An Undergraduate Lab (on-a-Chip): Probing Single Cell Mechanics

on a Microfluidic Platform

CHRISTOPHER MORAES,1,2 KRISTINE WYSS,1,2 EMMA BRISSON,2 BRYAN A. KEITH,2 YU SUN,1,2

and CRAIG A. SIMMONS1,2,3

1Department of Mechanical & Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, ON M5S 3G8,Canada; 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto,

ON M5S 3G9, Canada; and 3Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada

(Received 14 February 2010; accepted 4 May 2010; published online 14 May 2010)

Associate Editor Jin-Yu Shao oversaw the review of this article.

Abstract—The study of the mechanical behavior of cells is anactive area of academic research and as such, is an increas-ingly important component of biomedical engineering edu-cation. However, in delivering engineering courses, practicalexperience with experimental cell mechanics is often chal-lenging to provide for untrained undergraduate students, asthe lab work involved is expensive, delicate and usuallyrequires substantial experimental skill. This article reportsthe development of a novel lab experience for seniorundergraduate students, for which a microfabricated systemwas designed and constructed to conduct micropipetteaspiration of single cells. Application of the microfabricatedsystem to measure the elastic modulus of primary heart valvefibroblasts produced results comparable to those made with aconventional micropipette aspiration system. The relativesimplicity and affordability of the system made it accessibleto undergraduate students in a laboratory course, whojudged the lab as a strongly positive learning experience.

Keywords—Cellular biomechanics, Engineering education,

Micropipette aspiration, Microfabrication, Cell modulus.

INTRODUCTION

The internal mechanical structure of a cell iscomplex and dynamic. The cellular cytoskeleton con-sists of a network of protein filaments that mechani-cally support the cell membrane, and reorganizes inresponse to chemical signals,28 external mechanicalforces,24 and cell–cell contacts.4 Because the cytoskel-eton plays a role as a mechanotransducer,30 changes inorganization can result in changes in gene expression,

adhesion, secretion, and metabolic activity.14,21 Thesecytoskeletal changes alter the mechanical propertiesbetween cell phenotypes,6,13,25 at various stages of stemcell differentiation,27 and may lead to alterations innormal physiological behavior,10 indicating or eventriggering pathogenic responses. Hence, the ability toevaluate single cell biomechanics is an importantexperimental technique, and may provide greaterinsight into how cells receive and integrate regulatorysignals from the surrounding environment.

Evaluating the mechanical properties of a single cellcan be achieved through several methods. Experi-mental techniques such as atomic force microscopy,magnetic microbead rheometry, compressive testing,optical microbead manipulation, and optical stretchingare typically employed,7 but are expensive, experi-mentally complex and require extensive operatortraining before use. Micropipette aspiration (MA) is arelatively robust and simple technique capable ofevaluating the structural and mechanical response ofsingle cells.12,18 MA simply uses suction pressure topartially aspirate a cell into a small-diameter glasstube. Geometric constraints and the resulting defor-mation as a function of applied pressure can then beused with relevant analytical16,23 or computa-tional1,35,36 models to determine the mechanicalproperties of a single cell.

Modern engineering problems have no clear disci-plinary boundaries. Training engineers to address theseproblems requires an educational curriculum whichintegrates disciplinary knowledge and experience.22

Undergraduate courses in biomechanics are well-suitedto this task, as they can be designed to demonstrate theimportance and application of traditional mechanicalengineering principles to complex problems in physi-ology, rehabilitation, medicine, and tissue engineering.Despite the importance of hands-on experimentation

Address correspondence to Craig A. Simmons, Department

of Mechanical & Industrial Engineering, University of Toronto,

5 King’s College Road, Toronto, ON M5S 3G8, Canada. Electronic

mail: simmons@mie.utoronto.ca, c.simmons@utoronto.ca

Cellular and Molecular Bioengineering, Vol. 3, No. 3, September 2010 (� 2010) pp. 319–330

DOI: 10.1007/s12195-010-0124-0

1865-5025/10/0900-0319/0 � 2010 Biomedical Engineering Society

319

in engineering education,8 such courses often lack apractical, experiential component for studies in bio-mechanics at the cellular scale. This is primarily due tothe delicate, challenging, and expensive nature of thework involved, making them impractical for a rela-tively short educational experience. Even in a simpleMA system, students would first be required to handleextremely fragile glass micropipettes; and second,skillfully operate an expensive micromanipulator,which would require extensive user training.

To address these issues and meet the need for apractical component in cellular biomechanics educa-tion, we have developed a simple and cost-effectivemicrofabricated system to conduct single cell MAexperiments. The integrated Micropipette Aspiratorchip (iMAchip) eliminates the need for students tohandle micropipettes, or access and operate a micro-manipulator. Using the iMAchip, we developed a cost-effective, practical and hands-on undergraduate labexperience for senior mechanical and biomedicalengineering students. The lab was delivered in the fallsemesters of 2008 and 2009 at the University ofToronto, and we have evaluated the impact of thisexperience by means of a student survey.

MATERIALS AND METHODS

Unless otherwise stated, all chemicals and reagentsfor cell culture were purchased from Sigma-Aldrich(Oakville, ON, Canada). Fluid connectors, valves, andadapters were purchased from Cole-Parmer Canada(Montreal, QC). Other equipment and materials wereobtained from Fisher Scientific Canada (Ottawa, ON).

iMAchip Overview

In order to improve the robustness and eliminate theneed for an expensive micromanipulator in an MAsetup, a microfabricated approach was developed(Fig. 1). The primary purpose of the microdevice is toput cells in close proximity to a fixed micropipette tip.Large pressure differentials applied to the micropipettecan then be used to attract nearby cells to the tip,where well-controlled pressures can then be used toaspirate cells into the pipette. Once a measurement ismade, the cell can be expelled from the micropipette;and the system flushed and reused.

The system was designed to flush a cell suspensionthrough a microfabricated U-shaped channel, 500 lmin width. A straight flowing channel could be used justas effectively as the shape of the channel is not a crit-ical design parameter for device operation. A secondchannel narrows to 20 lm in width and connects to

the apex of the U. The purpose of this channel is toallow a standard micropipette to access cells in theconfined space of the U channel. The micropipette ispermanently fixed into place within the channel, sothat the experimental system is robust, portable andself-contained.

Mold Master Fabrication

Fabrication of microchannels was conducted usingsoft lithography, in which a photoresist based ‘master’is used as a negative relief template for polymer cast-ing. To create the masters, SU-8 25 (Microchem;Newton, MA, USA) was spin-coated at 2000 RPMonto a 3¢¢ 9 2¢¢ glass slide. The glass slide was previ-ously coated with an unpatterned SU-8 seed layer toenhance adhesion between the microstructures and theglass. A masked pattern was printed on a transparencyfilm at 20,000 DPI (Cad/ART Services; Bandon, OR,USA). The master was soft-baked, patterned byselectively photo-crosslinking the resist by exposurethrough the mask, hard-baked and developed accord-ing to the manufacturer’s specifications. The resultingSU-8 features were measured on a Wyko optical sur-face profilometer (Veeco Instruments Inc.; Woodbury,NY, USA) and found to be 30–35 lm thick. The masters

FIGURE 1. (a) Schematic side-view of the device, demon-strating fabrication and operation of the iMAchip device. Themicropipette is manipulated into the PDMS device, andepoxied into place on the Petri dish. The slip-mountedmicromanipulator fitting is withdrawn, and the device isprimed, loaded with a cell suspension, and connected to apressure source for cell aspiration. (b) Fabricated iMAchipsystem (pictured with capillary syringe used to prime themicropipette with deionized water prior to use; colored dyeused for clarity).

MORAES et al.320

were then treated with a silanization agent, (Trideca-fluoro-1,1,2,2-tetra-hydrooctyl)-1-trichlorosilane (Uni-ted Chemical Technologies, Bristol, PA, USA), undervacuum conditions, to facilitate mold-release of thepatterned elastomeric polymer slabs.

iMAchip Fabrication

Sylgard 184 poly(dimethylsiloxane) kits (PDMS;Dow-Corning, purchased through A.E. Blake SalesLtd.; Toronto, ON, Canada) were obtained and themonomer and crosslinker reagents were mixed in thestandard 10:1 ratio. The uncured polymer was pouredover the microfabricated master, and placed undervacuum to remove any trapped air bubbles. PDMSwas cured in an oven for at least 4 h at 80 �C. Theresulting patterned PDMS slabs were then removed,peeled away from the master, and trimmed to size.A 1/8¢¢ punch was used to core out the fluid inlet andoutlet ports. The patterned PDMS surface and a3¢¢ 9 1.5¢¢ glass slide were treated in an oxygen plasmacorona for 30 s each, generated by a hand-held coronadischarge unit (Electrotechnic Products; Chicago, IL,USA). The substrates were then placed in conformalcontact such that the base of the Y-channel was withinmillimeters of the glass edge. The device was heated ona hotplate at 80 �C for 20 min to permanently bondthe two materials (Fig. 1).11 A 0.2% w/v solution ofPluronics F127 surfactant was then flushed into thechannel, to minimize adhesion between the channelsurfaces and the cell samples. The Pluronics solutionwas removed and the device channels were flushed withdeionized water twice, before allowing them to dry atroom temperature.

A 1/8¢¢ diameter hole was drilled into the base of a160 mm Petri dish approximately 2 inches from theedge of the dish. A quick-drying epoxy was used tobond the device to the Petri dish, such that the nexus ofthe Y-channel was centered over the hole in the Petridish.

Micropipettes of varying diameters were preparedfrom glass capillaries using a commercial pipette puller(Model 700C, David Kopf Instruments; Tujunga, CA,USA), and the tips were cut using a De Fonbrunemicroforge (Warner Instruments; Hamden, CT, USA).Pipettes with tips having an inner diameter between 6and 10 lm were selected for use in the iMAchip setup.In order to reduce non-specific adhesion of the cells tothe micropipette during device operation, Sigmacotewas applied to the micropipettes prior to use. Micro-pipette tips were dipped into a vial of Sigmacote for1–2 s and removed. Air was then pushed through themicropipette to clear the tip of any excess solution.Pipettes were allowed to dry for several hours in afumehood before use.

For each iMAchip manufactured, a micropipettewas manipulated and fixed within the channel, as perthe schematic in Fig. 1a. A selected micropipette wasmounted in a micromanipulator (Siskiyou; MissionViejo, CA, USA), on a slip-fit tube mount alignedparallel to the microscope stage (Olympus IX71;Olympus Microscopes; Markham, ON, Canada), towhich the Petri dish containing the fixed PDMS-glassmicrochannels was clamped. The micropipette wascarefully manipulated into the base arm of theY-channel until the pipette tip protruded ~20 lm intothe main channel. Epoxy was used to bond themicropipette into the base of the Y-channel, and thesystem was left untouched for 20 min while the epoxyhardened. The slip-fit tube mount was then withdrawn,leaving the micropipette embedded in the microchan-nel base. A section of Intramedic polyethylene tubing(Clay Adams PE190) was used as a step adapterbetween the micropipette and a 1/16¢¢ ID PVC tube,which was in turn connected via a barbed adapter to afemale luer lock port. Epoxy was applied to the con-nections to ensure there was no leakage. The com-pleted iMAchip was then stored before use.

Immediately prior to running the aspiration exper-iments, the channels were primed with liquid media.To do this, a long capillary (MF34G-5 MicroFil cap-illaries; World Precision Instruments; Sarasota, FL,USA) was used to inject deionized water into themicropipette via the luer port connection (Fig. 1b).The capillary is carefully inserted as far downthe pipette as possible, and deionized water isinjected slowly as the capillary is drawn out of themicropipette.

Pressure Source Peripherals

Two ranges of fluid pressures are required to oper-ate the iMAchip. In order to apply well-regulated,small changes in pressure for cell aspiration, the heightof liquid in a fixed reservoir (at atmospheric pressure)was adjusted. A ruler was clamped vertically in a retortstand beside the microscope stage. The open reservoir,which consisted of a 60 mL syringe with the plungerremoved, was mounted directly to the ruler usingelastic bands, at approximately the level of themicroscope stage. During the experiment, the level offluid was adjusted by carefully adding to or removingwater from the reservoir.

In order to attract cells toward the pipette tip andclear the pipette of aspirated cells, larger pressures arerequired. These are manually applied using a closed,filled syringe. Switching between these two pressuresources required a three-way valve with luer attach-ments. The three-way valve was connected to the openfluid reservoir, a closed 5 mL syringe and to the

Micropipette Aspiration on-a-Chip 321

iMAchip device, via tubing and a male luer-to-barbconnector (Fig. 2).

Immediately prior to the experiment, the pressuresources were filled with water. Approximately 40 mLof water was dispensed into the reservoir. The valvewas set to isolate the iMAchip from the reservoir andsyringe, and water was drawn into the syringe. Thevalve was then used to isolate the syringe, and waterwas allowed to fill the tubing between the iMAchip andthe reservoir. Once filled, the tubing was connected tothe primed iMAchip device, using a droplet mergingtechnique to prevent air bubbles being trapped in thepressure line.33 Briefly, a small excess of liquid wasadded to the primed iMAchip device, such that adroplet bulged over the luer-lock connector edge.Liquid in the tubing was controlled to form a similardroplet at the connector. The two droplets weremerged, and the connectors locked together, eliminat-ing air bubble trapping during this tube connectionprocess.

Cell Culture

Primary porcine aortic valvular interstitial cells(PAVICs) were isolated by enzymatic digestion aspreviously described,32 expanded in Dulbecco’s Mod-ified Eagle Medium (DMEM) supplemented with 10%fetal bovine serum, and frozen at passage five, prior touse. Immediately prior to running the lab session,frozen cells were thawed, mixed with 10 mL ofDMEM, centrifuged and resuspended in 1 mL of freshsupplemented DMEM. Cell suspensions were storedon ice during the lab, and replaced with freshly thawedcells every 45 min.

Experimental Procedures

At the beginning of the experimental session,primed and filled iMAchip devices were placed on themicroscope stage, and connected to the pressure source

as previously described. The microscope and cameraimages were first calibrated to a standard length scal-ing factor, using a hemacytomer (Fisher ScientificCanada; Ottawa, ON).

Cells were flushed into the large channels byapplying a small pressure via a standard Pasteur pip-ette bulb.20 Ten microliters of well mixed cell suspen-sion were pipetted into the inlet port of the device, anda pipette bulb was placed around the port. Gentlysqueezing the pipette bulb resulted in a pressure dif-ferential across the channel, and the cell suspensionwas flushed into the channel (Fig. 3). 7–8 lL of cellsuspension was then deposited into the outlet port, tominimize the pressure differential created due to theheight difference of fluid in the ports, so that the sys-tem reached equilibrium more quickly.

When the flow of cells within the microchannel hadsubsided, the equipment was ready for aspirationexperiments. The water reservoir was adjusted suchthat the water level was a few millimeters below theexperimental equipment. This height difference wasrecorded as the ‘tare pressure height’, and serves toapply a small suction pressure to the cells, forming aseal between the cell and the micropipette rim. Thevalve system on the pressure sources was then adjustedto isolate the reservoir, such that the syringe-basedpressure source could be used to attract and repel cellsfrom the micropipette tip. This required some experi-mental skill and practice, but was achievable by stu-dents in a lab session. A steady application of pressurewas used to draw cells close to the pipette tip. Imme-diately on contact, the pressure source was switched tothe reservoir system, where the preset tare pressureformed a seal between the cell and the pipette tip. After60 s, an image was taken of the cell, slightly aspiratedinto the pipette. Liquid was then removed from thereservoir with a pipette, creating larger suction pres-sures at the micropipette tip. The height differencebetween the water level and the microscope stage wasagain recorded as the ‘aspiration pressure height’.

FIGURE 2. (a) Schematic diagram of pressure system. The three-way valve is used to switch between applying large, uncontrolledpressures via the syringe, and applying small well controlled pressures via the liquid column in the reservoir. Adding or removingliquid to the reservoir changes the pressure applied. (b) Experimental setup for iMAchip peripherals (green dye used for thisdemonstration).

MORAES et al.322

Cells were allowed to deform for at least 60 s, untilthey reach their equilibrium aspiration length, before apicture was taken.

Analytical Model for Micropipette Aspiration

The most common mechanical model for MA, thehalf-space model, approximates the cell as an incom-pressible elastic half-space, where aspiration length isproportional to the applied suction pressure and in-versely proportional to the elastic modulus.26 Despite anumber of simplifying assumptions, this half-spacemodel is widely used in current MA studies.3,15,26 Theelastic modulus is given as:

E ¼ 3Up � DP � Rp

2p � Lasp

where E is the Young’s modulus, Lasp is the differencebetween the tare and final aspiration lengths, Rp is thepipette radius, DP is the difference between the tareand final aspiration pressures, and Fp is a dimension-less constant dependent on the ratio between pipettewall thickness and pipette radius. The Lasp and Rp

parameters were measured directly from the images ofaspirated cells, using image analysis software (ImageJ;NIH). A schematic diagram of an aspirated cell(Fig. 4) is provided to indicate the required measure-ments. The value of Fp is taken as 2.1 for mostexperiments.26 DP is calculated using the equation:

DP ¼ q � g � Dh

where q is the density of the water column (1 g/mL), gis the acceleration due to gravity (9.8 m/s2), and Dh isthe difference between the tare and aspiration heightsof the liquid column.

EXPERIMENTAL RESULTS AND DISCUSSION

In order to validate the iMAchip setup, we repeateda characterization experiment previously performed byour group using a standard MA system,35 on primarysub-cultured PAVIC cells. MA measurements could beinfluenced by cell type, operating temperature, analysisprocedure, applied pressure levels, micropipette diam-eter, and micropipette wall chamfer radius,35 all of

FIGURE 3. Loading cell samples (green dye used for clarity) into the iMAchip devices. (a) Cell suspension is pipetted into themicrochannel access port. (b) A Pasteur pipette bulb is placed over the port and (c) gently squeezed, (d) driving fluid flow throughthe channel.

Micropipette Aspiration on-a-Chip 323

which were kept constant between the two studies.Aspiration measurements were made on eight separatePAVICs, using two iMAchip setups. Sample images ofa cell being aspirated are shown in Fig. 5. The results,shown in Fig. 5c, indicate a mean elastic modulus of927 ± 256 Pa (standard deviation). The values for cellmodulus obtained in this experiment were not signifi-cantly different from those reported by Zhao et al.35

(738 ± 222; p = 0.052 by t-test), who used a standardMA system and the half-space model. The slight dif-ferences observed could have been due to heterogeneityin the cell populations measured5; the use of passagedcells, which stiffens PAVICs35; or differing extent ofvalvular disease between the animals from whichthe cells were harvested. To date, no other grouphas conducted MA experiments on porcine valve

interstitial cells. However, Merryman et al., have usedthis technique to characterize ovine valve interstitialcells.17 Their results (346 ± 142 Pa) are significantlydifferent from our studies (p< 0.001 by t-test), likelydue to differences in the animal model, culture condi-tions, and experimental parameters, which can impactmodulus measurements.35

For this validation experiment, these data pointswere obtained by an operator familiar with using astandard micropipette aspiration system. Qualitatively,the measurements were made in approximately thesame amount of time required using a standard MAsetup (~7–8 min per cell). Untrained undergraduatestudents were not as quick in data collection. Due totheir limited time in the lab and the learning curvenecessary to understand and correctly operate theequipment, students were only able to generate datafor 1–2 cells per group. Though qualitatively similar tothe data collected in our validation studies, student-generated data is not reported here, as comparisonwith these limited datasets would be ineffective.

Besides eliminating the cost and complexity of themicromanipulator from an MA system, there are otheradvantages to this microfluidic platform. The operatorappreciated the fact that the exclusion of the micro-manipulator from this setup eliminated vibration ofthe pipette tip, which made it easier to form an ade-quate tare pressure seal around the cell for aspiration.The operator, previously inexperienced with micro-fluidics, was also able to quickly learn how to manip-ulate fluids within the system, using the Pasteur pipette

FIGURE 4. Cell aspiration schematic indicating key param-eters in the elastic half space analytical model.

FIGURE 5. (a, b) Sample images of cells being aspirated at a tare pressure and at a higher aspiration pressure. (c) Young’smodulus for eight individual single cells, calculated using the elastic half-space model. Mean modulus value represented as adotted line.

MORAES et al.324

bulb. In contrast with previous attempts to conductMA experiments in microfluidic channels with rectan-gular cross sections,19 the ability to use circular mic-ropipettes in this system was a distinct advantage, as itallowed the use of well-established analytical models toextract the stiffness of the cell from the MA parame-ters. For these experiments, we used the half-spacemodel to determine cell modulus, because it was cov-ered in the course curriculum and has been shown tobe reasonably accurate for this cell type.35 This doesnot preclude the use of other more complex models,12

or the use of a video capture camera to determinethe time-dependent deformation (viscoelasticity) ofsingle cells, by analytical or inverse finite elementapproaches.35 However, measurements of viscoelas-ticity will require careful interpretation, due to thepotentially confounding effects of the large initialsuction pressure used to attract cells to the pipette tip.This should not be a concern for the reported equi-librium measurements, as the final aspiration length isindependent of the rate of pressure application for thiscell type.35

Though it was not utilized or controlled for in thislab, the use of a microfluidic system could have addi-tional advantages in controlling temperature of thesystem. Because the liquid volumes in the channel aremuch smaller than those in standard MA systems, thetemperature of the system can be controlled quiteprecisely using a heated stage. Temperature has beenshown to play a role in cytoskeletal biomechanics,31

and though it was not controlled for in these experi-ments, could be an additional parameter to include inthis lab experience.

In designing the iMAchip system to be appropriatefor an undergraduate lab, certain design compromiseshad to be made. The iMAchip system is certainly notas versatile as a standard MA system in terms of thetypes of experiments that can be performed. Further-more, a key potential disadvantage is the possibility forcells to attach to and clog the micropipette, in spite ofthe chemical surface treatments applied to both thepipette and to the microchannels. In a traditional MAsystem, the micropipette would be quickly discardedand replaced with a fresh one, and this is not an optionin this microfluidic version. While chemically cleaningthe channels is relatively simple, it does take time, andmay impact workflow, especially in a time-limitedundergraduate lab situation. We made sure to producea large number of extra devices, so that equipmentcould quickly be swapped out in this instance, and wefound that keeping the cells on ice and replacing themevery 45 min helped address this issue. We also foundthat using a cell line, such as the L929 or 3T3 fibro-blasts substantially reduced unwanted cell adhesion,and we will use these cell lines for future labs.

IMPLEMENTATION IN AN UNDERGRADUATE

LAB

Course Background

The lab has been incorporated for the past two yearsinto the curriculum of MIE439, a single-semestersenior course in Biomechanics, offered by the Depart-ment of Mechanical & Industrial Engineering at theUniversity of Toronto. The course is a capstone designcourse for undergraduate students in the BiomedicalEngineering Option in the Division of EngineeringScience, and for those in the bioengineering streams ofthe Mechanical and Chemical Engineering programs.The course is primarily focused on applying principlesof mechanical engineering, including solid and fluidmechanics, to living systems, and covers topics rangingfrom cellular and tissue biomechanics to human gaitand locomotion. Course enrollment ranges from 45 to80 undergraduate students. Course activities includethree hours of lecture per week, biweekly tutorials,three lab sessions, and a capstone group design project.On-going assessment of student learning is conductedusing homework assignments, lab reports, surveys andformative feedback during lectures and tutorials, and amid-term exam. A final exam and a written technicalreport and presentation complete the course evaluativeprocedures. Though there are no formal prerequisites,the nature of the course requires students to have abasic understanding of solid and fluid mechanics,including linear stress–strain models and viscoelastic-ity. These concepts are reviewed in lecture, whilesimultaneously introducing various models of cellularmechanics.7 The integrative synthesis of these inter-disciplinary concepts is what makes this course uniqueand appealing, and hence is an ideal environment inwhich to conduct this micropipette aspiration lab.

Laboratory Logistics and Procedures

The laboratory was conducted in the Undergradu-ate Teaching Laboratory facilities, maintained by theInstitute of Biomaterials and Biomedical Engineering(IBBME) at the University of Toronto. The teachinglab is designated Biosafety Level 2 (although this lab-oratory required only Biosafety Level 1), and isequipped with basic tools for sterile cell culture, and 12microscopy stations which were used extensively dur-ing this lab. Each microscopy station consists of aphase contrast microscope equipped with three airobjectives (109, 209, and 409), a movable stage,digital video camera and a data collection computer,running basic image acquisition and analysis software.

Prior to lab day, students formed groups and signedup for a lab session. The finite capacities of the

Micropipette Aspiration on-a-Chip 325

teaching lab and practical considerations in coursescheduling were limiting factors in the number of labsessions we were able to conduct. In the first year ofthis lab, we ran three lab sessions, each one hour long.In order to address some of the concerns raised by thefirst group of students regarding the severely limitedtime available for the lab, two lab sessions, each twohours in length were arranged for the second year. Inboth years, group sizes were limited to four people,assigned to a single microscopy station.

During the week before the lab, students were pre-pared through lectures and distributed course material.Lectures covered concepts including cell mechanics,mechanical characterization techniques, and analysismethods. Traditional micropipette aspiration tech-niques were outlined, and a half-space model used toanalyze micropipette aspiration data and extract cel-lular stiffness parameters was discussed in detail andincluded as part of a problem set assignment. A labmanual was also provided to the students (availableonline at www.introductorybiomechanics.com) inorder to familiarize them with the new microfluidictechniques developed in this lab to facilitate micropi-pette aspiration, and to prepare them for the upcomingexperiment. In addition to the experimental proceduresprovided in the lab manual, appendices detailing imageacquisition and analysis procedures were provided.Simultaneously, iMAchip experimental setups werebeing prepared and tested by a teaching assistant. Cellswere also expanded and frozen to ensure sufficientquantities for student use.

On the day of the lab, each microscope station wasequipped with a primed iMAchip device and completepressure setup. The lab manager, course instructor andteaching assistant were all present for the full durationof both lab sessions. Their roles included assistingstudents in equipment setup, solving technical prob-lems, providing basic background knowledge andinformation, monitoring student progress, providingformative feedback, and ensuring that safe lab prac-tices were being followed. Following a brief introduc-tory talk in which key points of the lab equipment andprocedure were highlighted, students were providedwith thawed and resuspended cell solutions, and beganwork on the provided equipment. During the lab,students relied on help from the staff, notes taken inlecture and provided in the lab manual, and assistancefrom each other, depending on their individual expe-riences and backgrounds in either mechanical systemsor cell handling techniques.

After completing the lab, students were expected toanalyze the data collected in their groups, using imageanalysis software. Sample images collected by theteaching assistant were also made accessible, for thosestudents who were unable to obtain usable data during

the limited time available for the experiment. Studentswere also asked to answer three post-lab discussionquestions, designed to encourage reflection on the labexperience. In answering these questions, students wereexpected to identify and evaluate the sources of errorpresent in this microfluidic system, and compare theirimpact with traditional micropipette aspiration sys-tems. For example, cells flowing in the microfluidicchannels can be at non-atmospheric pressures at thetime of aspiration, which can impact pressure differ-entials and hence, aspiration lengths. Students wereasked to quantify the impact of this effect and fur-thermore, to design methods and techniques to mini-mize this source of error. Likewise, students wereasked to re-examine the experimental design to addresssome of other challenges and problems they encoun-tered while performing the experiments. As these labswere considered ‘experimental’ (in the ‘new’ as well asin the ‘practical’ sense), no formal lab reports wererequired, but students were all made aware that thematerials covered would be included as a componentof the final exam.

Material costs for this lab were relatively small,given access to appropriate teaching laboratories, softlithography facilities, and a pipette puller and micro-forge. Given the availability of these facilities, materialcosts for device fabrication and cell culture for 20devices and pressure setups totaled approximatelyCAD $300. If a pipette puller and forge are unavail-able, commercially available micropipettes can beused, but add ~$10 to the cost of each device. Oncethe initial design, development and characterizationiterations were complete, a total of 20 h were requiredon the part of the teaching assistant to prepare for theactual operation of the lab. These preparations pri-marily covered fabrication of a batch of 20 devices;aspiration system setup; and cell culture and handling.Barring catastrophic failure of the devices, they can bereused for future labs by carefully flushing the chan-nels and micropipette with a mild acid and rinsingthoroughly with deionized water. Minor repairs to theepoxy sealants may also need to be conducted witheach use.

Laboratory Pedagogy

This lab was designed to fulfill several learningobjectives. The primary course-based objectives wereto reinforce the concepts of cell mechanics andmicropipette aspiration delivered in lecture. Thoughcell mechanics comprises approximately 25% of thecourse material, these topics were only presented asabstract concepts in lectures without a related hands-on learning experience. The lab experience describedhere was designed to complement the lecture material,

MORAES et al.326

and provide a visual, sensory experience of thesechallenging abstract concepts for those students withalternative learning style preferences.9

In addition to building physical intuition for thecourse material, the lab was more broadly designed toallow students to experience the uncertainties ofexperimental research. Hence, students were explicitlytold about the experimental nature of this lab: usingnovel techniques can often result in unexpected chal-lenges that the experimentalist needs to solve. Framingthe exercise in this way resulted in some interestinginnovations developed by the students themselves. Forexample, one of the challenges with physically loweringthe water reservoir to apply small changes in pressureis that this can generate pressure waves, unless doneextremely carefully and smoothly. The students iden-tified a solution in which water was gradually removedfrom the reservoir with a pipette, achieving the sameeffect and minimizing experimental skill required. Webelieve that the process of experiencing, identifyingand solving these practical problems is an absolutelyessential learning experience for budding researchers.Furthermore, this approach forces students to developan experimental approach to learning: overcoming thenatural fears involved with trying different things whenthe outcome is not necessarily assured is challengingand requires these guided, yet flexible, student-definedexperiences. Because of the team-based nature of thelab, this teaching approach also encouraged studentsto interact with each other and utilize the team mem-bers’ individual experiences to solve experimentalissues. This provided an opportunity for students toengage with each other and develop collaborative skillsfor future team-based projects.

Aside from essential details included in the labmanual, students were not explicitly taught about themicrofabrication aspect of this lab, as this was not thefocus of this course. However, such technologies arequickly growing in importance within biomedicalengineering, and one of the key benefits of conductingthis lab was to allow students to observe and experiencehow novel microfabricated tools can be designed toaddress questions and solve problems in the biologicalsciences. By providing a clear example of the integrativenature of biomedical engineering and other associatedcourses of study, we hope to motivate interest in thisemerging interdisciplinary field. To this end, thislearning experience was further augmented by invitingguest lecturers with expertise in both cellular biome-chanics and microfabrication, and through an addi-tional practical lab that utilizes microfluidic channels torecreate and explore the Fahraeus–Lindqvist effect, anon-intuitive microscale-related change in hematocritthat occurs when blood cells flow in small-diametervessels like arterioles, venules, and capillaries.34

Following the lab, student requirements were lim-ited to analyzing data and answering questions in thelab manual, without the requirement for a formal labreport. This was to allow students to focus onlearning the core physical and experimental concepts,without being concerned about a mark for a writtendocument.2,29 In order to ensure student learning,however, the answers to questions in the lab manualwere not explicitly provided, and a final exam ques-tion based on the lab experience was included. As aresult of this approach, the course staff noticed aquantitative and qualitative improvement in staff–student interaction, as students tried to prepare forthe exam by actively thinking about and questioningthe lab material.

Student Feedback and Discussion

Feedback was requested from students in the Fall2009 course, in the form of a voluntary in-classsurvey conducted immediately after the lab session.Eighty-seven percent of students enrolled in the classresponded, and the categorical survey results aresummarized in Fig. 6. Feedback was generally verypositive, with 73% of the students moderately orstrongly agreeing that the lab was beneficial in rein-forcing the concepts discussed in lecture. Oddly en-ough, more than 90% of the students agreed thatthey had a better understanding of the detailsinvolved in performing measurements of cellmechanics, which suggests that certain aspects of thelecture material were more immediately and stronglyreinforced than others. This might be a result ofconducting the survey immediately after the lab,when students may not have had the time to analyzethe collected data or contemplate the discussionquestions included in the lab manual. Even at thisearly stage, however, at least 84% of the students feltthat this was a fitting and useful component of abiomechanics course curriculum, and were enthusi-astic about the experience.

Comments were also solicited with the survey, andthese were also generally positive. Students particularlyliked the ‘‘translation from lecture to practice’’, andthe fact that the ‘‘we were expected to perform theexperiments ourselves, not as a bystander watching theTA’’. More than 85% of survey respondents agreedthat the lab was a fun and practical hands-on learningexperience. Other appreciative comments included amajority of observations that the lab was ‘‘not plug-and-play’’, and an ‘‘individual research experience foreach group’’. The majority of students also appreciatedthe chance to ‘‘look at real cells, and see how theybehave mechanically’’. Interestingly, a small section ofthe class made thematically identical comments and

Micropipette Aspiration on-a-Chip 327

considered these negative experiences: ‘‘looking at realcells is frustrating as they die easily’’, and that ‘‘the labmanual was unclear about procedures’’. One studentbest summarized this dichotomy with the comment:‘‘Liked: to get to know what it is like to do realexperimental work. Disliked: that it is sometimes dis-appointing’’. A number of students also appreciatedthe opportunity to work with ‘‘cutting-edge’’, ‘‘newlydeveloped’’ microdevices to do this work, and amajority of students were more interested in micro-fabricated technologies for life science studies after thisexercise.

A large number of survey respondents were enthu-siastic about the lack of a formal report, giving them‘‘no pressure to perform, and freedom to experimentand learn’’ with the provided systems. Students alsodid very well on the lab-related question included inthe final exam, suggesting that this pedagogicalapproach works well for this group of students.However, no conclusions can be drawn as similarquestions were not asked in previous years, and thedirect impact of this exercise on student performancecannot be assessed.

Criticism for the lab focused mainly around thelogistics of running the lab, and certain technicaldetails as to the experiment. Most of these negativecomments concerned the need for more space in thelab, as areas around each microscope station werefairly limited for four people. Many students also feltthat the lab would better be conducted with two orthree students per group. In spite of the increased

two-hour length of the lab, several students felt thatmore time would have been very useful. In terms oftechnical details, students in the first lab session wereprimarily frustrated by pipettes being clogged by deador dying cells. These pipettes were difficult to clear in atimely manner, and hence prevented some studentsfrom collecting data within the lab session. This issuewas addressed for the second lab session, when thestaff began keeping the suspended cells on ice throughthe duration of the lab, and replenishing cells withfresh supplies every 45 min. This prevented cell deathin the channels and prevented pipette clogging. Othercriticisms were related to equipment issues: a mal-functioning camera, and a burnt-out microscope illu-mination lamp.

CONCLUSIONS

A microfabricated system was developed to conductmicropipette aspiration experiments to measure themechanical properties of individual cells. The simpleand affordable system was designed for use byundergraduate students as a practical component of acourse in biomechanics. As such, it eliminates the needfor students to access and operate expensive and deli-cate equipment, while obtaining a hands-on experiencein studies of cellular mechanics. Students applied thesystem to probe the stiffness of a primary cell popu-lation, and the results produced were consistentwith observations made with a conventional system.

FIGURE 6. Summary of student feedback collected from a voluntary in-class survey. Bars represent means 6 standard devia-tions for 61 student responses per question.

MORAES et al.328

The usefulness of the lab as a teaching tool was eval-uated by means of a student survey, which indicated astrongly positive learning experience.

ACKNOWLEDGMENTS

We acknowledge microfabrication support from theEmerging Communications Technology Institute andthe Toronto Microfluidics Foundry. We thank theDepartments of Mechanical and Industrial Engineer-ing; Electrical and Computer Engineering; and theInstitute of Biomaterials and Biomedical Engineeringat the University of Toronto for administrative andfinancial support. We acknowledge financial supportfrom the Ontario Graduate Scholarship program toCM; the University of Toronto Division of Engineer-ing Science and the Bioengineering Minor to EB; andthe Canada Research Chairs in Micro and NanoEngineering Systems to YS, and in Mechanobiology toCAS.

REFERENCES

1Baaijens, F. P., W. R. Trickey, T. A. Laursen, andF. Guilak. Large deformation finite element analysis ofmicropipette aspiration to determine the mechanicalproperties of the chondrocyte. Ann. Biomed. Eng. 33(4):494–501, 2005.2Bella, D. A. Plug and chug, cram and flush. J. Prof. IssuesEng. Educ. Pract. 129(1):32–39, 2003.3Byfield, F. J., H. Aranda-Espinoza, V. G. Romanenko,G. H. Rothblat, and I. Levitan. Cholesterol depletionincreases membrane stiffness of aortic endothelial cells.Biophys. J. 87(5):3336–3343, 2004.4Chen, C. S., J. Tan, and J. Tien. Mechanotransduction atcell–matrix and cell–cell contacts. Annu. Rev. Biomed. Eng.6:275–302, 2004.5Chen, J. H., C. Y. Yip, E. D. Sone, and C. A. Simmons.Identification and characterization of aortic valve mesen-chymal progenitor cells with robust osteogenic calcificationpotential. Am. J. Pathol. 174(3):1109–1119, 2009.6Costa, K. D. Single-cell elastography: probing for diseasewith the atomic force microscope. Dis. Markers 19(2–3):139–154, 2003.7Ethier, C. R., and C. A. Simmons. Introductory Biome-chanics: From Cells to Organisms. Cambridge: CambridgeUniversity Press, p. 511, 2007.8Feisel, L. D., and A. J. Rosa. The role of the laboratory inundergraduate engineering education. J. Eng. Educ.94(1):121–130, 2005.9Felder, R. M., and L. K. Silverman. Learning and teachingstyles in engineering-education. Eng. Educ. 78(7):674–681,1988.

10Guck, J., S. Schinkinger, B. Lincoln, F. Wottawah,S. Ebert, M. Romeyke, D. Lenz, H. M. Erickson,R. Ananthakrishnan, D. Mitchell, J. Kas, S. Ulvick, andC. Bilby. Optical deformability as an inherent cell marker

for testing malignant transformation and metastatic com-petence. Biophys. J. 88(5):3689–3698, 2005.

11Haubert, K., T. Drier, and D. Beebe. PDMS bonding bymeans of a portable, low-cost corona system. Lab Chip6(12):1548–1549, 2006.

12Hochmuth, R. M. Micropipette aspiration of living cells.J. Biomech. 33(1):15–22, 2000.

13Huang, H. D., R. D. Kamm, and R. T. Lee. Cell mechanicsand mechanotransduction: pathways, probes, and physi-ology. Am. J. Physiol. Cell Physiol. 287(1):C1–C11, 2004.

14Ingber, D. E. Cellular mechanotransduction: putting all thepieces together again. FASEB J. 20(7):811–827, 2006.

15Jones, W. R., H. P. Ting-Beall, G. M. Lee, S. S. Kelley,R. M. Hochmuth, and F. Guilak. Alterations in theYoung’s modulus and volumetric properties of chondro-cytes isolated from normal and osteoarthritic humancartilage. J. Biomech. 32(2):119–127, 1999.

16Merryman, W. D., P. D. Bieniek, F. Guilak, and M. S.Sacks. Viscoelastic properties of the aortic valve interstitialcell. J. Biomech. Eng. 131(4):041005, 2009.

17Merryman, W. D., I. Youn, H. D. Lukoff, P. M. Krueger,F. Guilak, R. A. Hopkins, and M. S. Sacks. Correlationbetween heart valve interstitial cell stiffness and transval-vular pressure: implications for collagen biosynthesis. Am.J. Physiol. Heart Circ. Physiol. 290(1):H224–H231, 2006.

18Mitchison, J. M., and M. M. Swann. The mechanicalproperties of the cell surface. 1. The cell elastimeter. J. Exp.Biol. 31(3):443, 1954.

19Moraes, C., J. H. Tong, X. Y. Liu, C. A. Simmons, andY. Sun. Parallel micropipette aspirator arrays for high-throughput mechanical characterization of biological cells.In: Proceedings of the Micro Total Analysis SystemsConference, 2007, pp. 751–753.

20Moraes, C., Y. Sun, and C. A. Simmons. Connector-lessmanipulation of small liquid volumes in microchannels.Chips Tips (Lab on a Chip) 2010.

21Orr, A. W., B. P. Helmke, B. R. Blackman, and M. A.Schwartz. Mechanisms of mechanotransduction. Dev. Cell10(1):11–20, 2006.

22Rugarcia, A., R. M. Felder, D. R. Woods, and J. E. Stice.The future of engineering education I. A vision for a newcentury. Chem. Eng. Educ. 34(1):16–25, 2000.

23Sato, M., D. P. Theret, L. T. Wheeler, N. Ohshima, andR. M. Nerem. Application of the micropipette technique tothe measurement of cultured porcine aortic endothelial cellviscoelastic properties. J. Biomech. Eng. 112(3):263–268,1990.

24Smith, P. G., L. H. Deng, J. J. Fredberg, and G. N.Maksym. Mechanical strain increases cell stiffness throughcytoskeletal filament reorganization. Am. J. Physiol. LungCell. Mol. Physiol. 285(2):L456–L463, 2003.

25Suresh, S., J. Spatz, J. P. Mills, A. Micoulet, M. Dao, C. T.Lim, M. Beil, and T. Seufferlein. Connections betweensingle-cell biomechanics and human disease states: gastro-intestinal cancer and malaria. Acta Biomater. 1(1):15–30,2005.

26Theret, D. P., M. J. Levesque, M. Sato, R. M. Nerem, andL. T. Wheeler. The application of a homogeneous half-space model in the analysis of endothelial-cell micropipettemeasurements. J. Biomech. Eng. Trans. ASME 110(3):190–199, 1988.

27Titushkin, I., and M. Cho. Modulation of cellularmechanics during osteogenic differentiation of humanmesenchymal stem cells. Biophys. J. 93(10):3693–3702,2007.

Micropipette Aspiration on-a-Chip 329

28Trickey, W. R., T. P. Vail, and F. Guilak. The role of thecytoskeleton in the viscoelastic properties of human artic-ular chondrocytes. J. Orthop. Res. 22(1):131–139, 2004.

29Truax, D. D. Restructuring the undergraduate laboratoryinstructional process. J. Prof. Issues Eng. Educ. Pract.133(3):192–198, 2007.

30Wang, J. H., and B. P. Thampatty. An introductory reviewof cell mechanobiology. Biomech. Model. Mechanobiol.5(1):1–16, 2006.

31Waugh, R., and E. A. Evans. Thermoelasticity of red bloodcell membrane. Biophys. J. 26(1):115–131, 1979.

32Yip, C. Y., J. H. Chen, R. Zhao, and C. A. Simmons.Calcification by valve interstitial cells is regulated by thestiffness of the extracellular matrix. Arterioscler. Thromb.Vasc. Biol. 29(6):936–942, 2009.

33Young, E. W. K., A. R. Wheeler, and C. A. Simmons.Avoiding bubble injection by droplet merging. Chips Tips(Lab on a Chip) 2006.

34Young, E. W. K., and C. A. Simmons. ‘‘Student Lab’’-on-a-chip: integrating low-cost microfluidics into undergrad-uate teaching labs to study multiphase flow phenomena insmall vessels. Chem. Eng. Educ. 43(3):232–240, 2009.

35Zhao, R., K. Wyss, and C. A. Simmons. Comparison ofanalytical and inverse finite element approaches to esti-mate cell viscoelastic properties by micropipette aspiration.J. Biomech. 42(16):2768–2773, 2009.

36Zhou, E. H., C. T. Lim, and S. T. Quek. Finite elementsimulation of the micropipette aspiration of a living cellundergoing large viscoelastic deformation. Mech. Adv.Mater. Struct. 12(6):501–512, 2005.

MORAES et al.330