Surface‐Micromachined Microfiltration Membranes for Efficient Isolation and Functional Immunophenotyping of Subpopulations of Immune Cells

An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this "bulk" assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay ("AlphaLISA"), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples.

[1]  G. Pantaleo,et al.  Role of HIV-1-specific CD4 T cells , 2006, Current opinion in HIV and AIDS.

[2]  F. Re,et al.  Heterogeneity of TLR-induced responses in dendritic cells: from innate to adaptive immunity. , 2004, Immunobiology.

[3]  R. Siliciano,et al.  Analysis of host-virus interactions in AIDS with anti-gp120 T cell clones: Effect of HIV sequence variation and a mechanism for CD4+ cell depletion , 1988, Cell.

[4]  Z. Grossman,et al.  HIV preferentially infects HIV-specific CD4+ T cells , 2002, Nature.

[5]  Jonathan Cohen.  The immunopathogenesis of sepsis , 2002, Nature.

[6]  D. Venzon,et al.  Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency virus-seropositive individuals. , 1993, The Journal of clinical investigation.

[7]  F. Poulsen,et al.  A Luminescent Oxygen Channeling Immunoassay for the Determination of Insulin in Human Plasma , 2007, Journal of biomolecular screening.

[8]  A. Lackner,et al.  The mucosal immune system: primary target for HIV infection and AIDS. , 2001, Trends in immunology.

[9]  J. Flynn,et al.  An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection , 1993, The Journal of experimental medicine.

[10]  D. Remick,et al.  Pathophysiology of sepsis. , 2007, The American journal of pathology.

[11]  J. Lederer,et al.  Injury, sepsis, and the regulation of Toll‐like receptor responses , 2004, Journal of leukocyte biology.

[12]  A. Revzin,et al.  Biosensors for immune cell analysis-A perspective. , 2012, Biomicrofluidics.

[13]  Katsuo Kurabayashi,et al.  An integrated microfluidic platform for in situ cellular cytokine secretion immunophenotyping. , 2012, Lab on a chip.

[14]  Sylvia Janetzki,et al.  Measurement of cytokine release at the single cell level using the ELISPOT assay. , 2006, Methods.

[15]  G. Pantaleo,et al.  Functional signatures in antiviral T-cell immunity for monitoring virus-associated diseases , 2006, Nature Reviews Immunology.

[16]  G. Pantaleo,et al.  Immune-based interventions in HIV infection: doing the right studies, getting the right answers. , 2006, AIDS.

[17]  J. Convit,et al.  Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. , 1991, Science.

[18]  R. Germain,et al.  T cell heterogeneity: firmly fixed, predominantly plastic or merely malleable? , 2008, Nature Immunology.

[19]  K. Schütze,et al.  Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. , 2000, The American journal of pathology.

[20]  B. Boehm,et al.  Detection of low-frequency antigen-specific IL-10-producing CD4(+) T cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine. , 2003, Journal of immunological methods.

[21]  G. Pantaleo,et al.  Functional signatures of protective antiviral T‐cell immunity in human virus infections , 2006, Immunological reviews.

[22]  Mehmet Toner,et al.  Blood-on-a-chip. , 2005, Annual review of biomedical engineering.

[23]  Bo Lu,et al.  3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood , 2011, Biomedical microdevices.

[24]  Ruili Huang,et al.  Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells , 2011, Current chemical genomics.

[25]  E. Wherry,et al.  Heterogeneity and cell-fate decisions in effector and memory CD8+ T cell differentiation during viral infection. , 2007, Immunity.

[26]  A. Hill,et al.  A CD4+ T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum infection and disease , 2004, Nature Medicine.

[27]  M. Bielefeld-Sévigny.  AlphaLISA immunoassay platform- the "no-wash" high-throughput alternative to ELISA. , 2009, Assay and drug development technologies.

[28]  S. Gordon,et al.  Monocyte and macrophage heterogeneity , 2005, Nature Reviews Immunology.

[29]  S. Romagnani.  Th1 and Th2 in human diseases. , 1996, Clinical immunology and immunopathology.

[30]  Jianping Fu,et al.  Photolithographic surface micromachining of polydimethylsiloxane (PDMS). , 2012, Lab on a chip.

[31]  R. Hotchkiss,et al.  The pathophysiology and treatment of sepsis. , 2003, The New England journal of medicine.

[32]  R. Hotchkiss,et al.  Immunosuppression in patients who die of sepsis and multiple organ failure. , 2011, JAMA.

[33]  T. Molina,et al.  Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls. , 2011, American journal of clinical pathology.