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A Simple Microfluidic Platform for Long-Term Analysis and Continuous Dual-Imaging Detection of T-Cell Secreted IFN-γ and IL-2 on Antibody-Based Biochip.

Baganizi DR, Leroy L, Laplatine L, Fairley SJ, Heidmann S, Menad S, Livache T, Marche PN, Roupioz Y - Biosensors (Basel) (2015)

Bottom Line: The identification and characterization, at the cellular level, of cytokine productions present a high interest for both fundamental research and clinical studies.However, the majority of techniques currently available (ELISA, ELISpot, flow cytometry, etc.) have several shortcomings including, notably, the assessment of several cytokines in relation to individual secreting cells and the monitoring of living cell responses for a long incubation time.In the present work, we describe a system composed of a microfluidic platform coupled with an antibody microarray chip for continuous SPR imaging and immunofluorescence analysis of cytokines (IL-2 and IFN-γ) secreted by T-Lymphocytes, specifically, and stably captured on the biochip under flow upon continued long-term on-chip culture (more than 24 h).

View Article: PubMed Central - PubMed

Affiliation: University of Grenoble Alpes, INAC-SPRAM, Grenoble F-38000, France. marchep@ujf-grenoble.fr.

ABSTRACT
The identification and characterization, at the cellular level, of cytokine productions present a high interest for both fundamental research and clinical studies. However, the majority of techniques currently available (ELISA, ELISpot, flow cytometry, etc.) have several shortcomings including, notably, the assessment of several cytokines in relation to individual secreting cells and the monitoring of living cell responses for a long incubation time. In the present work, we describe a system composed of a microfluidic platform coupled with an antibody microarray chip for continuous SPR imaging and immunofluorescence analysis of cytokines (IL-2 and IFN-γ) secreted by T-Lymphocytes, specifically, and stably captured on the biochip under flow upon continued long-term on-chip culture (more than 24 h).

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On-chip immunofluorescence detection of IFN-γ and IL-2 secreted by captured peripheral blood T lymphocytes. (A) Phase Contrast Microscopy images (objective 10× + Σ100× digital magnification) of lymphocytes which are specifically and stably bound on the specific anti-CD3 antibodies (alone or in combination with anti-IFN-γ or anti-IL-2 antibodies). There is very low or no unspecific capture of T cells on anti-IFN-γ, anti- IL-2, or mouse IgG isotypic controls; (B) fluorescence images (objective 10×, gain ×4, and exposition time 0.04 s) of secreted IFN-γ or IL-2 after detection with secondary anti-cytokine antibodies labeled with biotine and revealed by (strept)avidin-phycoerythrin; and (C) fluorescence intensities of secreted IFN-γ and IL-2 (n = 5). [D] ELISA titration of cytokines in cellular supernatants after on-chip cell culture (24-hours). After each experiment, the culture medium was recovered from the microfluidic reaction chamber (~50–80 µL) and the cytokines dissolved in the bulk medium measured by ELISA (n = 5).
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biosensors-05-00750-f007: On-chip immunofluorescence detection of IFN-γ and IL-2 secreted by captured peripheral blood T lymphocytes. (A) Phase Contrast Microscopy images (objective 10× + Σ100× digital magnification) of lymphocytes which are specifically and stably bound on the specific anti-CD3 antibodies (alone or in combination with anti-IFN-γ or anti-IL-2 antibodies). There is very low or no unspecific capture of T cells on anti-IFN-γ, anti- IL-2, or mouse IgG isotypic controls; (B) fluorescence images (objective 10×, gain ×4, and exposition time 0.04 s) of secreted IFN-γ or IL-2 after detection with secondary anti-cytokine antibodies labeled with biotine and revealed by (strept)avidin-phycoerythrin; and (C) fluorescence intensities of secreted IFN-γ and IL-2 (n = 5). [D] ELISA titration of cytokines in cellular supernatants after on-chip cell culture (24-hours). After each experiment, the culture medium was recovered from the microfluidic reaction chamber (~50–80 µL) and the cytokines dissolved in the bulk medium measured by ELISA (n = 5).

Mentions: The Figure 2 illustrates the polydimethylsiloxane-based (PDMS) microfluidic platform subsequently developed for T cell on-chip capture and long-term culture. This system was built by using biocompatible materials highly adapted to cell analysis. In addition, HEPES buffer was added to the cell culture medium to maintain the physiological pH in the microfluidic chamber. The microfluidic device contained connections at the inlet and outlet of the microfluidic cell to further allow a homogenous distribution of cells in the microfluidic chamber. In order to maintain a physiological environment within the chamber, a heating system was added to the device to maintain the temperature at 37 °C. Only the temperature in the microfluidic chamber was regulated by spatially-localized heating (Figure 4). Peripheral blood T lymphocytes (106 cells) suspended in 100 μL AIM-V Serum Free Medium buffered with 25 mM HEPES and containing 100 ng·mL−1 of PMA and 500 ng·mL−1 of ionomycin for cell stimulation were injected. In this biochip format, T cells were specifically and stably captured on their specific anti-CD3 antibodies. This was observed in real-time thanks to the digital CCD camera mounted on the microscope. There was very low or no unspecific capture observed on anti-CD19 antibody or on mouse IgG controls (Figure 5 and Figure 7). This correlated the relative abundance of T versus B cell populations as determined by flow cytometry of cell samples prior to their use, yielding to over 95% of CD3 T cells and less than 5% of CD19 B cells. Furthermore, it was important to ensure that cell capturing on the biochip did not adversely affect cell viability and that cells bound on the biochip via antibody-antigen interactions remained viable and bioactive. Using a cell vitality assay, the fluorescence intensity corresponding to healthy and metabolically active cells was emitted by over 95% of captured cells unlike the fluorescence intensity corresponding to dead cells which was not detected (Figure 5). Moreover, the majority of cells recovered from the biochip by gentle pipetting showed good viability (>90%). This clearly showed that cell viability and activity were not affected by their capture and long-term culture on the biochip.


A Simple Microfluidic Platform for Long-Term Analysis and Continuous Dual-Imaging Detection of T-Cell Secreted IFN-γ and IL-2 on Antibody-Based Biochip.

Baganizi DR, Leroy L, Laplatine L, Fairley SJ, Heidmann S, Menad S, Livache T, Marche PN, Roupioz Y - Biosensors (Basel) (2015)

On-chip immunofluorescence detection of IFN-γ and IL-2 secreted by captured peripheral blood T lymphocytes. (A) Phase Contrast Microscopy images (objective 10× + Σ100× digital magnification) of lymphocytes which are specifically and stably bound on the specific anti-CD3 antibodies (alone or in combination with anti-IFN-γ or anti-IL-2 antibodies). There is very low or no unspecific capture of T cells on anti-IFN-γ, anti- IL-2, or mouse IgG isotypic controls; (B) fluorescence images (objective 10×, gain ×4, and exposition time 0.04 s) of secreted IFN-γ or IL-2 after detection with secondary anti-cytokine antibodies labeled with biotine and revealed by (strept)avidin-phycoerythrin; and (C) fluorescence intensities of secreted IFN-γ and IL-2 (n = 5). [D] ELISA titration of cytokines in cellular supernatants after on-chip cell culture (24-hours). After each experiment, the culture medium was recovered from the microfluidic reaction chamber (~50–80 µL) and the cytokines dissolved in the bulk medium measured by ELISA (n = 5).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4697143&req=5

biosensors-05-00750-f007: On-chip immunofluorescence detection of IFN-γ and IL-2 secreted by captured peripheral blood T lymphocytes. (A) Phase Contrast Microscopy images (objective 10× + Σ100× digital magnification) of lymphocytes which are specifically and stably bound on the specific anti-CD3 antibodies (alone or in combination with anti-IFN-γ or anti-IL-2 antibodies). There is very low or no unspecific capture of T cells on anti-IFN-γ, anti- IL-2, or mouse IgG isotypic controls; (B) fluorescence images (objective 10×, gain ×4, and exposition time 0.04 s) of secreted IFN-γ or IL-2 after detection with secondary anti-cytokine antibodies labeled with biotine and revealed by (strept)avidin-phycoerythrin; and (C) fluorescence intensities of secreted IFN-γ and IL-2 (n = 5). [D] ELISA titration of cytokines in cellular supernatants after on-chip cell culture (24-hours). After each experiment, the culture medium was recovered from the microfluidic reaction chamber (~50–80 µL) and the cytokines dissolved in the bulk medium measured by ELISA (n = 5).
Mentions: The Figure 2 illustrates the polydimethylsiloxane-based (PDMS) microfluidic platform subsequently developed for T cell on-chip capture and long-term culture. This system was built by using biocompatible materials highly adapted to cell analysis. In addition, HEPES buffer was added to the cell culture medium to maintain the physiological pH in the microfluidic chamber. The microfluidic device contained connections at the inlet and outlet of the microfluidic cell to further allow a homogenous distribution of cells in the microfluidic chamber. In order to maintain a physiological environment within the chamber, a heating system was added to the device to maintain the temperature at 37 °C. Only the temperature in the microfluidic chamber was regulated by spatially-localized heating (Figure 4). Peripheral blood T lymphocytes (106 cells) suspended in 100 μL AIM-V Serum Free Medium buffered with 25 mM HEPES and containing 100 ng·mL−1 of PMA and 500 ng·mL−1 of ionomycin for cell stimulation were injected. In this biochip format, T cells were specifically and stably captured on their specific anti-CD3 antibodies. This was observed in real-time thanks to the digital CCD camera mounted on the microscope. There was very low or no unspecific capture observed on anti-CD19 antibody or on mouse IgG controls (Figure 5 and Figure 7). This correlated the relative abundance of T versus B cell populations as determined by flow cytometry of cell samples prior to their use, yielding to over 95% of CD3 T cells and less than 5% of CD19 B cells. Furthermore, it was important to ensure that cell capturing on the biochip did not adversely affect cell viability and that cells bound on the biochip via antibody-antigen interactions remained viable and bioactive. Using a cell vitality assay, the fluorescence intensity corresponding to healthy and metabolically active cells was emitted by over 95% of captured cells unlike the fluorescence intensity corresponding to dead cells which was not detected (Figure 5). Moreover, the majority of cells recovered from the biochip by gentle pipetting showed good viability (>90%). This clearly showed that cell viability and activity were not affected by their capture and long-term culture on the biochip.

Bottom Line: The identification and characterization, at the cellular level, of cytokine productions present a high interest for both fundamental research and clinical studies.However, the majority of techniques currently available (ELISA, ELISpot, flow cytometry, etc.) have several shortcomings including, notably, the assessment of several cytokines in relation to individual secreting cells and the monitoring of living cell responses for a long incubation time.In the present work, we describe a system composed of a microfluidic platform coupled with an antibody microarray chip for continuous SPR imaging and immunofluorescence analysis of cytokines (IL-2 and IFN-γ) secreted by T-Lymphocytes, specifically, and stably captured on the biochip under flow upon continued long-term on-chip culture (more than 24 h).

View Article: PubMed Central - PubMed

Affiliation: University of Grenoble Alpes, INAC-SPRAM, Grenoble F-38000, France. marchep@ujf-grenoble.fr.

ABSTRACT
The identification and characterization, at the cellular level, of cytokine productions present a high interest for both fundamental research and clinical studies. However, the majority of techniques currently available (ELISA, ELISpot, flow cytometry, etc.) have several shortcomings including, notably, the assessment of several cytokines in relation to individual secreting cells and the monitoring of living cell responses for a long incubation time. In the present work, we describe a system composed of a microfluidic platform coupled with an antibody microarray chip for continuous SPR imaging and immunofluorescence analysis of cytokines (IL-2 and IFN-γ) secreted by T-Lymphocytes, specifically, and stably captured on the biochip under flow upon continued long-term on-chip culture (more than 24 h).

Show MeSH