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Nanowell approaches to measuring mucosal immune Practices in Mucosal... · PDF file 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr. Todd Gierahn Yuan Gong Dr....

Oct 09, 2020

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  • J. Christopher Love Koch Institute at MIT

    Dept. of Chemical Engineering

    [email protected] http://web.mit.edu/lovelab

    May 1, 2014

    Nanowell approaches to measuring mucosal immune responses

  • Sample Size! Cost/Sample!

    100,000,000!

    1,000,000! 1,000 -! 100,000!

  • Integrated single-cell analysis for clinical monitoring

    J.C. Love, AIChE J., 56, 2496 (2010)

  • Poly(dimethyl siloxane)!

    Cells in suspension!

  • Complete array (~250,000 30 µm wells

    or ~85,000 50 µm wells)

    ~ 1 / 20th of stamp

    25 x 75 mm2

    Single block (49 wells (50 µm))

    Automated Multi-Spectral Image Cytometry (MuSIC)

    T.Gierahn, A. Ozkumur, Y. Yamanaka

  • Imaging Cytometry for Functional Assays

    Y. Yamanaka

    On-chip cytolytic assays

    R. Contento

    Proliferation assays

    Dead P815 NK cell

  • Love, Ronan, et al., Nature Biotech. 24, 703-707 (2006) Ronan, et al., J. Immunol. Methods 340, 164-169 (2009)

    Ogunniyi et al., Nature Protocols 4, 767-782 (2009)

    Engraving plate!

    Microengraving: Single-cell secretome analysis

  • Microengraving for assessing immune cell responses

    multiplexed

    dynamic

    antigen-specific

    single-cell recovery

    Q. Han, et al., Lab Chip. (2010) Q. Han, N. Bagheri, et al. Proc. Natl. Acad. Sci. (2012)

    N. Varadarajan, D. Kwon, et al. Proc. Natl. Acad. Sci. (2012) A. Torres et al., Lab Chip (2013)

    A. Ogunniyi, et al., Vaccine (2014)

  • Form factor 25x75 mm2 slide SBS 24-well plate # nanowells per unit 1 x 85K – 250K 24 x 40K

    Max. samples/array 1-4 (subdivided) 24-96 (w/ barcoding)

    Automation friendly? No Yes MuSIC phenotyping Yes Yes

    Avg. Imaging time 4 Ch: 7-10 min 16 Ch: 70-90 min

    4 Ch: 3-4 min per macrowell 16 Ch: ~60 min

    Live-cell imaging? Yes (stage adaptor) Yes (enclosure)

    Microengraving (secretion) Yes (4-5 Ch) No Dynamic measures Serial secretion

    Cell motility Proliferation

    Cell-cell interaction

    Cell motility Proliferation

    Cell-cell interaction

    On-Chip PCR Yes No Single-cell (well) recovery? Yes Yes

  • Software tools for data extraction and analysis

    Crossword

    J. Proteome Res, 2014

    Microarray data extraction

    Enumerator

    Image cytometry data extraction

    •  # cells/well •  MFI per channel •  X-Y coordinates •  Well address

    MatchBox

    Data integration & analysis

  • Some questions: •  What are homing signals? •  Survival signals in tissue? •  Critical effector functions?

    T cell biology in female genital tract – many aspects still require further characterization

  • Nanowell-based process to analyze cervical leukocytes

    Advantages •  Minimal sample handling (single centrifugation step) •  Iterative analysis for deep phenotyping

    Isolate sample

    Label and deplete RBC &

    granulocytes

    Directly load sample Label cells

    Image Activate & Microengrave

    Spin

  • Deep phenotypic analysis of cervical CD45+ cells using MuSIC

    T. Gierahn, unpublished

  • Cervical CD4 T cells express CD69, but few are CD103+

    T. Gierahn, unpublished

    T Resident Memory Central/Effector Memory

    CD4+

    CD8+

  • Lessons learned for analysis of mucosal samples using nanowell-based technologies

    •  Less is more –  Minimal sample handling –  Direct processing in microtechnology –  1,000 cells – no problem

    •  More is better –  Fresh preferable (viability) –  Rare cells are still rare—sampling from finite numbers –  Multiple hypotheses for rare states limiting with few cells

    •  Better is better –  Open the spotlight – integrated measures of molecular profiles –  Embrace multidimensional analyses to generate hypotheses –  Minimize numbers of samples required from patient

  • Our Team Love Lab Viktor Adalsteinsson Dr. Rita Lucia Contento Dr. Todd Gierahn Yuan Gong Dr. Bin Jia Lionel Lam Denis Loginov Nicholas Mozdzierz

    Dr. Kartik Shah Brittany Thomas Yvonne Yamanaka Rachel Barry John Clark Thomas Douce Narmin Tahirova Ross Ziminsky Alumni

    Prof. Sangram Bagh (Presidents University, India) Dr. Jonghoon Choi (KIST) Dr. Qing Han Dr. Adebola Ogunniyi Dr. Ayca Yalcin Ozkumur Dr. Eliseo Papa Vasiliki Panagiotou (EPFL)

    Dr. Alexis Torres (Novartis

    Vaccines) Prof. Craig Story Dr. Qing Song Prof. Navin Varadarajan (U.

    Houston, ChE) Naren Tallapragada Narmin Tahirova

    MIT Karthik Shekar

    Arup Chakraborty

    Koch Institute

    Xiaosai Yao Dane Wittrup Neda Bagheri (NWU, ChemE) Douglas A. Lauffenburger Gregory Szeto Darrell Irvine

    U. California, Davis Barbara Shacklett Miriam Hospital Susan Cu-Uvin Ragon Institute

    Douglas Kwon Melis Anahtar Kenneth Law

    Fred Hutchinson Cancer Research Center

    Raphael Gottardo

    Yale University Steven Kleinstein Namita Gupta David A. Hafler

    Academic Medical Center Netherlands

    Paul L. Klarenbeek

    Marieke E. Doorenspleet

    Paul I.W. de Bakker