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

Delivering and Removing Stimuli

BRENDAN CASEY, LUCY HE, ALVIN K PAEYEH, 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 objectivesand showed promise for future usage in live cell

experiments, albeit with some modifications.

IntroductionThe 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 thecellular 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

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 RequirementsThe 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 allcells 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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Due to the many factors that lead to cell necrosis,

it is of utmost importance that the product maintains

a sterile environment for the cells. This could be

accomplished through disposable parts and/or

components that can be autoclaved.

Design AlternativesThe Slide:

This design would attempt to exploit a non-

viscous fluid’s tendency to “adhere” to surfaces over

which it flows, e.g. water flowing over rocks in a

stream. Cells would be plated on a clear plastic

material at an inclined angle. A stimuli stream would

be allowed to flow down the plane and over the cells,

effectively submerging them. The stimuli flow

would be driven by gravity and the runoff would be

collected and recycled for further use.

The major concern of this design would be the

amount of shear stress the cells would be exposed to,

from the direct contact with a moving flow field.

Another concern arising from the shear stress would

be cell detachment. A suggestion was made that a

membrane "cap" could be introduced over the cells,

thus allowing the stimuli stream to diffuse through

the membrane to the cells without exposure to the

flow field. However, due to monetary limitations,

this would necessitate the need for specially designed

membranes and/or cell inserts and would not be

feasible.

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, one for stimuli

inflow and the other for drainage. Along the outer

wall of the design, input spouts would feed into each

of the input holes of the inserts. Stimuli would be

simultaneously delivered to all inserts via an infusion

pump. The drainage hole, located near the bottom of

the insert, would be connected to a tube aligned at

45-degree angle. All the drainage tubes would

collect into a single reservoir and funnel the stimuli

away from the cells. Ideally, the rate of stimuli

funneling could be user controlled.

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,

leading to extraneous cellular responses.

Furthermore, each Transwell cell insert would need

to be modified to allow for stimuli input and

drainage. This would also cause numerous problems

in terms of sterilization, fabrication tools, and cost.

2(a) 2(b)

Figure 2: (a) Bird’s eye view of Funnel concept; (b) Side cut-away view

with holding reservoir and drain (red) shown

The Showerhead: The origins of this idea came from watching

the misting of produce in supermarkets to prevent

drying. This design would incorporate a showerhead-

like stimuli dispenser, immobilized over the cell

insert in a closed container. The showerhead would

generate a stimuli mist that would interact with the

cells, in the hopes that the mist would be gentle

enough to cause minimal shear stress on the cells.

The container would have user controlled drainage

holes near the bottom to allow for gravity driven

outflow.

The feasibility of engineering a hydraulics system

capable of generating a fine stimuli mist with

minimal shear stress would be of concern. 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. Finally, the

sterilization of the showerhead and connected parts

could prove to be a complicated endeavor.

Figure (3): Showerhead concept shown with insert in closed container



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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. To achieve this, a soccer

ball (buckyball) would be created where the

hexagonal shapes that make up the exterior of the

device would be cell culture inserts. Ideally the

inserts could be easily removed and inserted. The

stimulus would be delivered, via a spray-like device,

to the cell culture inserts from the centroid of the

device, thus allowing for even distribution.

It soon became apparent to the team that the

design would not be feasible in accomplishing the

design requirements. The interior of the design

would have to mimic an environment similar to that

in a bioreactor, due to the lack of open gas exchange,

in order to sustain the cells, unnecessarily

complicating the product design. Delivery of

stimulus in a controlled and efficient manner would also be extremely difficult and require some type of

shower-spray like device. A final drawback of the

system was the fact that the modeling of the kinetics

of the delivery would be nearly impossible.

Figure (4): Buckyball concept partial cut away showing inserts

The Injection Model:

This design utilizes a round cup-like

structure, into which the insert sits. 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, with flow rate controlled by an

external pump. Due to the orientation of the spouts,

the overall flow of fluid would be towards the center

of the well. A gravity induced drain would also exist

near the bottom of the well that could be connected to

additional tubing for fluid removal.

A concern would be that direct fluid flow

over the cells could induce significant fluid shear

stress. Also, a highly controlled injection rate would

have to be utilized to minimize superfluous cellular

response and cell detachment. Furthermore, an

uneven stimuli concentration may result from the

flow streams directed towards the center of the cup.

Figure (5): Side cut away view of injection model with partslabeled

The Dialysis Cup:

The idea for the dialysis cup arose from observing

how the kidneys extract specific solutes through the

Loop of Henle.4

To mimic this biological process,

the design would require a cup-like shell that would

hold a set of media-delivery tubes and a set of

stimuli-delivery tubes. The cell insert would sit

within the cup reservoir and rest over a dialysis array.

The media would be delivered through the tubes

lining the inside wall of the shell and be expelled intothe reservoir below the insert. The stimuli would

perfuse from the input tube of the shell, through the

dialysis array, and exit through the output tube. The

close proximity of the stimuli in the dialysis array to

the cells would quickly transport the stimuli via

diffusion.

Modeling for the transport of stimuli across

the insert membrane to the cells generated the

equation below, incorporating basic mass transport

ideas.

The team liked the concept of using dialysis to as

an efficient means of stimuli transport. However,

given the team’s resources and time limit, thecomplexity of this design requiring microfluidics for

the dialysis array would be extremely difficult.



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Figure (6): (far top) Side cut away view of The Dialysis Cup

model, cell insert and flow tubes marked; (near top) Bird’s eye

view with dialysis array marked.

( )

=

C

T

R Rr

t C Dr J

ln

The River:

This design approach was based on the method of solute removal in the human body. The design would

consist of a continuous "river" flow of stimuli

beneath the cell insert membrane. The concentration

gradient created would drive the diffusion transport

of stimuli across the membrane to the cells. Because

the stimuli river would make direct contact with the

membrane, diffusion times would only be limited by

membrane resistivity. The removal of stimuli could

be achieved, without disrupting fluid flow, by

running stimuli-free cell media through the river.

Initial mathematical modeling of the system to

determine the time required for diffusion was based

off a quasi-steady state assumption (Truskeyet al.

)

5

The versatility and adaptability of the river design to

live cell experiments was apparent. The lack of

direct contact between the cells and fluid flow would

limit the shear stress on the cells, thus decreasing the

possibility of extraneous cell responses and

detachment from the membrane. However, 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.

Figure (8): The River flow field schematic with insert and cells

shown (insert not drawn to scale)

ln2C

1C

0

C 0

= 2 A

m D

mt

VL

9(b)

Figure 9: (a) schematic of semi-infinite system; (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. The

compulsory requirements allowed us to narrow down

the number of design alternatives considered in the

J(r): radial flux across dialysis array

D: diffusion coefficientC: stimuli concentrationt: time

r: radial distance from center of dialysis arrayR T: dialysis tubing radiusR c: cell radius

Cell insert

Green Flow: Serum

Blue Flow: Stimuli

Serum &

Stimuli

Flow

Field

Cells

Figure (7): Dialysis cup flux equation used to modelsystem

9(a)

Am: Membrane areaDm: Diffusion coefficient

: Coefficient of permittivity

t: Time C1 Concentration of side 1

C0: Initial concentration

V: VolumeL: Thickness



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design matrix. The use of the design matrix was the

final assessment in selecting a design.

The compulsory requirements were (1) the use of

unmodified Transwell ™ cell inserts and (2) cell

exposure to the air. Any design selected must be able

to maintain cell life; the most efficient manner to

achieve this would be to provide the cells with a

sterile point of contact. The Transwell™ cell inserts

come in a sterilized package and any modification to

them after opening would result contamination.

Given the team resources and time limitations, the

only possible way to achieve adequate gas exchange

would be to leave the cells open to air. Only the

injection model, dialysis cup, and the river designs

fulfilled the two requisites and were considered in the

decision matrix.

The decision matrix included the remaining design

requirements. Weighting of these requirements was

performed with a pair-wise comparison matrix. The

requirement rankings (from most to least important)

were as follows: even stimuli distribution, quick stimuli delivery, minimization of shear stress, and

stimuli removal. The ranking weights were scaled 1-

4, with 4 for the most important and 1 for the least

important.

The injection model failed to evenly distribute

stimuli due to the orientation of the input spouts and

flow field. Additionally, due to the direct fluid flow

over the cells, the injection model was not able to

adequately minimize shear stress. These two factors

contributed to the low score the design received.

The decision matrix showed a tie between the

dialysis cup and river designs, with scores of 44. The

dialysis cup and river designs both employed diffusive transport of stimuli to the cells, therefore

minimizing shear stress on the cells. Furthermore,

both designs allowed for the even distribution of

stimuli. The design concepts and results of the

decision matrix were shown to the client, Dr. Setton,

who directed the design team to go forward with the

river design due to feasibility of prototype fabrication

and ease of mathematical modeling.

Goals Stimuli

Removal Shear

Stress Quick

Delivery Even

Distribution Total

StimuliRemoval

----- 0 0 0 0

Shear Stress 1 ----- 0 0 1

Quick

Delivery 1 1 ----- 0 2

Even

Distribution 1 1 1 ----- 3

Figure 6: Pariwise Comparison Matrix, scores are totaled by

row

Design

Requirements Weight Injection

Model

Dialysis

CupRiver

Quick Stimuli

Removal1 4 4 4

Minimize

Shear Stress 2 2 4 4

Quick Stimuli

Delivery

3 5 4 4

Even Stimuli

Distribution 4 2 5 5

Total --- 31 44 44

Figure 7: Decision Matrix

Design FabricationIn order to take the river from design concept to

actual product, the design team decided to focus on

three design components: (1) the chamber, (2) fluid

flow, and (3) fluid mixing.

Prototype 1:

The initial prototype was fabricated out of two-25mL cell culture flasks (CCF). One CCF was

used for the main chamber, with three 5mm holes

drilled into the flat end and one 13mm hole drilled on

top. The second CCF was cut to separate the

triangular section, which was then adhered to the flat

end of the first CCF. This overall chamber consisted

of a rectangular shape, the ends of which taper to fit

the inflow and outflow tubes. An o-ring was adhered

over the 13mm hole to ensure the creation of a fluid-

tight interface between the Transwell™ insert and

chamber. All adhered components also received a

layer of plumbing calk to prevent leakage. The cap

of one CCF with a 5mm cut hole was used as thedrain for the chamber. The maximum dimensions of

this prototype were (LxWxH) 7cm x 3.75cm x 2.5cm,

with a total volume of 46mL.

Figure (10): Prototype 1 experimental setup

Media & Stimulant Tanks

Chamber

Insert

Length: 7 cm

Height: 2.5 cmVolume: 46 mL



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To achieve high fluid flow rates, gravity pumps

were used for both media and stimuli delivery. This

included the use of two 2.5-gallon Dear Park water

tanks each connected to 5/8" inner diameter tubing.

Both tanks were held at the same height above the

chamber. Tubing from the stimuli and media tanks

were joined together by a plastic "T" junction, the

outflow end leading to the chamber. Plastic tubing

clamps were used to control what fluids entered the

chamber.

For experimentation, the chamber was placed on

the microscope platform to allow for imaging while

the fluid tanks were housed on a shelf above the

microscope. Initially, the stimuli tube was clamped,

thus allowing only media to flood the chamber. To

begin the experiment, the stimuli tube was

unclamped while simultaneously clamping the media

tube, thus allowing only stimuli to flow through the

chamber. Termination of the experiment required

stopping stimuli flow and reinitiating media flow.

High volumetric flow rates were necessary toensure 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.

However, it became apparent that these flow rates

necessitated unreasonably large volumes of media

and stimuli for live cell experiments. Finally, the

prototype with cell insert was found to obstruct

proper objective lens focusing on a regular confocal

microscope. We feared this problem would also

translate to the inverted confocal microscope in Dr.

Setton’s lab. Therefore, it was determined that a

second prototype was necessary to address theconcerns with chamber dimensions and volume

constraints.

Final Prototype

The chamber’s base was constructed out of a

clear, polycarbonate rectangular case. A

Rubbermaid™ Tupperware lid, cut to fit the base,

with a 13.5mm hole drilled into its center was used as

the top of the chamber. The two pieces were adhered

together with Plumber's Goop (a toluene based

adhesive and sealant). An o-ring (inner diameter

13.5mm) was adhered over the predrilled hole on the

top piece with the same adhesive. All parts were

allowed to dry for 24 hours. Drilling a single 1/16”

hole into one end of the chamber created the input.

The input hole was fitted with a straight

polypropylene tube connector (Cole-Parmer, Model

6365-90, Vernon Hills, IL). Drilling three 1/16”

holes at the opposite end of the input hole created

drainage outlets. A large sheet of Parafilm® was

wrapped around the end of the chamber to create a

funnel that directed outflow into a drainage container.

The final prototype employed a different

experimental setup, from what was used previously.

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. The Dear

Park® tank used to hold stimuli was replaced by a

Harvard Apparatus Infusion Pump (Holliston, MA).

In conjunction with the pump, we used a 60mL

plastic syringe (BD, Franklin Hills, NJ) connected to

1/16” inner diameter tubing. The stimuli and media

tubing were joined together by a 3-way stopcock

(Cole-Parmer). The stopcock was connected to the

chamber inlet with 7cm of 1/16” diameter tubing.

Figure(11): Final Prototype setup

The final prototype mixed stimuli and mediatogether prior to entering the chamber. To

accomplish this mixing, it was necessary that

turbulent flow (Re>104) be established in the section

of tubing between the stopcock and the chamber.

Two separate pressure drops were created, one

between the media bag and stopcock and the second

between the stopcock and the chamber, which also

facilitated mixing.

To use the prototype, a stimuli infusion rate

was first determined to ensure constant stimuli

delivery throughout the experiment. First, the

stopcock was orientated to allow for media flow only

into the chamber. To begin the experiment, theinfusion pump was started and the stopcock was

adjusted to allow for both stimuli and media flow.

Termination of the experiment was achieved

immediately by stopping stimuli flow while

maintaining media flow.

Our final prototype considerably decreased

the fluid volumes necessary to run the experiment

through decreased tubing size and drainage rate.

These alterations made the final prototype more

Chamber

Insert

Media Container

Infusion Pump& Stimuli

3-way stopcock

Length: 12 cm

Height: 1 cmVolume: 54 mL



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feasible for live cell experiments. However, due to

stimuli-media mixing prior to entering the chamber,

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. See

Appendix I. Performance Assessment

After finalizing our prototype, we conducted a

series of experiments in order to elicit the capabilities

of our design. A proof of concept experiment was

conducted with pH solutions to determine if there

was indeed diffusion of fluid across the insert

membrane. The second experiment used FITC, a

fluorescent dye, to quantify the rate of diffusion

across the membrane. The final experiment was

conducted in the Setton lab to determine the

capabilities of our prototype with live cells.

However, the need for high cytokine concentration

and fluid volumes made conducting the same cell

experiment too costly. A cheap and readily available

stimuli surrogate was used instead of cytokine. For

all experiments the inserts were pre-wetted with

media prior to insertion into the chamber.

Transwell™ insert used contained a polyester

membrane, 12mm in diameter with 0.4um pore size,

at a pore density of 4*106

pores/cm2.

pH Procedure:

For these experiments a buffer solution of pH 4

was used as the stimuli, and water was used as the

media. Water was allowed to flow through the

chamber for 5 minutes before the infusion of the

buffer began. After 10 minutes, a 10uL fluid samplewas taken from the inside the well. The collected

fluid samples were analyzed using litmus paper. This

experiment was repeated 3 times.

FITC Procedure:

A solution of 0.3mM FITC (Fisher Scientific)

was used as the stimuli and water used as the media.

Due to the differential ratios of volumetric flow, the

concentration of FITC in the chamber was 0.3uM.

Water was allowed to flow through the chamber for 5

minutes prior to FITC infusion. After 1 minute, the

infusion of FITC was then terminated and the

chamber flushed with water. A fluid sample wasimmediately taken from inside the well. Water was

allowed to flush the chamber for an additional 5

minutes. The procedure was repeated for varying

time intervals of FITC infusion to collect data points

for 2, 3, 4, etc. minutes. The collected fluid samples

were analyzed using a spectrophotometer (Genesys)

at wavelength of 525nm. These trials were repeated

three times.

Live Cell Experiment (LCE) Procedure:

In this experiment deionized water was the

stimuli and 0.5mM PBS solution was the media. T-4

mice fibroblast cells were pre-injected with calcein-

AM dye in a PBS solution and incubated for 15

minutes. Calcein-AM fluoresces in living cells, due

to metabolic processing, and extinguishes during cell

death. When enveloped in a hypo-osmotic (pure

deionized water) environment, cells take up dH20,

rapidly swell, and lyse. First, PBS solution flowed

through the chamber for 3 minutes. Next, PBS

solution flow was terminated and the dH2O was

flowed through the chamber. Using the inverted

confocal microscope and a digital camera,

fluorescence images of the cell culture were taken

approximately every 2 minutes. The procedure was

repeated for a total of 3 trials. A control experiment

was also conducted by imaging cell fluorescence

after dH2O was directly administered to the cells.

Performance Results & Analysis pH Results

All three samples showed a pH level of 5, a

significant decrease in acidity from that of pure

water. These results verified our fundamental

assumption that the continuous flow field generated

in the chamber allows for diffusion across the

membrane. However, 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.

Figure 12: pH test results using Litmus paper

FITC Results

Absorbance data from the spectrophotometer

was converted to concentration via Beer’s Law,

Cl A = . The path length assumed to be 11mm, the

width of the cuvette, and extinction coefficient 105,

taken from the “Fisher Scientific FITC Product

Information.”

Pure H2O Insert Sample

Legend:4 5 6 7



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As expected, the overall concentration of the

FITC within the well increased with increasing

duration of time. Several trials showed data points

that did not follow the expected trend. Trial 1

showed a high outlier data point believed to be

caused by improper usage of the spectrophotometer.

Trial 3 showed concentration levels significantly

dropping off after 7 minutes. We believed that

multiple sample extractions, with the pipette,

comprised the integrity of the membrane leading to

membrane detachment from the outside of the insert.

Consequently, this allowed water from the chamber

to mix with the insert fluid leading to lower FITC

concentrations. These data points were removed

prior to analysis. See Appendix II.

Figure 13: FITC concentration results

It was initially expected that the concentration

would asymptotically approach a maximum value.This saturation would have been due to a near zero

concentration gradient caused by continuous

diffusion of FITC across the membrane. However,

no concrete steadying of concentration levels were

seen across three trials. We believe that this lack of

saturation was due a high membrane resistivity,

which acted to slow the diffusion of FITC across the

membrane.

One of our initial assumptions with the river

design was that diffusion time, while slower than

directly adding stimuli to the cells, would be fast

enough to expose the cells to stimuli within 1 minute.

However, based on the FITC concentration results, itnow appears that diffusion time could take upwards

of 10 minutes. Protein stimuli are often 1000 times

larger than FITC, which could correlate to an even

larger amount of time necessary to transfer stimuli to

the cells.

LCE Results

The process of cell lysing is seen as a slowly

expanding fluorescent circle that eventually

dissipates when the cell explodes. The control trial

allowed us to observe cell lysing. After 7 minutes, it

was apparent that the majority of cells had lysed due

to the significant decrease in fluorescence density.

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.

14(a)

14(b)

Figure 14: (a) Control trial; (b) Experimental Results

The three experimental trials showed similar

results to that the control, albeit within a much longer

time frame. The average time for definitive

fluorescence decrease was on the order of 25

minutes. 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. Additionally, a large of

amount dH20 was necessary to create the hypotonic

capable of inducing cell lysing. Taking this into

account, 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. See Appendix III.

Another important finding of this experiment

was that no cell detachment occurred; indicating the

amounts of fluid shear stress on the cells wasminimal. The minimization of shear stress in our

prototype serves to decrease the likelihood of

superfluous cellular response, making response data

robust.

The results of the live cell experiments proved

that a sufficient amount of stimuli was crossing the

barrier. Even with transport impediments (membrane

resistance, flow fields, etc.) the design should be able

t=0 t=7min

t=0 t=26min



9/19

to deliver stimuli (e.g. cytokines), which initiate cell

a response on a 1:1 ratio, within a matter of a few

minutes.

ConclusionsOverall, the results of the three experiments

confirmed that it was possible to transport stimuliacross a membrane by means of orthogonal diffusion.

The live cell data indicated that even with high

membrane resistivity, a significant amount of stimuli

should reach the cells within 10 minutes. Finally, the

live cell experiment confirmed that the prototype did

not cause cell detachment and minimized shear

stress, two important product requirements.

In addition to the minimization of shear stress

and cell detachment, the prototype was also

successful in controlling the delivery and removal of

stimuli via infusion and gravity pumps. The

prototype was also compatible with the use of an

inverted confocal microscope and Transwell® cell

insert. The cells were also exposed to open air,

allowing for adequate gas exchange. However, due

to time constraints, the sterilization of the prototype

by an autoclave went untested.

Prototype testing and assessment illuminated

several limitations. The differential volumetric flow

rates between the media and stimuli tube requires a

very high initial stimuli concentration, which is

highly expensive. While the volume requirements

for the final prototype were considerably reduced

from prototype 1, these amounts were still too large

to allow for the product’s use with cytokine and cell

serum.

Possible solutions to this problem include the useof a recycling system to collect fluid runoff from the

drain and redelivering it to the chamber; an

automated injection and suction system to directly

deliver stimuli to the cells while minimizing shear

stress; and the simplest solution is the creation of a

much smaller chamber to limit fluid flow

requirements and waste. With proper machinery a

microfluidics delivery array could also be used to

deliver a small amount of stimuli to individual cells,

however this requires technology beyond our

capabilities.

Another limitation we found was that the product

was not directly able to control the rate of stimulidelivery to the cells. We were able to control the

delivery rate and concentration of stimuli in the

chamber, however the diffusion of stimuli across the

membrane did not meet our initial assumptions. We

had assumed that diffusion would take place

instantaneously after the membrane came into contact

with the moving stimuli flow field. However, as the

FITC and live cell experiments show, diffusion times

are on the order of 10-20 minutes and therefore

unable to directly control stimuli delivery to the cells.

The slow diffusion time also prevents us from

instantaneously terminating cell-stimuli interactions.

While flooding the chamber with media can

immediately decrease stimuli concentrations, stimuli

already diffused into the membrane require additional

time to diffuse back across the membrane into the

fluid field for removal.

Our assumptions, based on fast diffusion across

the membrane, lead us to believe that so long as

stimuli delivery and termination to the chamber could

be controlled, we would also control the rate of

stimuli delivery to and from the cells. However,

experimental results show the fast diffusion

assumption to be untrue, leaving our product only

able to control stimuli delivery and removal to the

chamber.

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 differentmembrane material altogether to shorten diffusion

times. Faster fluid flow within the chamber may also

aid in decreasing diffusion times. However, the most

effective solution would be to create a product that

does not rely on diffusion to deliver stimuli to the

cells. Not relying on diffusion raises concerns about

increasing shear stress and the possibility of cell

detachment.

In summary, the design team was successful in

creating a chamber with controlled stimuli delivery

and removal. However, due to the constraints of

diffusion across the Transwell® membrane, we are

not directly able to control the rate of stimuli-cellinteractions at this time. Our next steps would be to

recommend more research into alternative methods

of stimuli delivery while keeping in bounds with the

shear stress requirements; reduce the overall setup

size to decrease volume and concentration needs;

automate fluid flow; and improve draining and

sealing.

AcknowledgementsThe authors would like to thank the Setton Lab,

Chris Gilchrist, Dr. Setton, Professor Gimm, and

Professor Boyd.



10/19

Appendix I: Final Prototype Flow Calculations

The schematic below shows the media and stimuli volumetric flows, QM and QS respectively, mixing together in

the tubing leading in the chamber, QC. For a given pressure drop of 70cm between the media bag and 3-way

stopcock, along with Bernoulli’s equation and the definition of Reynold’s number (ratio of inertial to viscous flows),

the eFunda Reynold’s number calculator 6

was used and obtained a value of Re=105

in the tubing. The ratios of

volumetric flows, QS/QM was found to be 0.001.

DC = 0.16 cm

QS, Co

DS = 0.16 cm

QC

QC =

QM+QS

QM,

DM = 0.4 cm



11/19

Appendix II: FITC Experiment Complete Data

The table below lists absorbance and concentration data as a function of time. Note that erroneous and outlier

data points have been taken out.

Trial 1 Trial 2

Time (min) Absorbency Concentration (uM) Time (min) Absorbency Concentration (uM)

1 0.04 3.636363636 2 0.019 1.7272727272 0.044 4 3 0.024 2.181818182

3 0.04 3.636363636 4 0.072 6.545454545

5 0.042 3.818181818 5 0.199 18.09090909

7 0.046 4.181818182

8 0.059 5.363636364

9 0.051 4.636363636

10 0.037 3.363636364

Trial 3

Time (min) Absorbance Concentration (uM)

1 0.047 4.272727273

2 0.049 4.454545455

3 0.064 5.818181818

4 0.07 6.363636364

7 0.086 7.818181818

8 0.063 5.727272727

9 0.061 5.545454545



12/19

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=

0t=25min

t=0 t=26min

t=0 t=20min



13/19

The figures below show the control trial and cell lysing in situ.

t=0 t=7min

Cell lysis



14/19

The River:

Product Design Specifications

Jenna OlsonLucy He

Brendan Casey

Alvin KpaeyehClient: Dr. Setton

04/30/05



15/19

Function

The client needs a device to quickly deliver a stimulus to cells without inducing any cellular responses. The system must deliver said stimulus evenly and simultaneously to all cells in an

experiment. The device must allow for measuring cellular responses due to the stimulus via a

confocal microscope. The system should allow the cells to be exposed to air and should allowfor timely removal of the introduced stimulus.

Client requirements

• The device must deliver stimulus quickly and maintain a specific concentration of

stimulus on the cells.

• The system should not elicit any cellular responses.

• The device should be exposed to air.

• The system should allow quantification of stimulus-induced responses my means of aninverted confocal microscope.

• The device must be easily sterilized or disposable.

Design requirements

a) Performance Requirements:

• The system should deliver stimulus to cell culture plated on Transwell insert.

• The system should not induce any biological responses on the cells.

b) Accuracy and reliability:

• System should reproduce similar results based on similar inputs in order to beviable in a research capacity.

c) Safety:

• The device should be user safe.• The device must be completely enclosed in order to prevent damage to

microscope and endanger user (microscope is electric).d) Life in Service:

• Cellular responses may begin within 100 micro-seconds and may continue for

up to 10 minutes.

• After cellular responses system should return cells to original state, in order to

reuse cells thereby saving money and cutting down amounts of cells needed.e) Operating Environment:

• Cellular bath within device should be exposed to air throughout experiment.

• An inverted confocal microscope should easily scan the device in order toobserve cellular responses.

f) Shelf Life:

• System should either be reusable or extremely cheap and easy to manufacture.

g) Size:

• The system should fit easily on a confocal microscope and all parts should beable to fit onto a reasonably sized lab bench.



16/19

• It is preferred but not required that the system fit multiple Transwell insertsand their varying sizes.

j) Materials:

• Permanent parts of the device need to be auto-clavable to ensure sterilizationand or to make use of disposable and reasonably priced parts.

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. This would require of course a different microscope that could visualize and measure multiple points on the same plane at the same time.



17/19

BME 265/227 INVOICEPrepared by Brendan Casey

Brendan=blue

Lucy=redAlvin=greenJenna=yellow

Description Store Date Bought Amount (dollars)

Cathetor tubing,syringes

Medical SupplySuperstore

4/12/05 34.13

Vinyl tubing, elbow joint

Home Depot 3/02/05 5.63

Plastic Cement,

Exacto Knife

Michaels 3/30/05 3.30

Fluid Container Walmart 3/02/05 5.47

Fluid Containers

(Various)

Walmart 3/06/05 19.61

Vinyl Tubing, BrassConnector

Home Depot 3/19/05 19.52???

Digital pH Tester ProfessionalEquipment

3/21/05 45.90

DigitalThermometer

Technika 3/21/05 22.96

Plastic Check

Valve, tubingclamps

US Plastics 4/02/2005 18.45

2nd

Check Valve,

with 3 more clamps

US Plastics 4/01/05 16.76

Aqua Epoxy Home Depot 4/12/05 3.79

TOTAL $195.52



18/19

WARNING!!!

FLOWING RIVER

DO NOT LEAVE UNATTENDED! CONTAINS MOVING LIQUID, CAN CAUSE DAMAGE TO LAB

EQUIPMENT. MAINTAIN CONSTANT LIQUID LEVEL



19/19

References:

1 Chen J., Baer AE, Paik Py, Yan W, Setton LA. Matrix protein gene expression in intervertebral disc cells

subjected to altered osmolarity. Duke University. Biochemical and Biophysical Research Communications. 293

(2002) 932-938.2 Lieu DK, Pappone PA, Barakat AL. Differential membrane potential and ion current responses to different types of

shear stress in vascular endothelial cells. Dept of Mechanical and Aeronautical Engineering, University of California. Am J. Physiol. Cell Physiol. 2004 Jun; 286(6):C1367-75. Epub 2004 Feb 04.3

Lehoux S, Tedgui A. Signal Transduction of Mechanical Stresses in the Vascular Wall. INSERM U141. Paris,

France. 20044 Anonymous1. Dialysis. National Instittue of Health.

http://kidney.niddk.nih.gov/kudiseases/pubs/hemodialysis/index.htm5 Truskey, G., Yuan, F., & Katz, D. (2004). Transport Phenomena in Biological Systems. New Jersey: Pearson

Prentice Hall, 2004.6 Anonymous2. Reynold’s Number Calculator. Engineering Formulas.

http://www.efunda.com/formulae/fluids/calc_reynolds.cfm

river finalrep

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