nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/best practices...
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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