12 research outputs found
The Influence of Biomaterials on Cytokine Production in 3D Cultures
As
a result of improved relevance to in vivo physiology, in vitro
studies are increasingly performed in diverse, three-dimensional (3D)
biomaterials. However, materialācell type pairing effects on
cytokine availability remain unclear. We cultured five cell types
in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene
glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned
media, and we identified differences in measured cytokine levels that
were cell-type- or material-dependent. We further evaluated our data
using principal component analysis. Interestingly, component one identified
two classes of biomaterials with characteristic cytokine expression
levels. Component two identified cell-type-dependent differences in
cytokines related to the wound response. Although elements of soluble
cytokine availability are shared despite parameter differences, material
and cellular properties variably influenced cytokine levels, underlining
the influence of biomaterialācell type pairings on in vitro
assay outcomes. Relationships between material properties, cellular
responses, and cytokine availability in 3D in vitro models warrant
further investigation
Morphologies and cytoskeletal structure for HT-1080 fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) in synthetic extracellular matrix (ECM).
<div><p>(<b>A</b>) Schematic representation of synthetic extracellular matrix (synthetic ECM) formed through āthiol-eneā photopolymerization chemistry to couple norbornene C=C bonds on 4-arm poly(ethylene glycol) (PEG) molecules with thiol (-SH) bonds of cysteine-containing peptides. Crosslinks were formed using matrix metalloproteinase (MMP)-degradable peptides with cysteine groups on each end while adhesion was promoted using pendant RGD-containing peptides (C<b>RGDS</b>) with a single cysteine (2.5 or 3 wt% by mass PEG-NB + MMP-degradable crosslinking peptide, shear moduli = 140 Pa or 220 Pa respectively). Constant total pendant peptide was maintained using non-bioactive C<i><b>RDGS</b></i> (1500 Ī¼M active C<b>RGDS</b> + non-active C<i><b>RDGS</b></i>). </p>
<p>(<b>B</b>-<b>I</b>) Projected z-stack immunofluorescence (IF) images illustrating hDFs and HT-1080s seeded in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). (<b>B</b>,<b>C</b>) Overview to illustrate morphological differences (F-actin, red; Nuclei, blue) for (<b>B</b>) hDFs and (<b>C</b>) HT-1080s. (<b>D</b>-<b>F</b>) IF images illustrating myosin IIb expression for hDFs: (<b>D</b>) Overlay image (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>E</b>) F-actin and (<b>F</b>) Myosin IIb. White arrows point to actomyosin filaments. (<b>G</b>-<b>I</b>) IF images illustrating myosin IIb expression for <b><i>HT-1080s</i></b>: (<b>G</b>) Overlay (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb. </p>
<p>(<b>J</b>-<b>L</b>) Comparison of quantified mean (<b>J</b>) circularity and (<b>K</b>) elongation for hDFs and HT-1080s calculated using Nikon NIS Elements software (n > 150 cells, ā„ 6 hydrogels, at least two separate experiments; *** = p<0.001). (<b>L</b>) Fraction of elongated (Elongation ā„ 3.0), middle (2.0 ā¤ Elongation < 3.0), and rounded (Elongation < 2.0) cells. Differences in fraction of elongated and rounded hDFs compared to HT-1080s were each statistically significant (N ā„ 6 total gels, at least two separate experiments; *** = p<0.001). </p></div
Matrix influences on migration and morphologies for HT-1080s in synthetic ECM.
<p>(<b>A</b>) Cell speed and (<b>B</b>) directionality (DTO/TD) for HT-1080s as a function of matrix conditions (ā„ 6 gels, ā„ 40 cells, at least two separate experiments; * = p<0.05; ** = p<0.01). X-axis: Modulus in Pa / RGD concentration in Ī¼M. <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Error bars for DTO/TD represent standard error of the mean for individual cells. There is also a statistical difference in cell speed for the 140 Pa (1500 Ī¼M RGD) and 220 Pa (250 Ī¼M RGD) conditions (p<0.05, not shown on graph for clarity). (<b>C</b>) A comparison of the fraction of rounded HT-1080s (Elongation Index < 2.0) as a function of synthetic ECM conditions (x-axis: Modulus in Pa / RGD concentration in Ī¼M; white bar = 140 Pa; black bars = 220 Pa). Error bars represent standard deviation for fraction of rounded cells per gel (ā„ 6 gels, at least two separate experiments; * = p<0.05; ** = p<0.01; *** = p<0.001). </p
Contractile movement for HT-1080s in 3D culture.
<div><p>(<b>A</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS; 10 min / frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s014" target="_blank">Movie S3</a>). (<b>B</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in collagen (1.7 mg/mL; 15 min./frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s020" target="_blank">Movie S9</a>). Scale bars = 25 Ī¼m.</p>
<p>(<b>C</b>-<b>G</b>) Z-projected immunofluorescence images illustrating myosin IIb expression for an HT-1080 in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS): (<b>C</b>) Overlay image illustrating myosin IIb (green), counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nucleus, blue). Single channel images (grayscale) illustrate (<b>D</b>) F-actin and (<b>E</b>) Myosin IIb. Profile plots generated using ImageJ āInteractive 3D Surface Plotā function (āSpectrumā intensity scale, projection for middle 3 planes) illustrate (<b>F</b>) F-actin and (<b>G</b>) Myosin IIb. Two consecutive single plane images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb.</p></div
A comparison of quantified migration and morphologies for HT-1080s in synthetic ECM and collagen.
<div><p>Migration and morphologies were compared for HT-1080s cultured in lower modulus synthetic ECM (140 Pa, 1500 Ī¼M CRGDS) and collagen (1.7 mg/mL, acid-extracted, rat tail collagen, BD Biosciences). Time-lapse microscopy (15 min / frame) illustrating HT-1080s migrating in (<b>A</b>) synthetic ECM (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s026" target="_blank">Movie S15</a>) and (<b>B</b>) collagen (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s020" target="_blank">Movie S9</a>). Scale bars = 25 Ī¼m.</p>
<p>HT-1080s migrated in lower modulus synthetic ECM and collagen with statistically equivalent (<b>C</b>) speed and (<b>D</b>) directionality (DTO/TD). <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Differences in HT-1080 morphology were determined by comparing quantified (<b>E</b>) circularity and (<b>F</b>) fraction of rounded cells (Elongation < 2.0). Circularity was calculated for individual cells (3 separate experiments, N > 200); Fraction of rounded cells was calculated per gel (at least two separate experiments, N ā„ 3 gels), with error bars representing standard deviation (*** = p < 0.001). </p></div
Qualitative and quantitative migration for HT-1080s and hDFs.
<div><p>Time-lapse images (10 min./frame) illustrating migration for (<b>A</b>) an hDF and (<b>B</b>) an HT-1080 in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS). Image sequences in (<b>A</b>,<b>B</b>) are contrast and brightness enhanced for better display (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s013" target="_blank">Movies S2, S3</a> for unaltered images, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s015" target="_blank">Movie S4</a> for overview comparison of many cells). (<b>A</b>) Migration movement for hDF is characterized by: 1. Front end extension, 2. Attachment, 3. Cell body contraction, and 4. Rear-end release. (<b>B</b>) HT-1080 movement illustrates cell body contraction (white dashed lines and arrow) and simultaneous front end extension (red dashed lines and arrow). </p>
<p>(<b>C</b>-<b>E</b>) Comparison of quantified 3D migration for HT-1080s and hDFs in synthetic ECM (220 Pa and 1000 Ī¼M CRGDS): (<b>C</b>) Cell speed (adjusted by a factor of ā3/2, which was a 3D correction for analysis on 2D minimum intensity z-projections), (<b>D</b>) directionality (DTO/TD), and (<b>E</b>) fraction migrating cells. DTO/TD is a dimensionless parameter that provides a measure of directional motility analogous to persistence time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s001" target="_blank">Fig. S1A</a>) that is calculated as the distance-to-origin (DTO) after 6 hours divided by the total path length (total distance, TD) (shown schematically, panel to right of C). Migration was calculated from images collected in 15 min. increments for 6 hours, with dividing or interacting cells excluded (ā„200 cells, ā„ 3 separate experiments, ā„ 9 total hydrogels). <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Values for DTO/TD represent the mean for all cells while fraction migrating represents the mean for replicate experiments (N ā„ 3). Significance was calculated for hDF relative to HT-1080 migration for each parameter (*** = p<0.001). </p></div
Rounded proteolytic migration modes for tumor cells in synthetic ECM.
<p>Rounded tumor cells migrating in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS): (<b>A</b>) Time-lapse images (15 min / frame; See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s022" target="_blank">Movie S11</a>) for a rounded HT-1080 after overnight swelling (day 1). (<b>B</b>) Ī²1-integrin expression (immunofluorescence) for a rounded HT-1080. (<b>C</b>) Time-lapse images (10 min / frame; See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s023" target="_blank">Movie S12</a>) for a rounded HT-1080 shortly after encapsulation (Initial migration, Day 0). (<b>D</b>) Time-lapse images (30 min / frame) for a rounded WM239a melanoma cell after overnight swelling (day 1). Scale bars = 25 Ī¼m unless otherwise noted.</p
Comparison of adhesion properties for HT-1080s and hDFs.
<div><p>(<b>A</b>-<b>M</b>) Projected z-stack immunofluorescence (IF) images for hDFs and HT-1080s in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). </p>
<p>(<b>A</b>-<b>D</b>) IF images illustrating vinculin expression for <i><b>hDFs</b></i> (boxed region from A shown in B-D): (<b>A</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). White arrows point to regions enriched with vinculin. (<b>B</b>) Overlay (Vinculin, green; F-actin, red); Single channel images (grayscale) illustrate (<b>C</b>) F-actin and (<b>D</b>) Vinculin. (<b>E</b>-<b>G</b>) IF images illustrating vinculin expression for <b><i>HT-1080s</i></b>: (<b>E</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). Single channel images (grayscale) illustrate (<b>F</b>) F-actin and (<b>G</b>) Vinculin. </p>
<p>(<b>H</b>-<b>J</b>) IF images illustrating Ī²1-integrin expression for <i><b>hDFs</b></i>: (<b>H</b>) Overlay (Ī²1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>I</b>) F-actin and (<b>J</b>) Ī²1-integrin (White arrows point to punctate Ī²1-integrin features; Also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s003" target="_blank">Fig. S3</a>). (<b>K</b>-<b>M</b>) IF images illustrating Ī²1-integrin expression for <b><i>HT-1080s</i></b>: (<b>K</b>) Overlay (Ī²1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>L</b>) F-actin and (<b>M</b>) Ī²1-integrin. </p>
<p>(<b>N</b>) Comparison of attachment for hDFs and HT-1080s on RGD-SAMs as a function of RGD density. Attachment was statistically significant (cell number > 0 RGD control spots) for HT-1080s on surfaces with ā„ 0.19% mol fraction RGD and for hDFs on surfaces ā„ 0.007% mol fraction RGD. Error bars represent standard error of the mean (SEM) for array spots at given RGD density. Significance for cell attachment on individual spots was calculated for hDFs relative to HT-1080s (* = p<0.05; ** = p<0.01; *** = p<0.001). </p></div
HT-1080s and WM239a cells are characterized by rear-end MCAM-expressing uropod like structures.
<p>Illustration of uropod-like features and MCAM expression for tumor cells in synthetic ECM (220 Pa, 1000 Ī¼M CRGDS): (<b>A</b>-<b>C</b>) Z-projected immunofluorescence images for a WM239a melanoma cell; (<b>A</b>) Ī²1-integrin (green), counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nucleus, blue); Single channel images (grayscale) illustrate (<b>B</b>) Factin and (<b>C</b>) Ī²1-integrin. (<b>D</b>) Time-lapse images for a GFP-MCAM transfected WM239a melanoma cell (30 min / frame; See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s024" target="_blank">Movie S13</a>); Brightfield (top) and GFP-MCAM expression (bottom). (<b>E</b>-<b>G</b>) Z-projected immunofluorescence images for a WM239a melanoma cell; (<b>E</b>) Melanoma cell adhesion molecule (MCAM, green), counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nucleus, blue); Single channel images (grayscale) illustrate (<b>F</b>) Factin and (<b>G</b>) MCAM. (<b>H</b>) Time-lapse images for a GFP-MCAM transfected HT-1080 cell (15 min / frame; Background pixel intensity subtracted using the āMathā function in ImageJ and uniformly applied to all GFP images for better display); Brightfield (top) and GFP-MCAM expression (bottom).</p