this idea came from watching the misting of produce in supermarkets to prevent drying. this would necessitate the need for specially designed membranes and/or cell inserts and would not be feasible.Due to the many factors that lead to cell necrosis. each Transwell cell insert would need to be modified to allow for stimuli input and drainage. leading to extraneous cellular responses. water flowing over rocks in a stream.

via a spray-like device. incorporating basic mass transport ideas.4 To mimic this biological process. Due to the orientation of the spouts. through the dialysis array. To achieve this. 3/19 . given the team’s resources and time limit. A concern would be that direct fluid flow over the cells could induce significant fluid shear stress. The cup has multiple input spouts around the circumference and the flat bottom portion would be made of clear plastic to allow for inverted microscope imaging. Fluid could be added to the reaction well through the secretion spouts. A final drawback of the system was the fact that the modeling of the kinetics of the delivery would be nearly impossible. a highly controlled injection rate would have to be utilized to minimize superfluous cellular response and cell detachment. an uneven stimuli concentration may result from the flow streams directed towards the center of the cup. the design would require a cup-like shell that would hold a set of media-delivery tubes and a set of stimuli-delivery tubes. and exit through the output tube. However. The cell insert would sit within the cup reservoir and rest over a dialysis array. It soon became apparent to the team that the design would not be feasible in accomplishing the design requirements. unnecessarily complicating the product design. Also. Delivery of stimulus in a controlled and efficient manner would also be extremely difficult and require some type of shower-spray like device. The Dialysis Cup: The idea for the dialysis cup arose from observing how the kidneys extract specific solutes through the Loop of Henle. The stimuli would perfuse from the input tube of the shell. The media would be delivered through the tubes lining the inside wall of the shell and be expelled into the reservoir below the insert.The Buckyball: The buckyball design originated from the idea that quick and even stimuli delivery could be achieved by placing all cells equidistance from the source of stimuli delivery. with flow rate controlled by an external pump. The stimulus would be delivered. Ideally the inserts could be easily removed and inserted. thus allowing for even distribution. due to the lack of open gas exchange. the overall flow of fluid would be towards the center of the well. to the cell culture inserts from the centroid of the device. Modeling for the transport of stimuli across the insert membrane to the cells generated the equation below. into which the insert sits. in order to sustain the cells. Furthermore. the complexity of this design requiring microfluidics for the dialysis array would be extremely difficult. The close proximity of the stimuli in the dialysis array to the cells would quickly transport the stimuli via diffusion. The interior of the design would have to mimic an environment similar to that in a bioreactor. a soccer ball (buckyball) would be created where the hexagonal shapes that make up the exterior of the device would be cell culture inserts. A gravity induced drain would also exist near the bottom of the well that could be connected to additional tubing for fluid removal. The team liked the concept of using dialysis to as an efficient means of stimuli transport. Figure (5): Side cut away view of injection model with parts labeled Figure (4): Buckyball concept partial cut away showing inserts The Injection Model: This design utilizes a round cup-like structure.

thus decreasing the possibility of extraneous cell responses and detachment from the membrane. (near top) Bird’s eye view with dialysis array marked.  2C  C0   2 Am Dm t  ln  1  =    VL  C0    Am: Membrane area Dm: Diffusion coefficient  : Coefficient of permittivity t: Time C1 Concentration of side 1 C0: Initial concentration V: Volume L: Thickness 9(b) Figure 9: (a) schematic of semi-infinite system. diffusion times would only be limited by membrane resistivity. Initial mathematical modeling of the system to determine the time required for diffusion was based off a quasi-steady state assumption (Truskey et al. cell insert and flow tubes marked. The design would consist of a continuous "river" flow of stimuli beneath the cell insert membrane. The removal of stimuli could be achieved. without disrupting fluid flow. Figure (8): The River flow field schematic with insert and cells shown (insert not drawn to scale) J (r ) = D C t R  r ln  TR  C   J(r): radial flux across dialysis array D: diffusion coefficient C: stimuli concentration t: time r: radial distance from center of dialysis array RT: dialysis tubing radius Rc: cell radius 9(a) Figure (7): Dialysis cup flux equation used to model system The River: This design approach was based on the method of solute removal in the human body.Serum & Stimuli Cell insert Flow Field Cells Green Flow: Serum Blue Flow: Stimuli Figure (6): (far top) Side cut away view of The Dialysis Cup model. However. The concentration gradient created would drive the diffusion transport of stimuli across the membrane to the cells. questions remained on how to house a continuous flow field in a product that would fit on the platform of a microscope and allow for imaging.) 5 The versatility and adaptability of the river design to live cell experiments was apparent. by running stimuli-free cell media through the river. (b) diffusion equations to model the River Selection Matrix The objective evaluation of design alternatives was completed in a two-step process: (1) fulfillment of compulsory requirements and (2) fulfillment of weighted design matrix requirements. Because the stimuli river would make direct contact with the membrane. The compulsory requirements allowed us to narrow down the number of design alternatives considered in the 4/19 . The lack of direct contact between the cells and fluid flow would limit the shear stress on the cells.

scores are totaled by row Figure (10): Prototype 1 experimental setup 5/19 . The maximum dimensions of this prototype were (LxWxH) 7cm x 3. The compulsory requirements were (1) the use of unmodified Transwell ™ cell inserts and (2) cell exposure to the air. and (3) fluid mixing. the ends of which taper to fit the inflow and outflow tubes. the most efficient manner to achieve this would be to provide the cells with a sterile point of contact. All adhered components also received a layer of plumbing calk to prevent leakage. The decision matrix showed a tie between the dialysis cup and river designs. Furthermore. and the river designs fulfilled the two requisites and were considered in the decision matrix. The injection model failed to evenly distribute stimuli due to the orientation of the input spouts and flow field. due to the direct fluid flow over the cells. Any design selected must be able to maintain cell life. dialysis cup. The ranking weights were scaled 14. quick stimuli delivery. Only the injection model. These two factors contributed to the low score the design received. Media & Stimulant Tanks Insert Goals Stimuli Removal Shear Stress Stimuli Shear Quick Even Total Removal Stress Delivery Distribution ----1 0 ----0 0 0 0 0 1 Chamber Length: 7 cm Height: 2. who directed the design team to go forward with the river design due to feasibility of prototype fabrication and ease of mathematical modeling. with 4 for the most important and 1 for the least important.5cm. therefore minimizing shear stress on the cells. The dialysis cup and river designs both employed diffusive transport of stimuli to the cells. Weighting of these requirements was performed with a pair-wise comparison matrix. An o-ring was adhered over the 13mm hole to ensure the creation of a fluidtight interface between the Transwell™ insert and chamber.75cm x 2. with scores of 44. the injection model was not able to adequately minimize shear stress. Setton. (2) fluid flow. minimization of shear stress.design matrix. The cap of one CCF with a 5mm cut hole was used as the drain for the chamber. Design Requirements Quick Stimuli Removal Minimize Shear Stress Quick Stimuli Delivery Even Stimuli Distribution Total Weight 1 2 3 4 Injection Model 4 2 5 2 Dialysis Cup 4 4 4 5 River 4 4 4 5 44 --31 44 Figure 7: Decision Matrix Design Fabrication In order to take the river from design concept to actual product. The requirement rankings (from most to least important) were as follows: even stimuli distribution. This overall chamber consisted of a rectangular shape. the design team decided to focus on three design components: (1) the chamber. The decision matrix included the remaining design requirements. with three 5mm holes drilled into the flat end and one 13mm hole drilled on top. Dr. which was then adhered to the flat end of the first CCF. Given the team resources and time limitations. The second CCF was cut to separate the triangular section. The design concepts and results of the decision matrix were shown to the client. The use of the design matrix was the final assessment in selecting a design. both designs allowed for the even distribution of stimuli.5 cm Volume: 46 mL Quick 1 1 ----0 2 Delivery Even 1 1 1 ----3 Distribution Figure 6: Pariwise Comparison Matrix. Additionally. The Transwell™ cell inserts come in a sterilized package and any modification to them after opening would result contamination. and stimuli removal. with a total volume of 46mL. One CCF was used for the main chamber. Prototype 1: The initial prototype was fabricated out of two-25mL cell culture flasks (CCF). the only possible way to achieve adequate gas exchange would be to leave the cells open to air.

The stimuli and media tubing were joined together by a 3-way stopcock (Cole-Parmer). Plastic tubing clamps were used to control what fluids entered the chamber. the outflow end leading to the chamber. the infusion pump was started and the stopcock was adjusted to allow for both stimuli and media flow. the chamber was placed on the microscope platform to allow for imaging while the fluid tanks were housed on a shelf above the microscope. These alterations made the final prototype more 6/19 . Both tanks were held at the same height above the chamber.5mm) was adhered over the predrilled hole on the top piece with the same adhesive. cut to fit the base. thus allowing only stimuli to flow through the chamber. the prototype with cell insert was found to obstruct proper objective lens focusing on a regular confocal microscope.To achieve high fluid flow rates. Franklin Hills. which also facilitated mixing. thus allowing only media to flood the chamber. The two pieces were adhered together with Plumber's Goop (a toluene based adhesive and sealant). This included the use of two 2. Model 6365-90. In conjunction with the pump. with a 13. NJ) connected to 1/16” inner diameter tubing. a stimuli infusion rate was first determined to ensure constant stimuli delivery throughout the experiment. All parts were allowed to dry for 24 hours. Infusion Pump & Stimuli Media Container 3-way stopcock Chamber Insert Length: 12 cm Height: 1 cm Volume: 54 mL Figure(11): Final Prototype setup The final prototype mixed stimuli and media together prior to entering the chamber. it was necessary that turbulent flow (Re>104) be established in the section of tubing between the stopcock and the chamber. the stimuli tube was clamped. Finally. polycarbonate rectangular case. Termination of the experiment required stopping stimuli flow and reinitiating media flow. For experimentation. the stopcock was orientated to allow for media flow only into the chamber. Termination of the experiment was achieved immediately by stopping stimuli flow while maintaining media flow. the stimuli tube was unclamped while simultaneously clamping the media tube. The final prototype employed a different experimental setup. A Rubbermaid™ Tupperware lid. To begin the experiment. Drilling a single 1/16” hole into one end of the chamber created the input. gravity pumps were used for both media and stimuli delivery. Two separate pressure drops were created. Vernon Hills.5-gallon Dear Park water tanks each connected to 5/8" inner diameter tubing. Tubing from the stimuli and media tanks were joined together by a plastic "T" junction. it was determined that a second prototype was necessary to address the concerns with chamber dimensions and volume constraints. An o-ring (inner diameter 13. We feared this problem would also translate to the inverted confocal microscope in Dr. Drilling three 1/16” holes at the opposite end of the input hole created drainage outlets. it became apparent that these flow rates necessitated unreasonably large volumes of media and stimuli for live cell experiments. one between the media bag and stopcock and the second between the stopcock and the chamber. from what was used previously. Therefore. The stopcock was connected to the chamber inlet with 7cm of 1/16” diameter tubing. IL). we used a 60mL plastic syringe (BD. First. Our final prototype considerably decreased the fluid volumes necessary to run the experiment through decreased tubing size and drainage rate. MA). To begin the experiment. The Dear Park® tank used to hold stimuli was replaced by a Harvard Apparatus Infusion Pump (Holliston. To use the prototype. High volumetric flow rates were necessary to ensure a step function-like change in concentration gradient at t=0. The use of large diameter tubing allowed for the desired high flow rate and maintenance of constant fluid level in the chamber. Initially. A large sheet of Parafilm® was wrapped around the end of the chamber to create a funnel that directed outflow into a drainage container. To accomplish this mixing.5mm hole drilled into its center was used as the top of the chamber. Final Prototype The chamber’s base was constructed out of a clear. However. The input hole was fitted with a straight polypropylene tube connector (Cole-Parmer. Setton’s lab. A 5L saline bag replaced the large Dear Park® water tank as the media container and was connected to 5/16” inner diameter clear plastic tubing.

” 7/19 . Next. For all experiments the inserts were pre-wetted with media prior to insertion into the chamber. a fluorescent dye. Calcein-AM fluoresces in living cells. Water was allowed to flow through the chamber for 5 minutes prior to FITC infusion. we conducted a series of experiments in order to elicit the capabilities of our design. 4. a 10uL fluid sample was taken from the inside the well. The procedure was repeated for varying time intervals of FITC infusion to collect data points for 2. the need for high cytokine concentration and fluid volumes made conducting the same cell experiment too costly. Performance Results & Analysis pH Results All three samples showed a pH level of 5. However. A = Cl . taken from the “Fisher Scientific FITC Product Information. When enveloped in a hypo-osmotic (pure deionized water) environment. minutes. Pure H2O Insert Sample Legend: 4 5 6 7 Figure 12: pH test results using Litmus paper FITC Results Absorbance data from the spectrophotometer was converted to concentration via Beer’s Law. The final experiment was conducted in the Setton lab to determine the capabilities of our prototype with live cells. The path length assumed to be 11mm. After 10 minutes. These trials were repeated three times. A fluid sample was immediately taken from inside the well. 12mm in diameter with 0. Transwell™ insert used contained a polyester membrane. Water was allowed to flush the chamber for an additional 5 minutes.3mM FITC (Fisher Scientific) was used as the stimuli and water used as the media. After 1 minute. A control experiment was also conducted by imaging cell fluorescence after dH2O was directly administered to the cells. rapidly swell.5mM PBS solution was the media. the width of the cuvette. Water was allowed to flow through the chamber for 5 minutes before the infusion of the buffer began. See Appendix I. due to stimuli-media mixing prior to entering the chamber. the concentration of FITC in the chamber was 0. First. Live Cell Experiment (LCE) Procedure: In this experiment deionized water was the stimuli and 0. A cheap and readily available stimuli surrogate was used instead of cytokine. T-4 mice fibroblast cells were pre-injected with calceinAM dye in a PBS solution and incubated for 15 minutes. cells take up dH20. PBS solution flow was terminated and the dH2O was flowed through the chamber. pH Procedure: For these experiments a buffer solution of pH 4 was used as the stimuli. a significant decrease in acidity from that of pure water. to quantify the rate of diffusion across the membrane. The collected fluid samples were analyzed using litmus paper. However. The second experiment used FITC. due to metabolic processing. 3. Performance Assessment After finalizing our prototype. Due to the differential ratios of volumetric flow. PBS solution flowed through the chamber for 3 minutes. and lyse. FITC Procedure: A solution of 0. the results pH experiment may not accurately reflect the same rates of diffusion for large proteins like the cytokines used in actual cell response experiments. fluorescence images of the cell culture were taken approximately every 2 minutes. The procedure was repeated for a total of 3 trials. and extinction coefficient 105. at a pore density of 4*106 pores/cm2. it was found that initial concentration of stimuli in the syringe would need to be on the order of 1000 times greater than necessary for cell stimulation.feasible for live cell experiments. Using the inverted confocal microscope and a digital camera. However. A proof of concept experiment was conducted with pH solutions to determine if there was indeed diffusion of fluid across the insert membrane. The collected fluid samples were analyzed using a spectrophotometer (Genesys) at wavelength of 525nm. and water was used as the media. This experiment was repeated 3 times.4um pore size. and extinguishes during cell death. These results verified our fundamental assumption that the continuous flow field generated in the chamber allows for diffusion across the membrane. etc. the infusion of FITC was then terminated and the chamber flushed with water.3uM.

t=0 14(a) t=7min t=0 t=26min 14(b) Figure 14: (a) Control trial. After 7 minutes. However. it was apparent that the majority of cells had lysed due to the significant decrease in fluorescence density. Trial 1 showed a high outlier data point believed to be caused by improper usage of the spectrophotometer. These data points were removed prior to analysis. (b) Experimental Results Figure 13: FITC concentration results It was initially expected that the concentration would asymptotically approach a maximum value. dissipates when the cell explodes. flow fields. This roughly 5-fold increase in time required to observe the same amount of cell lysing is caused by slow diffusion as a direct result of high membrane resistivity. Taking this into account. while slower than directly adding stimuli to the cells. However. The results of the live cell experiments proved that a sufficient amount of stimuli was crossing the barrier. making response data robust. Trial 3 showed concentration levels significantly dropping off after 7 minutes. The control trial allowed us to observe cell lysing. Another important finding of this experiment was that no cell detachment occurred. albeit within a much longer time frame. Protein stimuli are often 1000 times larger than FITC. Additionally. comprised the integrity of the membrane leading to membrane detachment from the outside of the insert. etc. The average time for definitive fluorescence decrease was on the order of 25 minutes. a large of amount dH20 was necessary to create the hypotonic capable of inducing cell lysing. indicating the amounts of fluid shear stress on the cells was minimal. would be fast enough to expose the cells to stimuli within 1 minute. based on the FITC concentration results. The “green haze” seen in the control trial images is in part due to multiple cell lysings at different depths and also due to UV bleaching of the calcein-AM dye. We believed that multiple sample extractions. with the pipette. We believe that this lack of saturation was due a high membrane resistivity. the 25 minutes for significant cell lysing using the river is comparable to the control trial where a hypotonic environment was immediately created with the addition of dH20. the overall concentration of the FITC within the well increased with increasing duration of time. it now appears that diffusion time could take upwards of 10 minutes. which acted to slow the diffusion of FITC across the membrane. See Appendix III. Several trials showed data points that did not follow the expected trend. See Appendix II. LCE Results The process of cell lysing is seen as a slowly expanding fluorescent circle that eventually The three experimental trials showed similar results to that the control. which could correlate to an even larger amount of time necessary to transfer stimuli to the cells. this allowed water from the chamber to mix with the insert fluid leading to lower FITC concentrations.As expected. no concrete steadying of concentration levels were seen across three trials. Consequently. This saturation would have been due to a near zero concentration gradient caused by continuous diffusion of FITC across the membrane. Even with transport impediments (membrane resistance. One of our initial assumptions with the river design was that diffusion time. The minimization of shear stress in our prototype serves to decrease the likelihood of superfluous cellular response.) the design should be able 8/19 .

experimental results show the fast diffusion assumption to be untrue. within a matter of a few minutes. the results of the three experiments confirmed that it was possible to transport stimuli across a membrane by means of orthogonal diffusion. due to time constraints. While flooding the chamber with media can immediately decrease stimuli concentrations. In summary. However. The live cell data indicated that even with high membrane resistivity. and the simplest solution is the creation of a much smaller chamber to limit fluid flow requirements and waste. the sterilization of the prototype by an autoclave went untested. The cells were also exposed to open air. Dr. In addition to the minimization of shear stress and cell detachment. Chris Gilchrist. Setton. With proper machinery a microfluidics delivery array could also be used to deliver a small amount of stimuli to individual cells. lead us to believe that so long as stimuli delivery and termination to the chamber could be controlled. The prototype was also compatible with the use of an inverted confocal microscope and Transwell® cell insert. however the diffusion of stimuli across the membrane did not meet our initial assumptions. However. The slow diffusion time also prevents us from instantaneously terminating cell-stimuli interactions. However. The differential volumetric flow rates between the media and stimuli tube requires a very high initial stimuli concentration. diffusion times are on the order of 10-20 minutes and therefore unable to directly control stimuli delivery to the cells.to deliver stimuli (e. stimuli already diffused into the membrane require additional time to diffuse back across the membrane into the fluid field for removal.g. Conclusions Overall. however this requires technology beyond our capabilities. two important product requirements. Prototype testing and assessment illuminated several limitations. However. Our assumptions. 9/19 . which initiate cell a response on a 1:1 ratio. a significant amount of stimuli should reach the cells within 10 minutes. due to the constraints of diffusion across the Transwell® membrane. and Professor Boyd. However. the live cell experiment confirmed that the prototype did not cause cell detachment and minimized shear stress. We were able to control the delivery rate and concentration of stimuli in the chamber. Our next steps would be to recommend more research into alternative methods of stimuli delivery while keeping in bounds with the shear stress requirements. leaving our product only able to control stimuli delivery and removal to the chamber. the design team was successful in creating a chamber with controlled stimuli delivery and removal. an automated injection and suction system to directly deliver stimuli to the cells while minimizing shear stress. as the FITC and live cell experiments show. the most effective solution would be to create a product that does not rely on diffusion to deliver stimuli to the cells. we are not directly able to control the rate of stimuli-cell interactions at this time. based on fast diffusion across the membrane. which is highly expensive. cytokines). the prototype was also successful in controlling the delivery and removal of stimuli via infusion and gravity pumps. Possible solutions to this problem include the use of a recycling system to collect fluid runoff from the drain and redelivering it to the chamber. these amounts were still too large to allow for the product’s use with cytokine and cell serum. and improve draining and sealing. Not relying on diffusion raises concerns about increasing shear stress and the possibility of cell detachment. Faster fluid flow within the chamber may also aid in decreasing diffusion times. While the volume requirements for the final prototype were considerably reduced from prototype 1. Another limitation we found was that the product was not directly able to control the rate of stimuli delivery to the cells. Professor Gimm. Finally. reduce the overall setup size to decrease volume and concentration needs. automate fluid flow. We had assumed that diffusion would take place instantaneously after the membrane came into contact with the moving stimuli flow field. Solutions to this problem could include the use of a different type of Transwell® membrane (with different pore sizes and/or pore density) or a different membrane material altogether to shorten diffusion times. we would also control the rate of stimuli delivery to and from the cells. Acknowledgements The authors would like to thank the Setton Lab. allowing for adequate gas exchange.

Co DS = 0.4 cm QC = QM+QS QC DC = 0.Appendix I: Final Prototype Flow Calculations The schematic below shows the media and stimuli volumetric flows.16 cm QS. QM and QS respectively. the eFunda Reynold’s number calculator6 was used and obtained a value of Re=105 in the tubing.001. QM. along with Bernoulli’s equation and the definition of Reynold’s number (ratio of inertial to viscous flows). QC.16 cm 10/19 . mixing together in the tubing leading in the chamber. QS/QM was found to be 0. For a given pressure drop of 70cm between the media bag and 3-way stopcock. DM = 0. The ratios of volumetric flows.

727272727 2.Appendix II: FITC Experiment Complete Data The table below lists absorbance and concentration data as a function of time.363636364 Absorbency 0.636363636 3.024 0.818181818 5.047 0.545454545 18.454545455 5.727272727 5.063 0.059 0.818181818 4.545454545 11/19 .072 0.037 Concentration (uM) 3. Note that erroneous and outlier data points have been taken out.636363636 4 3.086 0.199 Concentration (uM) 1.09090909 Absorbance 0. Trial 1 Time (min) 1 2 3 5 7 8 9 10 Trial 3 Time (min) 1 2 3 4 7 8 9 Trial 2 Time (min) 2 3 4 5 Absorbency 0.363636364 7.636363636 3.818181818 6.363636364 4.07 0.051 0.064 0.181818182 5.019 0.272727273 4.061 Concentration (uM) 4.04 0.046 0.044 0.042 0.04 0.049 0.181818182 6.

Appendix III: Live Cell Experiment The figures below show fluorescence results from all three trials in successive order: Trial 1. Trial 2. and Trial 3. t= 0 t=25min t=0 t=26min t=0 t=20min 12/19 .

t=0 t=7min Cell lysis 13/19 .The figures below show the control trial and cell lysing in situ.

Setton 04/30/05 14/19 .The River: Product Design Specifications Jenna Olson Lucy He Brendan Casey Alvin Kpaeyeh Client: Dr.

The device must allow for measuring cellular responses due to the stimulus via a confocal microscope. Accuracy and reliability: • System should reproduce similar results based on similar inputs in order to be viable in a research capacity. The system should not elicit any cellular responses. b) c) d) e) f) g) 15/19 . Client requirements • • • • • The device must deliver stimulus quickly and maintain a specific concentration of stimulus on the cells. • After cellular responses system should return cells to original state. Operating Environment: • Cellular bath within device should be exposed to air throughout experiment. Size: • The system should fit easily on a confocal microscope and all parts should be able to fit onto a reasonably sized lab bench. Safety: • The device should be user safe. The system should allow the cells to be exposed to air and should allow for timely removal of the introduced stimulus.Function The client needs a device to quickly deliver a stimulus to cells without inducing any cellular responses. The device must be easily sterilized or disposable. The device should be exposed to air. The system should allow quantification of stimulus-induced responses my means of an inverted confocal microscope. • The device must be completely enclosed in order to prevent damage to microscope and endanger user (microscope is electric). • The system should not induce any biological responses on the cells. Shelf Life: • System should either be reusable or extremely cheap and easy to manufacture. • An inverted confocal microscope should easily scan the device in order to observe cellular responses. Design requirements a) Performance Requirements: • The system should deliver stimulus to cell culture plated on Transwell insert. The system must deliver said stimulus evenly and simultaneously to all cells in an experiment. in order to reuse cells thereby saving money and cutting down amounts of cells needed. Life in Service: • Cellular responses may begin within 100 micro-seconds and may continue for up to 10 minutes.

• 16/19 . This would require of course a different microscope that could visualize and measure multiple points on the same plane at the same time. Production Characteristics: a) Target production cost: • System design and development should be cost around $200. Miscellaneous a) Customer: • Ideally a multiple well system would make experiments quicker and easier for users. j) Materials: • Permanent parts of the device need to be auto-clavable to ensure sterilization and or to make use of disposable and reasonably priced parts.It is preferred but not required that the system fit multiple Transwell inserts and their varying sizes.

45 US Plastics Home Depot 4/01/05 4/12/05 TOTAL 16.52??? 45.47 19.61 19. Brass Connector Digital pH Tester Digital Thermometer Plastic Check Valve. Exacto Knife Fluid Container Fluid Containers (Various) Vinyl Tubing.30 5. elbow joint Plastic Cement.52 17/19 .63 3. with 3 more clamps Aqua Epoxy Store Medical Supply Superstore Home Depot Michaels Walmart Walmart Home Depot Professional Equipment Technika US Plastics Date Bought 4/12/05 3/02/05 3/30/05 3/02/05 3/06/05 3/19/05 3/21/05 3/21/05 4/02/2005 Amount (dollars) 34.90 22. tubing clamps nd 2 Check Valve.76 3.BME 265/227 INVOICE Prepared by Brendan Casey Brendan=blue Lucy=red Alvin=green Jenna=yellow Description Cathetor tubing.13 5.96 18. syringes Vinyl tubing.79 $195.

MAINTAIN CONSTANT LIQUID LEVEL 18/19 .WARNING!!! FLOWING RIVER DO NOT LEAVE UNATTENDED! CONTAINS MOVING LIQUID. CAN CAUSE DAMAGE TO LAB EQUIPMENT.

F. Engineering Formulas. Paik Py. http://www.efunda. Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells. Dialysis.htm 5 Truskey. Yan W. Duke University. Dept of Mechanical and Aeronautical Engineering. 286(6):C1367-75. Pappone PA. France. Cell Physiol. Yuan.nih. (2004). Baer AE. Reynold’s Number Calculator. 293 (2002) 932-938. Epub 2004 Feb 04. Barakat AL. National Instittue of Health. Biochemical and Biophysical Research Communications. INSERM U141. 2004 Jun. G.com/formulae/fluids/calc_reynolds. Setton LA. http://kidney. 2004 4 Anonymous1. Am J. Paris. Matrix protein gene expression in intervertebral disc cells subjected to altered osmolarity. Tedgui A. Transport Phenomena in Biological Systems. & Katz. New Jersey: Pearson Prentice Hall..cfm 1 19/19 .niddk. Physiol.References: Chen J. 3 Lehoux S. Signal Transduction of Mechanical Stresses in the Vascular Wall.. D. 2004..gov/kudiseases/pubs/hemodialysis/index. 2 Lieu DK. University of California. 6 Anonymous2.

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