Biomedical Technologies: Microfluidic systems for three

June 25, 2014

Biomedical Technologies: Microfluidic gsystems for three-dimensional cell culture

and microenvironment

Karen C Cheung, PhDAssociate Professor

Department of Electrical and Computer EngineeringThe University of British Columbia

Vancouver, BC, Canada

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UBC ECE Research Day, Applied Materials, June 25, 2014

Biomedical Technologies: Convergence of Disciplines

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source: H. Pielartzik, Bayer MaterialScience AG, Leverkusen


Biomedical Microdevices (BioMEMS) fabrication

BioMEMS: in addition:Micro Electro Mechanical Systems BioMEMS: in addition:Nontraditional substrates

glass, polymers

Molding of polymers

Micro Electro Mechanical Systems(MEMS):semiconductor processing technologies Molding of polymers

hot embossing; injection moldingsoft lithography

Materials surface modification to

technologies Conventional: Thin film deposition, etching, photolithography, etc.New: Deep RIE, Thick Plating, electro Materials surface modification to

increase biocompatibilitydischarge machining, …

Form structures used in sensors and actuators

accelerometerProf. Edmond Cretu

microelectrodesProf. Ken Takahata

fiber endoscopes for in-vivo imagingProf. Shuo Tang

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Rapid prototyping infrastructure at UBCEmbedded Systems Canada (emSYSCAN): 5-year project worthEmbedded Systems Canada (emSYSCAN): 5-year project worth over $50 million; provides platform-based microsystems design and prototyping environments. Shared Infrastructure in Canada’s National Design Network.

Prof. Edmond Cretu

aerosol jet printeraerosol jet printer

In addition:3D printerSonoplot

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maskless lithographySonoplotLaser micromachining …


BioMEMS: Applications

BASHIR R (2004) BioMEMS: state-of-the-art in detection opportunities and

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BASHIR, R. (2004) BioMEMS: state of the art in detection, opportunities and prospects. Advanced Drug Delivery Reviews, 56, 1565-1586.


Biomedical Microdevices at UBC: drug delivery systems

Arrays of hollowArrays of hollow out-of-plane microneedles for vaccine injectionProf. Boris Stoeber

Stoeber et al 2013 J. Micromech. Microeng. 23 085011

Drug deliveryDrug delivery battery-less device to treat diabetes-

l t d i i lrelated vision lossProf. Mu Chiao

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Chiao et al Lab Chip, 2011,11, 2744-2752


Drug discovery and development pipeline

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A virtual space odyssey, Cath O'Driscoll, 2004.


Microfluidic systems for cell-based anticancer drug screening

Aim: 3-D cell culture for drug screeningAim: 3 D cell culture for drug screeningApproach:• microfluidic generation of cell-laden hydrogel beadsg y g• hydrogel supports three-dimensional cell cultureCollaboration:• C. Roskelley, M. Bally, BC Cancer Research Centre

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Motivation: hydrogel-based cell culturemicroenvironment affects cell phenotype, p yp ,proliferation, differentiation, and migration.

Even small spheroids (containing about 25‐50 cells) are more resistant to killing than

2-D cell culture50 cells) are more resistant to killing than monolayers.

~ 105 – 106 cellsvolumes: ~ 100s μLvolumes: ~ 100s μL

3 D ti

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3-D tissueP Olive and R Durand, Cancer and Metastasis Reviews, 1994


On-chip image-based drug screeningMulticellular drug resistanceMulticellular drug resistance

bead in microchannelbead in culture flaskmonolayer in culture flask

Fluorescence assay based on live/dead cell viability stain.

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L. Yu, MC Chen, KC Cheung, Lab on a Chip, 2010


One step process: Core/Shell beads (collagen, Matrigel, alginate)

10 days

Core: tumor cells in collagen and Matrigelproteins provide cues from natural extracellular

each bead ~ 8 nL volume

- proteins provide cues from natural extracellular environment; hydrogel scaffold supports cells- cells proliferate, uniformly sized spheroids

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L. Yu, C. Bayly, KC Cheung, MicroTAS 2013Shell: alginate


Two-photon microscopy and second harmonic generation

T h t iTwo-photon microscopy to visualize embedded cells.

breast epithelial cells expressing FUCCI cell cycle reporters (Sakaue-Sawano et al., Cell, 2008, , , ,132(3):487.)

Second harmonic generation signal g gvisualizes the collagen fibers.

imaged 4 days post-encapsulation.

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Microfluidic design controls oxygen environment; O2 sensorMost solid tumorsMost solid tumors contain regions of hypoxia.

blood vessel

Gradient of O2 in tissue.

Intermittent hypoxia.

low cell proliferation; hypoxia

O. Trédan et al., J Natl Cancer Inst, 2007

Intermittent hypoxia.

0 0 2 t 200 μm

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S. Grist, L. Yu, C. Bayly, KC Cheung, CMBES 20140 0.2 atm 200 μm


Model of intermittent hypoxia and re-perfusion

1.5

2

(%)

0 5

1

Oxy

gen

leve

l

5 mm

Alginate 200 400 6000

0.5O

Alginate shell Time (minutes)

Gaseous oxygen levels measured in situusing thin-film optical sensors.

100 μmBead trapCell-laden core bead

Model of intermittent hypoxia in tumours; re-perfusion and re-oxygenation after stroke.

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S. Grist, L. Yu, C. Bayly, KC Cheung, CMBES 2014100 μmBead trap core bead


3D culture and drug response

Effects of normoxic vs. hypoxic culture conditions on dose response.

Effects of 2D vs 3D culture on dose response.

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Inkjet patterning of living cells for tissue engineeringAim: precise patterning of living cellsAim: precise patterning of living cells

on substratesApproach:• inkjet dispensing• piezoelectric nozzle, drop-on-

demanddemand• hydrogels as tissue scaffoldCollaboration:Collaboration:• E. Cretu, B. Stoeber,

K. Walus

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S. Parsa, M. Gupta, F. Loizeau, K. Cheung, Biofabrication, 2010.


Inkjet patterning of living cells

500 μm

Challenges:

> 95% cell viability, 48 hrs after printing

• cell aggregation

• nozzle clogging

• poor reliability of printing precise numberspoor reliability of printing precise numbers of cells over long printing periods (hours)

S Parsa M Gupta F Loizeau K Cheung Biofabrication 2010

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S. Parsa, M. Gupta, F. Loizeau, K. Cheung, Biofabrication, 2010.D. Chahal, A. Ahmadi, KC Cheung, Biotechnology and Bioengineering, 2012.


Droplet break-off and cell behaviour: cell travel

high speed imaging:23,121 frames/second

refractive index matchedrefractive index matched holder

ll t l l tcell travels closer to nozzle orifice as expected

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E. Cheng, A. Ahmadi, KC Cheung, CMBES 2014


Droplet break-off and cell behaviour: cell reflection

high speed imaging:23,121 frames/second

refractive index matchedrefractive index matched holder

ll i fl t d f thcell is reflected further back into the nozzle after dispensing drop

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E. Cheng, A. Ahmadi, KC Cheung, CMBES 2014


Particle Image Velocimetry (PIV) study of droplet formationVelocity field at time = 10 μs shows the ejection of the fluid within the nozzle at the beginning of the droplet ejection cycle.

Equivalent bright-field images of the two frames to show the meniscus evolution during the time in which the PIV images were taken.

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E. Cheng, A. Ahmadi, KC Cheung, FEDSM 2014


Summary and Future Work

Microfluidically generated cell laden beads:Microfluidically generated cell-laden beads: 3-D culture environment supports spheroid formation.beads include elements of extracellular matrix.b d h ll t t i l t 3D ltbead core-shell structure improves long-term 3D culture.

Inkjet printing of living cells for tissue engineering: U P ti l I V l i t (PIV) t d t d flUse Particle Image Velocimetry (PIV) to understand flow field inside inkjet nozzle.Strategies for reducing cell reflection and ensure consistent dispensing of cellsdispensing of cells.

Future work: optimize imaging for automated image-based drug-optimize imaging for automated image based drugscreening.Integrated oxygen control and optical oxygen sensors to create and monitor cyclic hypoxic conditions.

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AcknowledgmentNSERCNSERCCIHRCFICMC Mi tCMC Microsystems

Dr. Linfen YuDr. Ali AhmadiEric ChengCynthia NiCynthia NiSamantha GristCarmen Bayly

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Biomedical Technologies: Microfluidic systems for three