12 research outputs found
A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles
Cellular labeling with inorganic
nanoparticles such as magnetic
iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles
is an important method for the noninvasive visualization of cells
using various imaging modalities. Currently, this is mainly achieved
through the incubation of cultured cells with the nanoparticles that
eventually reach the intracellular compartment through specific or
nonspecific internalization. This classic method is advantageous in
terms of simplicity and convenience, but it suffers from issues such
as difficulties in fully removing free nanoparticles (suspended in
solution) and the lack of selectivity on cell types. This article
reports an innovative strategy for the specific labeling of adherent
cells without the concern of freely suspended nanoparticles. This
method relies on a nanocomposite film that is prepared by homogeneously
dispersing nanoparticles within a biodegradable polymeric film. When
adherent cells are seeded on the film, they adhere, spread, and filtrate
into the film through the micropores formed during the film fabrication.
The pre-embedded nanoparticles are thus internalized by the cells
during this infiltration process. As an example, fluorescent silica
nanoparticles were homogeneously distributed within a polycaprolactone
film by utilizing cryomilling and heat pressing. Upon incubation within
physiological buffer, no silica nanoparticles were released from the
nanocomposite film even after 20 d of incubation. However, when adherent
cells (e.g., human mesenchymal stem cells) were grown on the film,
they became fluorescent after 3 d, which suggests internalization
of silica nanoparticles by cells. In comparison, the suspension cells
(e.g., monocytes) in the medium remained nonfluorescent no matter
whether there was the presence of adherent cells or not. This strategy
eventually allowed the selective and concomitant labeling of mesenchymal
stem cells during their harvest from bone marrow aspiration
EPCs under light microscopy.
<p>(<b>A</b>) An EPC colony, defined morphologically as a central cluster of rounded cells surrounded by multiple spindle-shaped cells (20x magnification). (<b>B</b>-<b>D</b>) Expression of VEGFR-2 (red) and CD34 (green) was assessed under laser scanning confocal microscopy (10x magnification). Double-positive colonies (yellow) were identified as EPC colonies. Scale bar represents 20 µm.</p
Water contact angle measurements of unmodified and modified POSS-PCU surfaces, using a sessile drop method.
<div><p>Biofunctionalization with anti-CD34 antibodies significantly reduces the mean water contact angle value compared to POSS-PCU and POSS-PCU-FS. Error bars: ± SD; * denotes <i>p</i> < 0.05.</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
Platelet adhesion assay.
<div><p>(<b>A</b>-<b>C</b>) SEM images (2000x and 5000x magnification) show the greatest number of platelets adhering to POSS-PCU surfaces. (<b>A</b>) POSS-PCU, (<b>B</b>) POSS-PCU-FS, (<b>C</b>) POSS-PCU-FS+CD34. (<b>D</b>) The degree of platelet adhesion, expressed as the Platelet Adhesion Index, was significantly reduced by incorporation of FS and subsequent conjugation of anti-CD34 antibodies. *denotes a significant difference (<i>p</i> < 0.05).</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
EPC extraction from peripheral blood.
<p>The buffy coat layer contains EPCs, which were cultured on test samples.</p
Detection of surface-immobilized anti-CD34 antibodies using quantum dots.
<div><p>When films were immunostained with red QD-IgG fluorescent labels, (A) control POSS-PCU and (B) POSS-PCU-FS did not exhibit fluorescence, as compared to (C-D) POSS-PCU-FS+CD34 films, which showed uniform immobilization of bound antibodies on the film even after washing by mechanical shaking for 24 and 72 hrs (10x magnification). (E) Measurements of samples’ residual fluorescent intensities after 24 and 72 hrs of washing demonstrates that antibodies remain stably bound. Error bars: ± SD; NS denotes no significant difference (<i>p</i> > 0.05).</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
Surface characterization of unmodified and modified POSS-PCU surfaces by scanning electron microscopy at 2000x magnification.
<div><p><b>A</b> POSS-PCU B POSS-PCU-FS C POSS-PCU-FS+CD34.</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
Platelet activation assay.
<div><p>SEM images (2000x magnification) of adhered platelets show distinctly different morphological appearances. (<b>A</b>) Collagen, (<b>B</b>) POSS-PCU, (<b>C</b>) POSS-PCU-FS, (<b>D</b>) POSS-PCU-FS+CD34. Adhered platelets found on collagen-coated surfaces (positive control, <b>A</b>), showed the highest degree of activation, with formation of distinct pseudopodia and hyaloplasm spreading. Platelets adhering on POSS-PCU (<b>B</b>) and POSS-PCU-FS (<b>C</b>) were mostly dendritic-spread, with prominent pseudopodia as well as some flattening. Those adhered to POSS-PCU-FS+CD34 (<b>D</b>) remained dendritic with a clear spherical body and without any evident flattening.</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
Representative TEG profiles of blood in contact with the modified and unmodified POSS-PCU surfaces.
<div><p>Standard unmodified TEG cups with citrated whole blood were used as positive controls, whereas standard cups containing citrated blood mixed with the anti-coagulant L-arginine were used as negative controls. Profiles demonstrate a similar cigar-shape, indicating functional hemostasis.</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div
EPC colony counts.
<div><p>Number of double-positive CD34<sup>+</sup> / VEGFR-2<sup>+</sup> EPC colonies formed per 10<sup>6</sup> cells plated was enumerated after 7 days of culture on the different surfaces. Error bars: ± SD; # denotes a significant difference (<i>p</i> < 0.05) between positive control and all other groups; * denotes a significant difference (<i>p</i> < 0.05) between POSS-PCU-FS+CD34 and POSS-PCU-FS+IgG. Scale bar represents 20 µm.</p>
<p>POSS-PCU-FS: POSS-PCU with fumed silica anchors, POSS-PCU-FS+CD34: POSS-PCU biofunctionalized with anti-CD34 antibodies.</p></div