J. Christopher LoveDept. of Chemical Engineering

Associate Member, Broad InstituteAssociate Faculty, Ragon Institute

clove@mit.eduhttp://web.mit.edu/lovelab

September 23, 2009

Microtools for multiparametric analyses of single cells

Chronic human diseases present a significantchallenge for public health in the 21st century

HIV/AIDS

Type 1 Diabetes/ Multiple Sclerosis

Allergies

Mapping complex immune responses requires understanding inter-cellular networks

Frankenstein et al. Biology Direct 2006 1:32

Multiple parameters needed to distinguish contributions from individual clones

Immunological manifestationof the Heisenberg uncertainty principle

What are the criteria for designing new tools to monitor immune responses?

• Measure multiple characteristics of live cells– Functional assays (secretions, cytotoxicity, proliferation)– Lineages (surface-expressed markers)

• Accommodate small numbers of cells (~104-106)

• Analyze close to ex vivo– Minimize bias from selective expansion

• Recover cells of interest– Clonal expansion– Gene expression

• Enable scalable processes– Minimize costs/cell screened and variability– Maximize number and diversity of samples

Arrays of microwells connect single-cell measurements on lineages, functions, and genotypes

1 mm

~ 224 x 384 wells

Microengraving captures proteinssecreted by single cells

Cross-section

Love, 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)

50 m

50 m

IgG IgM

CD19 (colonic biopsy)

Single-cell analyses yield multiparameter data

100 m

Rate of secretion

200 m

Epitope specificity

n ~ 1,500Proliferation

Affinity/Avidity

C. Story, E. Papa, et al., PNAS (2008)

Taking a snapshot of the antibody response

Single-cell data Collective profile Clonal analysis

C. Story, E. Papa, et al., PNAS (2008)

Temporary confinement enhances sensitivity and dimensionality of analysis for cellular responses

Quantitative capture

Q. Han

Multifunctional lymphocytes: An indicator of a healthy immune response?

Darrah et al. Nat Med (2007) 13 (7) 843-50

Quadriplexed simultaneous analysis of secretion from multifunctional cells

Q. Han

Can we resolve the state of the human immune system?

Summary and Conclusions

• Microtools enable detailed analyses of single cells– Miniaturization is necessary, but not sufficient– Complete lab-on-a-chip integration not required

• Applications in clinical research– Profile immune responses (in tissue samples)– Monitor vaccine efficacy– Well-suited for human immunology

• Challenges and opportunities– Real-time sensors for multiple functions/analytes– Massively parallel, deterministic cell positioning– Fast spectral imaging and processing

Our TeamLove Lab

Yuan Gong Qing Han Adebola Ogunniyi Vasiliki Panagiotou Yvonne Yamanaka Eli Papa

Dr. Qing Song Dr. Navin Varadarajan

Minna Du Mindy Du Jehnna Ronan Vinay Tripuraneni

Funding

Keck Foundation Dana Foundation Ragon Institute of MGH, MIT & Harvard SMART Infectious Disease IRG National Institutes of Health Deshpande Center for Innovation Texaco-Mangelsdorf

Career Development Chair

Brigham and Women’s Hospital Dr. Elizabeth Bradshaw Dr. Sally Kent Dr. Khadir Raddasi Prof. David A. Hafler

Ragon Institute Dr. Boris Juelg Dr. Hendrik Streeck Dr. Douglas Kwon Prof. Galit Alter Prof. Xu Yu Prof. Marcus Altfeld Prof. Bruce D. Walker

IAVI/Scripps Dr. Pascal Poignard Prof. Dennis Burton

Broad Institute Dr. Bjorn Nilsson

Gordon College Prof. Craig Story

Singapore-MIT Alliance Dr. Jae Hyeok Choi

Whitehead Institute Dr. Eduardo Guillen Prof. Hidde L. Ploegh

Why is human disease so difficult to study?