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Freely suspended cellular "backpacks" lead to cell aggregate self-assembly.

Swiston AJ, Gilbert JB, Irvine DJ, Cohen RE, Rubner MF - Biomacromolecules (2010)

Bottom Line: Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material.Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively.In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material. Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively. In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size. We investigate the efficiency of backpack-cell binding using flow cytometry and laser diffraction, examine the influence of backpack diameter on aggregate size, and show that even when cell-backpack complexes are forced through small pores, backpacks are not removed from the surfaces of cells.

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Confocal microscopy images and flow cytometry plots (FITC vs FSC) of aggregates formed under different cell to backpack ratios (R = 1−0.1). The diameter of each backpack is 7 μm. A higher magnification view of a cell−backpack aggregate is provided for R = 0.2. Scale bar is 100 μm (inset scale bar for R = 0.2 is 20 μm).
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fig3: Confocal microscopy images and flow cytometry plots (FITC vs FSC) of aggregates formed under different cell to backpack ratios (R = 1−0.1). The diameter of each backpack is 7 μm. A higher magnification view of a cell−backpack aggregate is provided for R = 0.2. Scale bar is 100 μm (inset scale bar for R = 0.2 is 20 μm).

Mentions: Figure 3 shows flow cytometry plots and confocal micrographs of cell−backpack (d = 7 μm) aliquots for R = 1−0.1 (for easier visualization, similar plots and micrographs for R = 10−3 may be found in the Supporting Information). Shown are FITC signal versus forward scatter (FSC) data from flow cytometry: cell aggregates are detected at higher FSC, and aggregates associated with one or more backpacks are detected at higher FITC values (since each backpack contains DiO in the PLGA region, which fluoresces almost identically to FITC). Thus, aggregates with backpacks are found in the upper right quadrant, and single cells with one or more backpacks are found in the upper left quadrant. We used confocal microscopy to directly observe aggregate size, which dramatically increases with decreasing R. For R > 1, we see very small aggregates (less than three cells), with primarily only one backpack associated per cell (see Supporting Information for confocal micrographs and flow cytometry plots). At R = 1, larger aggregates begin to form, and by R = 0.2, large complexes are found. At R = 0.1, a single aggregate formed in the dish; the micrograph in Figure 3 shows only the edge of this aggregate. To further quantify these aggregate structures, flow cytometry analysis of backpack fluorescence versus FSC on cell−backpack aliquots shows that as R decreases, the number of cells associated with a backpack increases. Because the flow cytometer passes the cell suspension through a small quartz capillary, aggregates break up before passing through the laser path for analysis. This limits analysis to small aggregates, single cells, and single backpacks (which are excluded from this analysis based on PI signal and FSC value), though the starting aliquot included large aggregates. As laser diffraction data indicates, the large aggregates seen in the optical images below are associated via both strong, specific, CD44-HA interactions and weak, nonspecific, cell−backpack binding. Small clusters, as seen in Figure 2, associate only via the strong CD44-HA interactions, and these are the FSChigh events shown in Figures 3 and 4. A detailed discussion of how different association strengths lead to large aggregates versus small cell clusters is presented along with the laser diffraction data below.


Freely suspended cellular "backpacks" lead to cell aggregate self-assembly.

Swiston AJ, Gilbert JB, Irvine DJ, Cohen RE, Rubner MF - Biomacromolecules (2010)

Confocal microscopy images and flow cytometry plots (FITC vs FSC) of aggregates formed under different cell to backpack ratios (R = 1−0.1). The diameter of each backpack is 7 μm. A higher magnification view of a cell−backpack aggregate is provided for R = 0.2. Scale bar is 100 μm (inset scale bar for R = 0.2 is 20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903013&req=5

fig3: Confocal microscopy images and flow cytometry plots (FITC vs FSC) of aggregates formed under different cell to backpack ratios (R = 1−0.1). The diameter of each backpack is 7 μm. A higher magnification view of a cell−backpack aggregate is provided for R = 0.2. Scale bar is 100 μm (inset scale bar for R = 0.2 is 20 μm).
Mentions: Figure 3 shows flow cytometry plots and confocal micrographs of cell−backpack (d = 7 μm) aliquots for R = 1−0.1 (for easier visualization, similar plots and micrographs for R = 10−3 may be found in the Supporting Information). Shown are FITC signal versus forward scatter (FSC) data from flow cytometry: cell aggregates are detected at higher FSC, and aggregates associated with one or more backpacks are detected at higher FITC values (since each backpack contains DiO in the PLGA region, which fluoresces almost identically to FITC). Thus, aggregates with backpacks are found in the upper right quadrant, and single cells with one or more backpacks are found in the upper left quadrant. We used confocal microscopy to directly observe aggregate size, which dramatically increases with decreasing R. For R > 1, we see very small aggregates (less than three cells), with primarily only one backpack associated per cell (see Supporting Information for confocal micrographs and flow cytometry plots). At R = 1, larger aggregates begin to form, and by R = 0.2, large complexes are found. At R = 0.1, a single aggregate formed in the dish; the micrograph in Figure 3 shows only the edge of this aggregate. To further quantify these aggregate structures, flow cytometry analysis of backpack fluorescence versus FSC on cell−backpack aliquots shows that as R decreases, the number of cells associated with a backpack increases. Because the flow cytometer passes the cell suspension through a small quartz capillary, aggregates break up before passing through the laser path for analysis. This limits analysis to small aggregates, single cells, and single backpacks (which are excluded from this analysis based on PI signal and FSC value), though the starting aliquot included large aggregates. As laser diffraction data indicates, the large aggregates seen in the optical images below are associated via both strong, specific, CD44-HA interactions and weak, nonspecific, cell−backpack binding. Small clusters, as seen in Figure 2, associate only via the strong CD44-HA interactions, and these are the FSChigh events shown in Figures 3 and 4. A detailed discussion of how different association strengths lead to large aggregates versus small cell clusters is presented along with the laser diffraction data below.

Bottom Line: Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material.Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively.In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material. Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively. In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size. We investigate the efficiency of backpack-cell binding using flow cytometry and laser diffraction, examine the influence of backpack diameter on aggregate size, and show that even when cell-backpack complexes are forced through small pores, backpacks are not removed from the surfaces of cells.

Show MeSH