Limits...
Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

Lee Szeto G, Van Egeren D, Worku H, Sharei A, Alejandro B, Park C, Frew K, Brefo M, Mao S, Heimann M, Langer R, Jensen K, Irvine DJ - Sci Rep (2015)

Bottom Line: However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells.Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ.Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH.

ABSTRACT
B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

No MeSH data available.


Related in: MedlinePlus

Microfluidic cell squeezing provides a robust, scalable method for macromolecule delivery to B-cells.A) Schematic representation of microfluidic squeezing for macromolecule delivery to cells. Constriction channels were X μm long (10 or 30 μm) and Y μm in diameter (4 or 5 μm); these values were 30 μm long and 4 μm in diameter unless otherwise indicated. B) Representative histograms and gating strategy for assessing device performance for delivery of small (3 kDa) and large (40 kDa) dextrans in B-cells under these antigen delivery conditions: untreated; endocytosis; SQZ=squeezed (mechano-poration). C) Quantitative analysis of macromolecule uptake into resting B-cells relative to endocytosis; cell viability following mechano-poration (n = 7 independent experiments). D) Quantitative analysis of intra-device delivery performance (n = 13 consecutive runs using 1 device; results representative of >3 independent experiments). E) Dextran delivery into resting B-cells as a function of cell density from 5-50 million B-cells/mL (n = 3 replicates; representative of>3 independent experiments). All data were represented as means±standard deviation. Pairs of conditions were tested in E) for statistically significant differences with ordinary 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, but none were found.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4441198&req=5

f1: Microfluidic cell squeezing provides a robust, scalable method for macromolecule delivery to B-cells.A) Schematic representation of microfluidic squeezing for macromolecule delivery to cells. Constriction channels were X μm long (10 or 30 μm) and Y μm in diameter (4 or 5 μm); these values were 30 μm long and 4 μm in diameter unless otherwise indicated. B) Representative histograms and gating strategy for assessing device performance for delivery of small (3 kDa) and large (40 kDa) dextrans in B-cells under these antigen delivery conditions: untreated; endocytosis; SQZ=squeezed (mechano-poration). C) Quantitative analysis of macromolecule uptake into resting B-cells relative to endocytosis; cell viability following mechano-poration (n = 7 independent experiments). D) Quantitative analysis of intra-device delivery performance (n = 13 consecutive runs using 1 device; results representative of >3 independent experiments). E) Dextran delivery into resting B-cells as a function of cell density from 5-50 million B-cells/mL (n = 3 replicates; representative of>3 independent experiments). All data were represented as means±standard deviation. Pairs of conditions were tested in E) for statistically significant differences with ordinary 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, but none were found.

Mentions: The process of mechano-poration for loading B-cells with antigen is illustrated in Fig. 1A. Live cells are passed through parallel microfluidic channels in a silicon device; in each channel, 1 or more constrictions create transient pores in the membranes of cells passing through the device. Macromolecular cargos present in the surrounding fluid can diffuse into the cell during this transient poration, leading to intracellular loading. Previously, we demonstrated that mechano-poration is effective for promoting cytosolic delivery of macromolecules into a wide variety of cell types including primary murine B-cells24; efficient delivery was achieved with 5 sequential constrictions with dimensions 30 μm length and 5 μm width. However, recent iterations in device design (increased number of parallel constriction channels to 75, longer entry region, reversibility, etc.)36 prompted us to revisit optimal mechano-poration parameters for protein delivery into B-cells and subsequent antigen presentation. Pilot optimization experiments showed that 30-4 × 1 microfluidics chips (1 constriction per channel that is 30 μm long and 4 μm in diameter) run at 120 psi were the most effective chip design for mechano-poration of murine B-cells with both efficient delivery and high cell viability at concentrations of 5 × 106 B-cells/mL (Supplementary Fig. S1A shows representative delivery of 30-5 × 5 vs. 30-4 × 1); these conditions were used for all subsequent delivery experiments. Cells at this pressure ran through a device at a rate of approximately 1 million cells per second. We performed a quantitative assessment of inter-device and intra-device variability in process efficacy for resting polyclonal murine B-cells, and compared the uptake of low and high molecular weight dextrans (3 and 40 kDa, respectively) delivered into B-cells via mechano-poration (condition abbreviated as “SQZ” in the figures). In parallel, control B-cells were incubated with the same polymers for the duration of mechano-poration (~30 min) to compare levels of internalization achieved by B-cells through steady-state fluid-phase endocytosis/pinocytosis (“endocytosis”). Representative histograms of dextran uptake by B-cells following mechano-poration (“SQZ”) vs. controls are shown in Fig. 1B. As shown in Fig. 1C (left), microfluidic squeezing promoted greatly enhanced dextran uptake compared to endocytosis, with internalization increased ~65-fold and ~25-fold for 3 and 40 kDa dextrans, respectively. This represented delivery to 75-90% of all cells for both dextrans; by comparison less than 10% of resting B-cells endocytosed detectable amounts of cargo (Fig. 1B,D). Viability of recovered cells after mechano-poration was ~95% (Fig. 1C; right), similar to endocytosis controls. The manufacturer-specified capacity for the maximum number of cells that can be passed through these disposable microfluidic chips ranged from 1-5 million cells per device; however, here devices were run with multiple aliquots of cells (1 million cells per run) until clogged, and the maximum number of cells run through each individual device in a given experiment was often in significant excess of 5 million cells. Importantly, we determined there was low variability in percentage of delivered cells from first run to clogging of devices, indicating that intra-device variability was minimal (Fig. 1D). We also found that delivery performance of multiple devices within the same experimental session (inter-device variability) was very consistent (Supplementary Fig. S1B). Recognizing that some applications may require higher numbers of B-cells, we also tested the efficacy of the microfluidic devices at different cell densities. The efficiency of intracellular dextran delivery was largely independent from cell concentration up to at least 50 × 106 cells per mL, indicating potential for robust scalability (Fig. 1E).


Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

Lee Szeto G, Van Egeren D, Worku H, Sharei A, Alejandro B, Park C, Frew K, Brefo M, Mao S, Heimann M, Langer R, Jensen K, Irvine DJ - Sci Rep (2015)

Microfluidic cell squeezing provides a robust, scalable method for macromolecule delivery to B-cells.A) Schematic representation of microfluidic squeezing for macromolecule delivery to cells. Constriction channels were X μm long (10 or 30 μm) and Y μm in diameter (4 or 5 μm); these values were 30 μm long and 4 μm in diameter unless otherwise indicated. B) Representative histograms and gating strategy for assessing device performance for delivery of small (3 kDa) and large (40 kDa) dextrans in B-cells under these antigen delivery conditions: untreated; endocytosis; SQZ=squeezed (mechano-poration). C) Quantitative analysis of macromolecule uptake into resting B-cells relative to endocytosis; cell viability following mechano-poration (n = 7 independent experiments). D) Quantitative analysis of intra-device delivery performance (n = 13 consecutive runs using 1 device; results representative of >3 independent experiments). E) Dextran delivery into resting B-cells as a function of cell density from 5-50 million B-cells/mL (n = 3 replicates; representative of>3 independent experiments). All data were represented as means±standard deviation. Pairs of conditions were tested in E) for statistically significant differences with ordinary 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, but none were found.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4441198&req=5

f1: Microfluidic cell squeezing provides a robust, scalable method for macromolecule delivery to B-cells.A) Schematic representation of microfluidic squeezing for macromolecule delivery to cells. Constriction channels were X μm long (10 or 30 μm) and Y μm in diameter (4 or 5 μm); these values were 30 μm long and 4 μm in diameter unless otherwise indicated. B) Representative histograms and gating strategy for assessing device performance for delivery of small (3 kDa) and large (40 kDa) dextrans in B-cells under these antigen delivery conditions: untreated; endocytosis; SQZ=squeezed (mechano-poration). C) Quantitative analysis of macromolecule uptake into resting B-cells relative to endocytosis; cell viability following mechano-poration (n = 7 independent experiments). D) Quantitative analysis of intra-device delivery performance (n = 13 consecutive runs using 1 device; results representative of >3 independent experiments). E) Dextran delivery into resting B-cells as a function of cell density from 5-50 million B-cells/mL (n = 3 replicates; representative of>3 independent experiments). All data were represented as means±standard deviation. Pairs of conditions were tested in E) for statistically significant differences with ordinary 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, but none were found.
Mentions: The process of mechano-poration for loading B-cells with antigen is illustrated in Fig. 1A. Live cells are passed through parallel microfluidic channels in a silicon device; in each channel, 1 or more constrictions create transient pores in the membranes of cells passing through the device. Macromolecular cargos present in the surrounding fluid can diffuse into the cell during this transient poration, leading to intracellular loading. Previously, we demonstrated that mechano-poration is effective for promoting cytosolic delivery of macromolecules into a wide variety of cell types including primary murine B-cells24; efficient delivery was achieved with 5 sequential constrictions with dimensions 30 μm length and 5 μm width. However, recent iterations in device design (increased number of parallel constriction channels to 75, longer entry region, reversibility, etc.)36 prompted us to revisit optimal mechano-poration parameters for protein delivery into B-cells and subsequent antigen presentation. Pilot optimization experiments showed that 30-4 × 1 microfluidics chips (1 constriction per channel that is 30 μm long and 4 μm in diameter) run at 120 psi were the most effective chip design for mechano-poration of murine B-cells with both efficient delivery and high cell viability at concentrations of 5 × 106 B-cells/mL (Supplementary Fig. S1A shows representative delivery of 30-5 × 5 vs. 30-4 × 1); these conditions were used for all subsequent delivery experiments. Cells at this pressure ran through a device at a rate of approximately 1 million cells per second. We performed a quantitative assessment of inter-device and intra-device variability in process efficacy for resting polyclonal murine B-cells, and compared the uptake of low and high molecular weight dextrans (3 and 40 kDa, respectively) delivered into B-cells via mechano-poration (condition abbreviated as “SQZ” in the figures). In parallel, control B-cells were incubated with the same polymers for the duration of mechano-poration (~30 min) to compare levels of internalization achieved by B-cells through steady-state fluid-phase endocytosis/pinocytosis (“endocytosis”). Representative histograms of dextran uptake by B-cells following mechano-poration (“SQZ”) vs. controls are shown in Fig. 1B. As shown in Fig. 1C (left), microfluidic squeezing promoted greatly enhanced dextran uptake compared to endocytosis, with internalization increased ~65-fold and ~25-fold for 3 and 40 kDa dextrans, respectively. This represented delivery to 75-90% of all cells for both dextrans; by comparison less than 10% of resting B-cells endocytosed detectable amounts of cargo (Fig. 1B,D). Viability of recovered cells after mechano-poration was ~95% (Fig. 1C; right), similar to endocytosis controls. The manufacturer-specified capacity for the maximum number of cells that can be passed through these disposable microfluidic chips ranged from 1-5 million cells per device; however, here devices were run with multiple aliquots of cells (1 million cells per run) until clogged, and the maximum number of cells run through each individual device in a given experiment was often in significant excess of 5 million cells. Importantly, we determined there was low variability in percentage of delivered cells from first run to clogging of devices, indicating that intra-device variability was minimal (Fig. 1D). We also found that delivery performance of multiple devices within the same experimental session (inter-device variability) was very consistent (Supplementary Fig. S1B). Recognizing that some applications may require higher numbers of B-cells, we also tested the efficacy of the microfluidic devices at different cell densities. The efficiency of intracellular dextran delivery was largely independent from cell concentration up to at least 50 × 106 cells per mL, indicating potential for robust scalability (Fig. 1E).

Bottom Line: However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells.Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ.Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH.

ABSTRACT
B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

No MeSH data available.


Related in: MedlinePlus