Micro-Scale Engineering –IIILab-on-a-Chip for Sorting Cells

Y. C. LeeDepartment of Mechanical Engineering

University of ColoradoBoulder, CO 80309-0427

leeyc@colorado.edu

February 4, 20141

Guest lecture on Thursday: Ramsey Zeitoun; Biofuel

High-throughput microfluidic single-cell RT-qPCR

A. K. White et al., "High-throughput microfluidic single-cell RT-qPCR," PNAS, 2011 Aug 23; 108(34):13999-4004.

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An Integrated Nanoliter DNA Analysis Device(Mark Burns et al., Science, 1998)

DNA solutionDNA fragmentsamplification site-specific oligonucleotides

Reagent solutionBso B1 restriction enzymesBst DNA polymerase enzymes

Outside sources:PressureElectronic controlOptical excitation source

Electronicsignals

Intercalatng dye

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47mm X 5mm X 1mm

Homework #2• Write a one-page report for the comparison by reporting:

1) summary of comparison2) cell capture and lysis3) reverse transcription4) PCR and real-time monitoring5) number of parallel processing units 6) another feature of your interest

• The purpose of this comparison is for you to appreciate the significant advancement in the lab-on-a-chip technology achieved during the last 15 years. Hope your independent project will identify a device that may make an impact 15 years from now.

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Single cell digital PCR device

White et al., Anal. Chem., 2013, 85 (15), pp 7182–7190 5

Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). 6

Antibodies latched onto the protein EpCAM

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Labelling Efficiency

For positive selection, pre-stained cell lines with low and high EpCAM expression were spiked into whole blood, incubated with various amounts of anti-EpCAM coated beads, and samples were mixed using either active magnetic mixing or passive mixing. Following mixing, samples were processed through a debulking array to remove RBCs and collected in a 24-well plate; target cells were identified based on their fluorescence and their bead loading was evaluated. 8

Complete System

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells (CTCs)

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013).

Positive depletion identifies CTCs using antibodies that latch onto the protein EpCAM, commonly found on the surface of CTCs. Current commercially available CTC-sorting devices are based on positive depletion, and the CTC-iChip also successfully isolated magnetically labeled CTCs with enhanced performance.

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Laminar and Turbulent Flows

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Re=umD/µ =1,000.00 kg/m³um= 0.050 m/secµ =0.001002 (N·s)/m2

D=100E-6 mRe= 5D=1,000E-6 mRe=50

Laminar Flow in a Pipe

Flow Past a Sphere

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Flow Past a sphere

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Re <11<Re<103

103<Re< 2E5

Re=umD/µ =1,000.00 kg/m³µ =0.001002 (N·s)/m2

D=10E-6 m (particle)um= 0.005 m/secRe= 0.05um= 0.0005 m/secRe= 0.005

Continuous Particle Separation Through Deterministic Lateral Displacement

(A) Geometric parameters defining the obstacle matrix. A fluid flow is applied in the vertical direction (orange arrow). (B) Three fluid streams (red, yellow, and blue) in a gap do not mix as they flow through the matrix. Lane 1 at the first obstacle row becomes lane 3 at the second row, lane 3 becomes lane 2 at the third row, and so on. Small particles following streamlines will thus stay in the same lane. (C) A particle with a radius that is larger than lane 1 follows a streamline passing through the particle's center (black dot), moving toward lane 1. The particle is physically displaced as it enters the next gap. Black dotted lines mark the lanes.

Lotien Richard Huang et al., Science 304, 987 (2004).

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Continuous Particle Separation Through Deterministic Lateral Displacement

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013). 17

Hydrodynamic size–based separation

Asingledebulking arrayhas24parallelchannelsandarraysare150μm indepth.Eachofthe24parallelchannelshastwoinputstreamsthatrunside‐by‐sideinlaminarflow.(A)Buildingonpreviouslyestablisheddesignprinciples,wedevelopedtwodifferentarrayconfigurations,Array1andArray2.Thegapsbetweenposts(g)were20and32μm forArrays1and2,respectively.Thecenter‐to‐centerdistancesbetweentheposts(λ)were35and56μm,respectively,andtherowshiftfraction(ε)was0.16forbotharraydesigns.(B)Cellretentionanddebulking efficiencieswerecharacterizedforbothofthearraysinmultipleexperiments(n>10).(C)Inourcurrentstudy,wemeasuredCTCdiametersbetween8and20μm (Fig.6).Therefore,weestimatedalowersizecut‐offof8μm,andlargersizerangegoingupto30μm.WeconcludedthattheapproximatedCTCsizerangeoverlapsbestwithArray2.

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Continuous inertial focusing,

ordering, and separation of particles in

microchannels

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Dino Di Carlo et al., Continuous inertial focusing, ordering, and separation of particles in microchannels, 18892–18897, PNAS, 2007.

Inertial Focusing

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013).

WBC

CTC

posCTC-iChip 21

Magnetic Deflection

Deflected

Undeflected

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Negative DepletionNegative depletion, in contrast, sorts out CTCs by eliminating all other “known” cells first. By magnetically labeling white blood cells rather than CTCs, the researchers were able to isolate a vast array of unlabeled tumor cells using CTC-iChip. Negative depletion allows for the detection of CTCs without having to know what type of tumor they came from beforehand and regardless of whether they produce EpCAM. Thus, negative depletion methods may be able to identify a greater variety of tumors across a broader range of development than positive depletion.

negCTC-iChip

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013).negCTC-iChip

WBC

CTC

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Overall system performance using cancer cell lines spiked into whole blood.

• SKBR3 human breast cancer cells• Human prostate PC3-9 cancer cells• MDA-MB-231, “triple-negative”

mesenchymal breast cancer• MCF10A breast cancer • LBX1: Epithelial-mesenchymal transition

(EMT) master regulator

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CTC isolation by posCTC-iChip in cancer patients

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The sensitivity of the CTC-iChip is particularlycritical in patients with a lower CTC burden.

CTCs with cytopathology and ICC stain

Immunocytochemistry (ICC)

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Variation of CTC sizes and morphologies

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Heterogeneity of RNA expression between CTCs isolated from a prostate cancer patient (qRT-PCR)

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

E. Ozkumur et al., Sci. Transl. Med. 5, 179ra47 (2013).negCTC-iChip

WBC

CTC

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Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells

• The CTC-iChip was able to sort CTCs from whole blood: - quicker than previously developed microfluidic devices, - allowing larger blood samples to be processed in a short amount of time more efficiently than other magnet-based sorting systems, - reducing the amount of materials required and increasing the sensitivity of the device - more effectively in samples with few EpCAM-producing CTCs compared to other sorting methods- more effectively in samples known to not express EpCAM, such as triple negative breast cancer and melanoma.

• By collecting CTCs in a way that allows them to be studied further, the CTC-iChip could also help clinicians identify important genetic differences between individual CTCs that may inform which targeted therapy is indicated. “It will enable, in the long run, [a physician] to treat the right patient with the right drug at the right dose at the right time.”

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