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Copyright 2014 American Scientic PublishersAll rights
reservedPrinted in the United States of America
ArticleJournal of
Biomedical NanotechnologyVol. 10, 10301040, 2014
www.aspbs.com/jbn
Filopodial Morphology Correlates to the CaptureEfciency of
Primary T-Cells on Nanohole Arrays
Dong-Joo Kim2 , Gil-Sung Kim2 , Jin-Kyeong Seol2, Jung-Hwan
Hyung2, No-Won Park1,Mi-Ri Lee2, Myung Kyu Lee4, Rong Fan35, and
Sang-Kwon Lee11Department of Physics, Chung-Ang University, Seoul
156-756, Republic of Korea2Basic Research Laboratory (BRL),
Department of Semiconductor Science and Technology, Chonbuk
National University,Jeonju, 561-756, Republic of Korea3Department
of Biomedical Engineering, Yale University, New Haven, CT 06511,
USA4Bionanotechnology Research Center, Korea Research Institute of
Bioscience and Biotechnology (KRIBB),Daejeon, 305-806, Republic of
Korea5Yale Comprehensive Cancer Center, New Haven, CT 06520,
USA
Nanostructured surfaces emerge as a new class of material for
capture and separation of cell populations includingprimary immune
cells and disseminating rare tumor cells, but the underlying
mechanism remains elusive. Although ithas been speculated that
nanoscale topological structures on cell surface are involved in
the cell capture process, thereare no studies that systematically
analyze the relation between cell surface structures and the
capture efciency. Herewe report on the rst mechanistic study by
quantifying the morphological parameters of cell surface
nanoprotrusions,including lopodia, lamellipodia, and microvilli in
the early stage of cell capture (
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Copyright: American Scientific Publishers
Kim et al. Filopodial Morphology Correlates to the Capture
Efciency of Primary T-Cells on Nanohole Arrays
nanometer-scale topography not only inuences diversecell
behavior such as cell adhesion, motility, prolifera-tion and
differentiation,1618 but represents a new routeto capture and
separate rare cell populations.1924 Theseinteresting ndings have
motivated us to develop a novelnano-platform for separating CD4
T-lymphocytes fromprimary mouse splenocytes via high afnity
streptavidin(STR)-biotin conjugation with immuno-functionalized
sil-icon nanowire arrays.19 Despite the remarkable improve-ment in
the capture efciency using nanostructure-basedcell capture devices,
the underlying mechanism that allowsnanostructured-surfaces to
achieve signicantly enhancedcell separation efciency as compared to
planar substrateremains elusive.Here we report on the study of how
the morphologies
of lopodia or microspikes, which are needle-like actin-rich
protrusions from the cell surface, are correlated tothe cell
capture efciency using a periodic 2D nanoholearray model surface.
Filopodia are involved in a wide vari-ety of functions including
absorption, secretion, cellularadhesion, and mechnotransduction.25
Because cell captureis a rapid process and the cells captured by
the immobi-lized antibodies can interact with the substrate surface
andrest/spread quickly within 20 min, we suspect the earlystage
cell-substrate interaction plays a critical role in thecell capture
process. We prepared a set of nanohole arrays(NHAs) with the hole
size ranging from 140 to 550 nm andobserved that the nanostructures
indeed directs the forma-tion of lopodia (100300 nm in diameter)26
in the caseof CD4+ T-lymphocytes in contact with
biofunctionalizedNHA substrates that further correlates to the
efciencyof CD4+ T-cell separation. While cross-sectional
imagingindicates that cell microvilli, which were previously
sus-pected to be playing a crucial role in enhancing cell cap-ture
efciency, were not observed at the interface betweencell and
nanostructured surface. Although this may notexclude the role of
microvilli in the process of initialcontact between immune T-cells
and the nanostructures,our results suggest that the subsequent cell
spreading andactin-rich lopodia formation is a more favorable
mech-anism that leads to signicant enhancement of rare cellcapture
efciency.
MATERIALS AND METHODSMaterialsColloidal polystyrene suspensions
(200, 300, 430, and750 nm in diameter) were purchased from Thermo
Scien-tic (Fremont, CA, USA). Cr etchant (CR-7) was suppliedby
Cyantek Corporation (Fremont, CA, USA). N -methyl-2-pyrrolidone,
(3-aminopropyl)-triethoxysilane (APTES),glutaraldehyde (GA),
streptavidin (STR), and osmiumtetroxide were purchased for
Sigma-Aldrich (St. Louis,MO, USA). C57BL/6/mice were supplied by
Nara-Biotech (Seoul, Republic of Korea). Biotinylated anti-CD4mAb
(clone GK 1.5), FITC (518 nm emission)-labeled
mAb-CD3, PE (575 nm emission)-labeled mAb-CD4(clone: RM4-4), and
PerCP (690 nm emission)-labeledmAb CD19, and uorescence dye
labeled-CD3, CD4,CD8, and CD19-mAbs were purchased from
eBioscienceInc. (San Diego, CA, USA). All other chemicals were
ofanalytical grade.
Nanohole Array (NHA) FabricationTo produce the NHA, the liquid
state of colloidalpolystyrene nanoparticles (PS NPs, 200, 300, 430,
and750 nm in diameter) monolayer was rst carefullydeposited on the
quartz (QZ) substrate using a modi-ed self-assembly technique (Fig.
1(A)) we developedpreviously.22 To create space between the
spin-coated PSNPs, their sizes were rst reduced by O2 plasma
(O2/Ar=35/10 sccm, RF power of 100 W and bias power of 50 W)for 10
s; 200 nm PS, 23 s; 300 nm PS, 35 s; 430 nm
Figure 1. (A) Schematic diagram of nanohole array
(NHA)fabrication using polystyrene (PS) nanoparticles on a pla-nar
quartz substrate. ((B)(E)) Scanning electron microscope(SEM) images
of NHAs (140, 200, 270, and 550 nm in diameter)prepared with four
different feature sizes of PS nanoparticles(200, 300, 430, and 750
nm in diameter). Scale bar is 0.5 m.Insets show the enlarged images
of each NHA. These guresalso represent the cross-section view
images of each NHAshown in the lower part of each SEM image. (F)
Schematic dia-gram showing sequent surface functionalization with
APTES,GA, and STR on NHA substrates, (G) showing cell suspension(30
l), which is containing CD4, CD8, B, NK, and NKT-cells,loading
process onto NHA substrates, and (H) revealing thewashing process
to remove unbound T-cells from the NHAsubstrates using a
3D-rocker.
J. Biomed. Nanotechnol. 10, 10301040, 2014 1031
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Filopodial Morphology Correlates to the Capture Efciency of
Primary T-Cells on Nanohole Arrays Kim et al.
PS, and 60 s; 750 nm PS, respectively, to control thediameter
and spacing for preparing the nanohole arraysperforated into
continuous metal lm. To fabricate thenanohole pattern, the reactive
ion etching (RIE, SoronaInc., Pyeongtaek, Republic of Korea)
process was thenperformed for 40 s (CF4/Ar = 40/5 sccm, RF power
of100 W, and bias power of 50 W) after 25-nm Cr metalonto the
coated surface of PS NPs, and the PS NPs weresubsequently removed
by unltrosonication in N -methyl-2-pyrrolidone. Finally, the Cr
metal layer was removed viaa lift-off process using Cr etchant
(Fig. 1(A)). The typi-cal diameters/widths of the resultant
nanoholes were about140/60, 200/100, 270/160, and 550/200 nm with
depthsof 260, 270, 320, an 330 nm, respectively as shown inFigures
1(B)(E). The period of hole-arrays was deter-mined by the initial
period of hcp nanospheres arrays andthe diameter of holes could be
easily tuned by the O2 etch-ing time. Moreover, our proposed method
provides a ex-ible and versatile route to the fabrication of QNP
arrayson a at QZ substrate, with potential application in
optics,electronics, sensing, and as building blocks for more
com-plex nanostructures.
NHA Surface FunctionalizationNHA substrates (7 mm 7 mm) were
carefully cleanedwith H2O2:H2SO4 (1:1) for 10 min to remove all
ofthe organic materials and impurities on the surface. Wethen
washed the substrates using a three-step clean-ing process
(acetone, isopropyl alcohol, and distilledwater) and dried them
with air. The NHA surface wastreated with O2 plasma for 20 s to
confer the hydroxylgroups on the NHA surface after piranha cleaning
process(96%H2SO4:30%H2O2 = 11) for 10 min. Next, the sur-face was
applied by a three-step surface functionalizationprocess using 1%
(v/v) (3-aminopropyl)-triethoxysilane(APTES) in ethanol for 30 min
at room temperature,12.5% (v/v) glutaraldehyde (GA) in distilled
water for4 hrs on a 3D-rocker, and 50 g/mL streptavidin (STR)
inphosphate buffered saline (PBS) overnight in an incubator(37 C,
5%CO2) as shown in Figure 1(F).
Cell PreparationThe CD4 T-lymphocytes to be separated were
mouseCD4+ T-cells from whole mouse splenocytes, which con-tain CD4+
T, CD8+ T, natural killer (NK), natural killer T(NKT), and B-cells.
These splenocytes were prepared fromthe spleens of C57BL/6/mice as
described previously.19
A certain quantity of cells (105 cells/mL) was countedusing a
conventional hemocytometer (Hausser Scientic,USA). Prior to loading
the cell suspension in the culturemedium, the cell population with
a nal volume of 30 lwas rst reacted with biotinylated anti-CD4 mAb
and incu-bated at 4 C for 20 min (Fig. 1(G)). Following
incubationfor 20 min with STR-conjugated NHA substrates at 4
C,unbound cells were removed by rinsing with phosphate
buffered saline (PBS), while separated CD4+ T cells couldbind to
STR-NHA surfaces due to adhesion enabled bythe STR-biotin
interaction. This process was repeated threetimes for 10 min on a
3-D rocker to completely removenon-specically unbound cells from
the NHA substrates(Fig. 1(H)). Remnant of unbound cells were then
collectedand transferred to the tube for enumerating the
populationby standard ow cytometry (Becton Dickinson, NJ, USA).
Fluorescence Activated CellSorter (FACS) AnalysisTo enumerate
the remnant of unbound cell population,FACS analysis was performed.
First, the non-specicallyunbound T-lymphocytes after STR-NHA
separation plat-form were collected by FACS falcon round-bottom
tubes(eBioscience, USA). The collected T-lymphocytes werethen
stained by FITC (518 nm emission)-labeled mAb-CD3, PE (575 nm
emission)-labeled mAb-CD4, andPerCP (690 nm emission)-labeled mAb
CD19 at 4 Cfor 20 min. After reaction, the stained
T-lymphocyteswere washed for removing the unbounded antibody
usingcentrifuge (1500 rpm, 5 min, twice) and then xed by4%
paraformaldehyde in PBS for 15 min. Finally thexed cell suspension
were transferred to the ow cytome-ter (FACS, Calibur, Beckton
Dickinson, USA) and ana-lyzed using CellQuest Pro software (BD
Bioscience,USA) for the separation efciency of the targeting
CD4+
T-lymphocytes.
Surface-Bound T-Cells Preparation UsingScanning Electron
Microscopy (SEM) AnalysisFor the FE-SEM analysis, a solution of the
cells conju-gated with biotin-conjugated CD4 mAbs in RPMI-1640(500
mL, Invitrogen, NY, USA) was pipetted onto STR-functionalized NHAs
(140, 200, 270, and 550 nm in diam-eter, 7 mm7 mm) and STR-planar
glass substrates withcell populations 105 cells/mL. After
incubation at 37 Cand 5% CO2 for 20 min, the separated and
immobilizedCD4+ T-cells on STR-functionalized NHAs and planarglass
substrates were rst xed with 4% GA in the refrig-erator for 2 h,
followed by a post-xing in 1% osmiumtetroxide for 2 h. The captured
T-cells on STR-conjugatedsubstrates were then dehydrated by
successive immersionin 25%, 50%, 75%, 95% and 100% ethanol for 5
min at4 C and followed by nal dehydration with 100% ethanoltwice
for 10 min at 4 C. Final dehydration needed toprocess twice.
Dehydrated T-cells were then frozen for3 h at 80 C. Subsequently,
the cells with substrateswere slowly dried for 24 h using a vacuum
desiccator.Subsequently, the cells with substrates were slowly
driedunder vacuum for 24 h. Once dried, the surface-boundT-cells
were then sputter-coated with a layer of platinum(56 nm) to prepare
conductive samples before perform-ing the FE-SEM measurement.
Quantitative characteriza-tion of the cellular morphologies
including lopodia and
1032 J. Biomed. Nanotechnol. 10, 10301040, 2014
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165.194.103.14 On: Mon, 10 Feb 2014 05:23:15
Copyright: American Scientific Publishers
Kim et al. Filopodial Morphology Correlates to the Capture
Efciency of Primary T-Cells on Nanohole Arrays
lamellipodia formation (length, width, and number of
theprotruded lopodia etc.) for the surface-bound T-cells
onSTR-functionalized NHAs and planar glass was performedon the
FE-SEM images.
RESULTS AND DISCUSSIONT-cell Capture Efciency on
STR-NHAsFluorescence activated cell sorter (FACS) revealed
thatwhole mouse splenocytes contained 42.7% CD4+T lymphocytes
(CD3+/CD4+ and 57.3% non-CD4+ T-cells (Fig. 2(A)). Each type of
cells (e.g., CD4,CD8-T, B, NK, and NKT cells) from the suspension
ofwhole mouse splenocytes was quantied using FACSmeasurement with
different surface markers (uores-cence dye labeled-CD3, CD4, CD8,
and CD19-mAbs).After completion of the cell separation process via
aSTR-NHA platform, the percentages of CD4+ T-cells in
Figure 2. (A) Flow cytometric (FACS) analysis results of CD4+
T-cells using cell suspensions, containing B, NK, NKT, CD4+
T and CD8+ T-cells after binding to STR-conjugated NHA
substrates ranging of 140 to 550 nm in diameter. (B) Average
cellseparation efciencies (top bar-graph) for four different sizes
of NHAs with the summary (bottom table). The cell
separationefciency evaluated from FACS is dened by the percentage
of cells captured (shown in right-part of (A)) to cells initially
loaded(shown in left-part of (A)), for different cell separation
STR-functionalized NHAs (140, 200, 270, and 550 nm in diameter).
Forthe comparison, the FACS results of STR-functionalized planar
glass wafer were also included. (C) Florescence images and
(D)purity distribution of CD4+ T-cells for stained with
4,6-diamidino-2-phenylindole and phycoerythrin using three
different NHAsamples (140, 200, and 270 nm in diameter). For the
comparison, the results from control sample are also presented in
(C) and(D). In (B) and (D), the error bars represent the standard
error of mean from four repeats (n = 4).
the cell suspension with four different NHAs (of 140,200, 270,
and 550 nm in diameter) decreased to 2.59,1.32, 3.10, and 3.99%,
respectively as shown inFigure 2(A). The cell-capture efciency,
which is denedas the percentage of the target CD4+ T-cells
successfullycaptured to the total number of cells in the cell
suspension(105 cells) initially loaded and analyzed by FACS,
isplotted for all four different NHAs in Figure 2(B) (topbar-graph)
and also summarized in the table of Figure 2(B)(bottom table). For
comparison, the STR-functionalizedplanar glass substrates as
control samples were also testedand the results are shown in Figure
2(B) (top-right ofbar graph). The result shows that all
STR-functionalizedNHA substrates (92.7%) show excellent
performance(a factor of 1.4) in cell capture efciency as comparedto
the control samples (65.2%), which is consistentwith previous
studies.1922 FACS results demonstrated thathigh yield separation of
CD4 T-cells was achieved with
J. Biomed. Nanotechnol. 10, 10301040, 2014 1033
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Filopodial Morphology Correlates to the Capture Efciency of
Primary T-Cells on Nanohole Arrays Kim et al.
all of the STR-functionalized NHAs, and the results
werecomparable to those from other nanostructure-based cellcapture
experiments previously reported.1922 The hypoth-esis why
signicantly elevated cell capture efciency canbe achieved on
STR-nanostructured surfaces as comparedto the planar glass
substrates is the following. First, it islikely due to the
relatively higher contact area betweenthe cells and the solid
surfaces compared to the planarglass substrates as we reported
previously.22 Second, itwas speculated to be associated with the
adhesive andtraction force of cells on the substrate, which is
uniquein the condition of higher degree of dimensionality inSTR-NHA
surface compared to the planar substrates. Ourprevious results
showed that the surface-bound T-cells onSTR-nanostructures have the
higher traction force (e.g.,adhesion force) due to the
three-dimensional (3-D) surfaceaccessibility (e.g., nanohole or
nanopillar etc.), whilethe cells on planar substrates exhibits a
lower adhesionforce at the same stage, corresponding to 2-D
behavior.27
Consequently, it results in elevated cell capture efciencyon the
STR-nanostructures with enhanced cell adhesionforce occurring at
the similar stages (Fig. 2(B)).
Purity of Captured T-Cells on STR-NHAsFurthermore, to
investigate the purity of the capturedT-cells on NHAs and also to
assess cell integrity, thecaptured CD4 T-cells were stained by
phycoerythrin (PE)-conjugated anti-CD4 mAb and 4-6-diamidino-2
phenylin-dole (DAPI, DB Bioscience, USA). DAPI (blue-350 nm)is a
nuclear dye that stains for cells with intact nuclei. Thestained
cells were subsequently enumerated for the CD4+
T-cells (PE+/DAPI+) out of total cells (DAPI+) boundto the
STR-functionalized NHA substrates by uorescencemicroscopy (FM)
analysis. Figure 2(C) shows uorescenceimages of both PE and
DAPI-stained CD4+ T-lymphocytesfor the STR-conjugated NHAs and
planar glass (controlsample). As shown in Figures 2(C), (D),
STR-conjugatedNHAs (140, 200, and 270 nm in diameter) were found
tohave a high purity (80633%), which is comparable tothat of the
control samples (planar glass, 77707%).
Cellular Morphology of Captured T-CellsTo quantify the
morphological properties of the capturedCD4+ T-lymphocytes bound to
STR-conjugated NHA sub-strates, FE-SEM analysis using a cell
freezing techniquewere performed. Quantitative characterization of
the cel-lular morphologies (number of extended lopodia andprotruded
length and width of lopodia etc.) on bothSTR-functionalized NHA
substrates and planar glass (con-trol sample) was performed via
analyzing the FE-SEMimages. Figure 3(A) shows representative FE-SEM
imagesof CD4+ T-lymphocytes bound on the surfaces of
fourdifferent-sized NHAs. It was observed that the capturedCD4+
T-cells possessed different forms of lopodia (0.10.3 m)26 on the
four NHAs with different nanohole sizes.
The formation of the at lamellipodia extending fromthe captured
T-cells was observed only on the surface ofNHAs exceeding 270 nm,
as shown in Figure 3(A).To further examine the interface and
binding propertiesbetween the captured CD4 T-cells and NHA
substrates,gallium ion (Ga+) milling of the captured CD4 T-cellswas
performed using focused ion beam (FIB). Figure 3(B)shows the
cross-sectional images of the captured CD4-Tcells on four different
NHAs, indicating that the capturedCD4 T-cells formed tight binding
and adhesion on STR-functionalized NHAs. However the insertion of
microvilliinto nanoholes, a mechanism speculated to account
forenhanced cell capture efciency, are surprisingly rare.Figure
3(C) shows the cell size, which was calculatedwithout considering
the area of lopodia and lamellipo-dia from the cells (n = 117)
captured on a 270 nm NHAsubstrate, displays a Gaussian
distribution, implying thatnon-specically bound cells were also
counted. Accordingto cell purity measurement (Figs. 2(C), (D)) of
cell sus-pension, 80.6% of cells bound on the NHAs substratescould
be CD4+ T-cells, indicating that approximately 80%of the bound
cells (2.84 m in cell-diameter) in thesize distribution graph could
be the right target cellsCD4+ T-lymphocytes. To ensure that the
evaluation of thelopodia morphology including the width, length,
featuresize and the number of the lopodial laments per cell inthe
early stage of cell adhesion (in our case, only 20 minincubation)
is statistically sound, we quantied more than55 cells, which were
80% of the total bound cells wecounted (n=70) (Figs. 3(D), (E) and
4(A)(D)). To jus-tify the signicance of our correlation results, p
valueswere calculated with neighboring column data. As shownin
Figs. 3(D), (E), the protruded lopodia width for 140-nm NHAs (80.2
nm in width) exhibits similar trend insize to that of the 200-nm
NHAs (83.1 nm in width)resulting in statically insignicant
difference (Fig. 3(D)and Table I). With further increasing the
diameter of NHAsfrom 270 to 550 nm, lopodia protruding from the
T-cellswere observed to increase in width (P < 00001, 126140 nm
in width, Figure 3(E) and Table I). This cor-relation develops even
at the very early stages of adhe-sion (20 min incubation). Hence,
this linear correlation(except below
-
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Copyright: American Scientific Publishers
Kim et al. Filopodial Morphology Correlates to the Capture
Efciency of Primary T-Cells on Nanohole Arrays
Figure 3. (A) Scanning electron microscope (SEM) images of CD4+
T-cells bound on four different sizes of NHA substrates (low-and
high-magnication top, tilt, and enlarged tilt view images). (B)
Cross-sectional SEM images of surface-bound CD4+ T-cells onfour
different NHAs (140, 200, 270, and 550 nm in diameter) with
enlarged images in selective area marked 1, 2, 3, and 4 in
rstcolumn of (B). The samples were prepared by Ga+ ion milling with
a focused ion beam (FIB). All of the CD4+ T-cells on the NHAsare
highlighted in yellow for easy differentiation. (C) Cell size
distribution (only cell-body excluding lopodia and lamellipodia)of
captured cells (n = 117) randomly bound on streptavidin
(STR)-functionalized NHA substrate (270 nm in diameter). Scale
baris 1 m. (D) Filopodia width distribution of CD4+ cells bound on
the four different STR-functionalized NHAs substrates after only20
min incubation at 4 C. P values of < 00001 () considered
statically signicant. An insignicant statistical difference
isrepresented as NS. (E) Selected lopodia width distribution in
which only 80% of lopodia width taken from (F) of the CD4+T cells
on NHAs substrates, indicating that the lopodia width was not
changed between the 140 and 200 nm in diameter andlinearly
increased with increasing of NHA diameters up to 550 nm.
J. Biomed. Nanotechnol. 10, 10301040, 2014 1035
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Filopodial Morphology Correlates to the Capture Efciency of
Primary T-Cells on Nanohole Arrays Kim et al.
Table I. The summary of evaluated lopodia morphology (number,
length, and width of lopodia) of CD4+ T-cells bound on thedifferent
nanohole arrays (NHA) using SEM analysis. For the comparison, the
results of control samples (planar glass substrates)were also
included.
Number of lopodia Filopodia length (nm) Filopodia width (nm)
Samples Average STDEV Average STDEV Average STDEV
Control 944 339 5475 2478 896 360NHA140 nm 639 194 6003 2304 802
129200 nm 667 201 6168 2590 831 392270 nm 511 160 6759 5713 1259
445550 nm 342 148 6734 2618 1412 521
Notes: STDEV: Standard deviation; Control: STR-functionalized
planar glass samples without nanostructure; NHA: Nanohole
arrays.
versus nanohole size shown in Figures 3(D), (E). Anotherpossible
explanation on the elevation in lopodia widthwith increasing the
NHA diameters (200550 nm) is theincreasing STR-conjugated surface
width (100270 nm).The amine containing in the STR-conjugated NHAs
pro-mote the actin extension to guide the development oflopodial
laments in the initial stage of the incubation.33
Subsequently, the lopodia of the captured T-cells startto
deliver more actin-rich lament along the shafts ofthe initial
lopodia for covering different widths of STR-NHAs.33 Therefore,
these results suggest that the lopo-dia of CD4+ T-cells interact
with the NHA substrate viaan initial high-afnity STR-biotin
conjugation19 followedby guided extension of lopodia on the
nanostructuresof the NHAs (Fig. 3(E)). Furthermore, we also
quanti-ed other morphological parameters such as the protru-sion
length of lopodia and the average number of thelopodial laments per
cell as summarized in Table Iand Figures 4(A)(D). In addition, the
cell-capture ef-ciency (n = 4) is plotted for the four different
NHAsin Figure 4(E). In Figures 4(A), (B), no statically sig-nicant
difference (NS, not-signicant) was observed inthe plot showing the
lopodial protrusion length of thecaptured CD4 T-cells versus
nanohole dimensions, indi-cating that the length of the protruded
lopodia is rela-tively independent of the dimensions of the NHAs in
theearly stage of cell capture (
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Kim et al. Filopodial Morphology Correlates to the Capture
Efciency of Primary T-Cells on Nanohole Arrays
Figure 4. (A) and (B) Protruded length of lopodia, (C) and (D)
the average number of extended lopodia per cell, and (E)
cellseparation yield (efciency) of mouse primary CD4+ T-lymphocytes
for four different sizes of STR-conjugated NHA substrates(140, 200,
270, and 550 nm). The protruded lopodia length and number of
lopodia per cell shown in (B) and (D) were takenfrom 80% of CD4+
T-lymphocytes of all bound cells shown in (A) and (C). To ensure
that the lopodia evaluation are staticallysignicant, at least 5262
cells, which are 80% of the bound cells (n = 7080) on NHAs
substrates, were used for the evaluationof lopodia morphology as
indicated in (C). In (E), the error bars represent the standard
error of mean from four repeats (n = 4).P values of < 00001 ()
considered statically signicant. An insignicant statistical
difference is represented as NS.
the STR-NHA substrates. This observation, which webelieve is
novel, can be explained by contact guidanceand cell-adhesion force.
When the T cells interact withSTR-conjugated NHA surfaces, they
develop lopodia tosense the surface, and then preferentially adhere
to theregion with features of similar dimensions. Afterwards
the
lopodia of the captured T-cells start to extend rapidly onthe
surface even within a short incubation time (20 min).
Filopodia Contact BehaviorThe length of lopodia including
lamellipodia (Fig. 4(A))protruding from primary mouse T-lymphocytes
on the
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Filopodial Morphology Correlates to the Capture Efciency of
Primary T-Cells on Nanohole Arrays Kim et al.
(A)
(B) (C)
(D)
Figure 5. (A) Fluorescence images and cell size histograms of
live primary CD4+ T-lymphocytes bound on four different
NHAsubstrates (140, 200, 270, and 550 nm in diameters) using
confocal microscopy. (B)(C) Cell size distribution (histogram) of
theCD4+ T-lymphocytes (n= 250300) on four different NHAs. The
results shown in (C) were taken from 80% of CD4+ T-lymphocytesof
all bound cells shown in (B). The actin and nuclei were stained by
rhodamine-conjugated phalloidin (Life technologies Co.,USA) and
4,6-diamidino-2-phenylindole dihydrochloride (DAPI, BD Bioscience,
USA), respectively. P values were calculated withneighboring column
data (P < 00282, P < 00061, and NS, not signicant). (D) The
summary of evaluated lopodia morphol-ogy (number, length, and width
of lopodia) of CD4+ T-cells bound on the different nanohole arrays
(NHA) using uorescencemeasurements. For the comparison, the results
of control samples (planar glass substrates) were also
included.
1038 J. Biomed. Nanotechnol. 10, 10301040, 2014
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Copyright: American Scientific Publishers
Kim et al. Filopodial Morphology Correlates to the Capture
Efciency of Primary T-Cells on Nanohole Arrays
Figure 6. Fluorescence images of live single CD4+
T-lymphocyte bound on NHA substrate (d = 270 nm) usingconfocal
microscopy. (A) Scanned uorescence images takenfrom the top to
bottom of the bound CD4 T-lymphocyte,showing the cross-sectional
view of the cell. ((B)(D)) and(F) show the enlarged images of
selected areas marked 1,2, 3, and 4, respectively, representing
clear actin-stainedlopodia shown in (F). (E) Schematic view of the
cells onNHA substrate for confocal scanning measurement. The
actinand nuclei were stained by rhodamine-conjugated phalloidinand
4,6-diamidino-2-phenylindole dihydrochloride (DAPI),respectively.
DAPI is a uorescent dye for labeling DNA.
NHA surface was 0.60 to 0.67 m determined by FE-SEM (Figs. 4(A),
(B)), which is relatively longer thanthe intrinsic nanoscale
structures (e.g., microvilli) on thesurface of human T cells
observed on planar cover-slip glass (0.25 to 0.78 m).35 The cell
lopodiaand microvilli are interrelated because the lopodia
ormicrospikes start to protrude when the cells contact thesubstrate
surface via microvilli and started to spread eventhough their sizes
were less than 1 m.25 However,in our study we observed that more
specically lopodia-substrate interaction is correlated to cell
capture efciencyand the microvilli at the interface of
cell-substrate werenot extensively observed. To further conrm the
develop-ment of the structural proteins of the actin-rich
lopodiafrom surface-bound CD4 T-cells on the NHA substrates,FM
analysis of live single CD4+ T-lymphocyte boundon NHA substrate (d
= 270 nm) was also performed(Figs. 6(A)(F)). Scanned FM images
taken from the topto bottom of the bound CD4 T-lymphocyte clearly
exhibitactin-rich lopodial protrusions in the early stage of
thecell-substrate interaction (
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Delivered by Publishing Technology to: Sang Ho JoIP:
165.194.103.14 On: Mon, 10 Feb 2014 05:23:15
Copyright: American Scientific Publishers
Filopodial Morphology Correlates to the Capture Efciency of
Primary T-Cells on Nanohole Arrays Kim et al.
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