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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
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Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Oct 09, 2020

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Page 1: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 2: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Sample Size!Cost/Sample!

100,000,000!

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

Page 3: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Integrated single-cell analysis for clinical monitoring

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

Page 4: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Poly(dimethyl siloxane)!

Cells in suspension!

Page 5: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 6: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Imaging Cytometry for Functional Assays

Y. Yamanaka

On-chip cytolytic assays

R. Contento

Proliferation assays

Dead P815 NK cell

Page 7: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 8: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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)

Page 9: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 10: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 11: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 12: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 13: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

Deep phenotypic analysis of cervical CD45+ cells using MuSIC

T. Gierahn, unpublished

Page 14: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

T. Gierahn, unpublished

T Resident Memory Central/Effector Memory

CD4+

CD8+

Page 15: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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

Page 16: Nanowell approaches to measuring mucosal immune responsespublic.hivmucosalgroup.org/Best Practices in Mucosal... · 2014. 5. 14. · Viktor Adalsteinsson Dr. Rita Lucia Contento Dr.

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