Download Advantages over Mechanical Dissociation of Cells

516
Full Paper
DOI: 10.1002/ppap.200600017
Summary: Previously, we compared the release of cell sheets
via low-temperature liftoff from ppNIPAM treated surfaces to
cell removal by enzymatic digestion and mechanical dissociation. We made two interesting observations, namely: 1)
mechanical dissociation of cells from surfaces leaves behind a
surface that promotes new cell growth, and 2) that ToF-SIMS
data from post-mechanical dissociation surfaces differed from
the other surfaces primarily due to the presence of hydrocarbon
species and a fragment at m/z ¼ 86 which could be attributed to
either amino acids (leucine/isoleucine) or lipid molecules. In
this study, we examine in detail the effect that mechanical
dissociation has on cell removal. Using PCA of the ToF-SIMS
data, we observe a clear separation of the post-mechanical
dissociation surfaces from controls. We find that postmechanical dissociation surfaces are primarily characterized
by the presence of high mass fragments (indicative of lipid
molecules), and the absence of certain low mass fragments
indicative of protein. These findings support the hypothesis
that the process of mechanical disruption ruptures cell walls,
leaving lipid molecules from the cell membrane on the surface.
Removal of adhered cultured cells by mechanical dissociation.
A Plasma-Deposited Surface for Cell Sheet
Engineering: Advantages over Mechanical
Dissociation of Cells
Heather E. Canavan,*1,3,5 Xuanhong Cheng,2,3 Daniel J. Graham,1,3 Buddy D. Ratner,1,2,3,4 David G. Castner1,2,3,4
1
National ESCA and Surface Analysis Center for Biomedical Problems, Box 351750, University of Washington, Seattle,
Washington 98195, USA
2
University of Washington, Engineered Biomaterials, Box 351750, University of Washington, Seattle, Washington 98195, USA
3
Department of Bioengineering, Box 351750, University of Washington, Seattle, Washington 98195, USA
4
Department of Chemical Engineering, Box 351750, University of Washington, Seattle, Washington 98195, USA
5
Department of Chemical and Nuclear Engineering, MSC 01 1120, University of New Mexico, Albuquerque,
NM 87131-0001, USA
E-mail: [email protected]
Received: March 7, 2006; Revised: May 30, 2006; Accepted: June 12, 2006; DOI: 10.1002/ppap.200600017
Keywords: cell culture; extracellular matrix (ECM); plasma polymerization; poly(N-isopropyl acrylamide) (pNIPAM);
secondary ion mass spectrometry (SIMS)
Introduction
Traditionally, the removal of cells cultured on tissue culture
polystyrene (TCPS) requires harsh methods such as enzymatic digestion or mechanical manipulation, which affect
the morphological appearance of the cells being harvested.
For instance, cell removal by enzymatic digestion tends to
yield disaggregated cells with a rounded appearance; cell
removal by mechanical dissociation is generally observed
to yield a few, isolated cells surrounded by a crystalline
matrix.[1–5] It follows that such readily observed effects on
Plasma Process. Polym. 2006, 3, 516–523
the cells harvested must be concurrent with damage to the
extracellular matrix (ECM) underlying the cells. In fact, it
has been observed that these methods affect the behavior
and chemical makeup of the cells themselves, as studied by
IR and lactate dehydrogenase assay.[6,7]
For these reasons, cell release using low-temperature liftoff
from poly(N-isopropyl acrylamide) (pNIPAM) treated surfaces is being investigated as a non-destructive cell harvest
alternative. Using pNIPAM, it is possible to rapidly recover
intact cell sheets from culture surfaces using only a modest
temperature drop as the means of detachment.[8–13] Surfaces
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treated with pNIPAM have been observed to undergo a transition above and below the lower critical solution temperature
(LCST) at 31 8C. Above the LCST (i.e., at the cell culture
temperature of 37 8C), the surfaces are observed to be more
hydrophobic (as observed by water contact angle), and many
cell types will adhere and proliferate. Below the LCST (i.e., at
room temperature) the surface rapidly hydrates, and cell
monolayers will spontaneously detach as a sheet. This behavior is surprising, as cells grown on regular TCPS will remain attached for hours or days, requiring enzymatic
digestion or physical scraping to detach them.[14] This sharp
property change due to a thermal transition around physiological temperatures has piqued the interest of many in the
biomaterials community, and pNIPAM has been used as a
culture platform for a variety of cell types. Urothelial, vascular
smooth muscle, liver, lung and spleen cells have all been
found to respond to pNIPAM by exhibiting confluent monolayer detachment.[15–19]
Although there are numerous methods of fabricating
pNIPAM-treated substrates,[9–11] it is advantageous to use
plasma polymerization of NIPAM (ppNIPAM), as it affords
a one-step, solvent-free method for producing conformal,
tightly adhering and ultrathin coatings that are compatible
with TCPS, the substrate traditionally used for cell culture.[20] Previously low-temperature liftoff of a monolayer
of bovine aortic endothelial cells (BAECs) from ppNIPAM
surfaces was found to remove the majority of the ECM
components, but leave some protein on the ppNIPAM surface.[21] In a comparison of the effect that different cell
removal methods (cell low-temperature liftoff, enzymatic
digestion, and mechanical dissociation), low-temperature
liftoff was found to be superior in terms of maintaining
intact cell sheets as well as ECM viability.[14]
In addition to those findings, mechanical dissociation of
cells from surfaces was found to leave behind a surface that
promoted new cell growth and those surfaces differed from
the post-enzymatic digestion and post- low-temperature
liftoff surfaces. X-ray photoelectron spectroscopy (XPS)
showed that mechanical dissociation of cells left behind a
surface with increased hydrocarbon character. Using
principal component analysis (PCA) to aid in the interpretation of time-of-flight secondary ion mass spectrometry
(ToF-SIMS) data confirmed that the majority of the
fragments obtained from post-mechanical dissociation
were from hydrocarbons, with the exception of a fragment
at m/z ¼ 86.
That publication mentioned, without elaboration, the
interesting effects of mechanical dissociation on cell removal.
Therefore, this study is focused on the combined use of PCA
with ToF-SIMS data to thoroughly examine mechanical
dissociation of cells on the ECM. We find that there is a clear
separation of the post-mechanical dissociation surfaces from
controls. Furthermore, it was demonstrated that this difference
is primarily due to the presence of lipid fragments on the postmechanical dissociation surfaces and NIPAM monomer
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fragments on the control surfaces. These findings support
the hypothesis that mechanical disruption ruptures the cell
walls during the cell removal process and deposits lipid
fragments onto the surface.
Experimental Part
Materials
Cell culture supplies were purchased from Gibco Invitrogen
Corporation (Carlsbad, CA) and filtered though 0.2 mm filters
before use. BAECs were a generous gift from Dr. Cecilia
Giachelli (University of Washington, Seattle, Washington).
TCPS 48-well plates were from Falcon (BD Biosciences,
Franklin Lakes, NJ). N-Isopropyl acrylamide (NIPAM, 97%þ)
monomer was purchased from Aldrich (Sigma-Aldrich, St.
Louis, MO), and used as received.
Methods
pNIPAM Deposition
There are many techniques to prepare NIPAM surfaces, including
by co-grafting pNIPAM with other polymers,[22] immobilizing
pNIPAM by photolithography,[23] and by polymerizing pNIPAM
with previously activated surfaces.[24] The pNIPAM surface
deposition used in this study is produced using a plasma polymerization technique to produce ppNIPAM.[20] This technique is
a one-step, solvent free and vapor-phase method for producing
conformal, sterile, tightly adhering and ultrathin coatings. Previously, we demonstrated that the transition of ppNIPAM occurs
at 31–32 8C, with the surface mechanical properties, wettability
and chemistry all changing in this temperature range.[25]
ppNIPAM deposition was carried out in a custom-built
reactor using the protocol described earlier.[20] In brief, there
are four primary components of the plasma polymerization
apparatus: reactor vessel, monomer delivery system, pumping
and pressure control system, and the radio frequency power
components.
The reactor vessel consists of a cylindrical glass chamber,
two external electrodes spaced 5 inches apart, and a glass
sample stage placed between the electrodes and parallel to the
direction of gas flow in the reactor. The monomer delivery
system consists of a 100 mL volume glass flask containing the
NIPAM monomer. The flask is connected to a metering needle
valve used to control the monomer delivery rate. Due to the low
volatility of NIPAM, the monomer flask and the monomer
delivery line are heated (at 72 and 80 8C, respectively) during
the deposition process. The pumping and pressure control
system consists of a mechanical pump used to evacuate the
reactor to the desired base pressure (low 103 Torr range), as
well as a liquid N2-cooled cold trap between the vacuum pump
and the reactor used to condense organic materials. The electronic components consist of a manual impedance matching
network and a 13.56 MHz radio frequency power source,
which is connected to the powered electrode (the other
electrode is grounded). As soon as the radio frequency power
is applied to the powered electrode, a uniform glow discharge is
observed primarily between the two electrodes.
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H. E. Canavan, X. Cheng, D. J. Graham, B. D. Ratner, D. G. Castner
The deposition process includes an 80 W methane plasma
deposition to form an adhesion-promoting layer, as well as
ppNIPAM plasma deposition occurring at stepwise decreasing
powers from 80 W to 1 W, with a processing pressure of 140
mTorr. After the deposition process is completed and the
residual organic vapors are pumped out of the reactor, the
substrates are removed from the chamber and rinsed three
times with cold deionized water to remove uncrosslinked
molecules before use in culture.
Cell Culture
BAECs were cultured in Dulbecco’s Modification of Eagle’s
Medium (DMEM) supplemented with 4.5 g L1 glucose, 10%
fetal bovine serum (FBS), 0.1 103 M MEM non-essential
amino acids, 1 103 M MEM sodium pyruvate, 100 U mL1
penicillin and 100 mg mL1 streptomycin. BAECs cells used
in the experiments were between passage 7 and 15. [The term
‘‘passage’’ refers to the number of times that a cell population
has been removed from the culture vessel and undergone a
subculture (or passage) process in order to keep the cells at a
sufficiently low density to stimulate further growth.] Cell
incubation was performed at 37 8C in a humidified atmosphere
with 5% CO2. The cells were dissociated from the culture
flasks with trypsin/ethylenediaminetetraacetic acid (EDTA)
and then washed with Dulbecco’s Phosphate Buffered Saline
(DPBS) before seeding onto 48-well plates.
Cell Removal
BAECs were plated from complete media containing 10% FBS
into either a 48 well plate at a cell density of 2.5 104 cells/
well (for enzymatic digestion and lift-off) or a 12-well plate at a
cell density of 5 104 cells/well (for mechanical dissociation)
and cultured at 37 8C. The wells were ppNIPAM-coated.
BAECs were cultured until confluence. Cell removal took
place by one of three methods: low-temperature lift-off, enzymatic digestion, or mechanical dissociation using a silicone
scraper. To test cell lift-off behavior, the culture media were
removed and replaced with serum free DMEM, and the cells
were incubated at room temperature for 2 h for cell sheet
detachment. To test the effect of enzymatic digestion with
trypsin, BAECs were washed with pre-warmed DPBS and
incubated with 0.25% trypsin containing 1 mM EDTA (GibcoInvitrogen, Carlsbad CA) at 37 8C for 5 min. The wells were
rinsed 3 times with DPBS to wash off the cells. To test the effect
of mechanical dissociation, BAEC layers were removed from
the surfaces with a rubber blade (Corning, Corning NY) in
DPBS buffer and rinsed with DPBS.
Sample Preparation for Surface Analysis
To obtain the samples used for analysis using high vacuum
techniques, the wells were washed with deionized water three
times, and soaked in water for at least 24 h to reduce free ions
remaining from the buffer. Next, the well bottoms were harvested by slicing them using a heated NiCr wire. The surfaces
were then packed in sealed, inert containers backfilled with N2,
and stored until analysis.
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Figure 1. High-resolution XPS C 1s spectrum of ppNIPAMtreated TCPS.
For each sample type, at least six replicates were prepared,
from which four were analyzed by ToF-SIMS. The remaining
two replicates were analyzed using XPS, and their surface
compositions were similar to previously reported values.[14,21]
The composition of the ppNIPAM-treated TCPS 48-well plates
(78.8% C, 14.0% O, and 7.2% N) differs slightly from that
predicted from the stoichiometry of the NIPAM monomer
(75.0% C, 12.5% O, and 12.5% N), and from ppNIPAM coatings on silicon chips produced using our method (76.2% 8C,
11.3% O, and 12.5% N). It is possible that this difference is due
to the geometry of the well preventing the gas species in the
plasma from coating the well bottom in an identical manner to
that on flat substrates. Whatever the source of this difference,
approximately 2/3 of the monomer structure is retained in
the plasma polymerized film, which is sufficient to yield the
temperature-dependant behavior of proteins[26] and cells,[14,21,26]
as well as in the surface wettability as determined by contact
angle measurements.[25] Figure 1 presents a representative highresolution C 1s spectrum from ppNIPAM-treated TCPS.
XPS Analysis
XPS data were acquired on Surface Science Instruments
X-Probe and S-Probe instruments. Each of these systems is
equipped with monochromatized aluminum Ka X-ray sources,
an electron flood gun for charge neutralization, and a hemispherical electron energy analyzer. All survey scans for compositional analyses were acquired at pass energy of 150 eVand
all high-resolution scans were acquired at a pass energy of
50 eV. Compositional analyses (0–1100 eV) and high resolution scans of the C 1s regions were carried out on all samples.
Data treatment was performed on the Service Physics
ESCAVB data reduction software. Binding energies for highresolution spectra were referenced to the C 1s (C–C/C–H) peak
at 285.0 eV to account for binding energy shifts inherent
to insulator samples. Core-level spectra were peak-fit using
the minimum number of peaks possible to obtain random
residuals. The binding energy shift of the C–N/C–OH
peak was constrained to þ1.5 eV from that of the C–H peak.
A 100 percent Gaussian line shape was used to fit the peaks, and
a Shirley function was used to model the background. At least
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A Plasma-Deposited Surface for Cell Sheet Engineering: Advantages over Mechanical Dissociation of Cells
two replicates were prepared with each sample, with three
spectra acquired on each replicate.
ToF-SIMS Analysis
A Model 7200 Physical Electronics instrument (PHI, Eden
Prairie, MN) with an 8 keV Csþ ion source and a reflectron
time-of-flight mass analyzer was used for static ToF-SIMS data
acquisition. Positive secondary ions mass spectra were acquired over a mass range from m/z ¼ 0 to 450. (Note: only those
masses in the range from m/z ¼ 0 to 250 are shown in the
loadings plot, as few peaks of interest were observed beyond
this range in this study.)
The area of analysis for each spectrum was 100 100 mm2,
and the total ion dose used to acquire each spectrum was less
than 2 1012 ions cm2. The mass resolution (m/Dm) of the
secondary ion peaks in the positive spectra was typically
between 4 000 and 6 000. The ion beam was moved to a different spot on the sample for each spectrum. The mass scales of
þ
the positive spectra were calibrated using the CHþ
3 , C2H3 ,
þ
þ
C3H5 , and C7H7 peaks. At least four replicates were prepared
for each sample type, with five spectra acquired on each replicate. ToF-SIMS spectra containing a sodium ion peak intensity
that was >1% of the total intensity of the selected peaks were
discarded due to matrix effects of the sodium ion on the SIMS
fragmentation process.[27]
PCA
A detailed discussion of PCA can be found in the work by
Jackson or Wold.[28,29] Briefly, PCA is a multivariate analysis
technique that analyzes the variance patterns within a data set
to find the directions of greatest variance. The variance in the
data set describes the differences (or spread) between the
samples within the set. PCA is the singular value decomposi-
tion of the variance covariance matrix of the data. In effect,
PCA is a matrix rotation that creates a new set of axes (principal
components, PCs) that define the directions of major variation
within the data set.
This process results in a reduction of variables as the original
variables are recombined to define the new principal component axis. Thus, a large data set with hundreds of variables
can be reduced to a few easy to manage variables that can be
interpreted using a series of simple plots. This decomposition
results in the creation of three new matrices: scores, loadings,
and residuals. The scores describe the relationship between the
samples. They represent the amount of the PC in each sample,
and are the projection of the samples onto the PC axes.
The loadings are defined as the direction cosines between the
original variables and the new PCs. Loadings define the
contributions of the original variables to the new PCs, and
describe which variables are responsible for the differences
seen within the samples. For example, variables with high
positive loadings on a given PC correspond, in general, with
samples with positive scores on the same PC. In the case of
ToF-SIMS data, this means that variables with high loadings
have higher relative intensities in the spectra for these samples.
The residual matrix describes the random variations not
described by the new PC axis and represents noise in the data.
PCA Data Processing
All ToF-SIMS spectral analysis was carried out using the PLS
Toolbox v. 2.0 (Eigenvector Research, Manson, WA) for
MATLAB (the MathWorks, Inc., Natick, MA). First, all spectra
were mean-centered prior to PCA analysis. Next, a limited
peak set was constructed to compare the positive ToF-SIMS
spectra from all sample types (ppNIPAM control; ppNIPAM
after cell removal by mechanical dissociation; ppNIPAM after
cell removal by enzymatic digestion; and ppNIPAM after cell
removal by low-temperature liftoff). This limited peak set was
constructed using unique amino acid fragmentation patterns of
Figure 2. Removal of adhered cultured cells (a) requires either enzymatic digestion (b), mechanical
dissociation (c), or cell sheet detachment by low-temperature liftoff from ppNIPAM (d).
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ToF-SIMS data previously identified for characterizing
proteins,[27] fragments previously identified by Winograd
et al., indicative of lipid species,[30,31] as well as those fragments indicative of NIPAM monomer[25] (see Table 1).
The peak areas for each spectrum were then normalized to
the intensity of the sum of the selected peaks to account for
fluctuations in secondary ion yield between different spectra.
PCA was then used to analyze the positive ToF-SIMS spectra,
generating the scores and loadings plots in Figure 3.
Table 1. Limited peak set used to compare control ppNIPAM
surfaces to surfaces from which cells were removed via
mechanical dissociation. The exact mass of each fragment in the
table is listed with the chemical species identified with that mass,
as well as the source that moiety is predicted to have arisen from.
Asterisk indicates that the fragment has multiple sources, and
therefore cannot be uniquely identified.
m/z
Species
Source
30.0352
43.054
55.0193
58.0295
58.06610
59.0498
60.0449
61.0103
68.0508
69.0358
70.0312
70.0672
71.015
72.0489
72.0841
73.0644
74.0646
81.0458
83.0513
84.0478
85.0438
86.0999
87.0582
88.0425
91.05590
95.0588
98.0252
100.089
107.051
110.0735
113.0362
114.09410
115.0549
120.0836
120.9768
121.0401
127.1005
130.068
132.0595
136.0803
142.0782
159.1213
166.0722
184.0873
224.1158
CH4N
C3H7
C3H3O
C2H4NO
C3H8N
CH5N3
C2H6NO
C2H5S
C4H6N
C4H5O
C3H4NO
C4H8N
C3H3O2
C3H6NO
C4H10N
C2H7N3
C3H8NO
C4H5N2
C5H7O
C5H10N
C3H5N2O
C5H12N
C3H7N2O
C3H6NO2
C7H7
C5H7N2
C4H4NO2
C4H10N3
C7H7O
C5H8N3
C4H5N2O2
C6H12NO
C4H7N2O2
C8H10N
C8H10N
C6H5N2O
C5H11N4
C9H8N
C9H8O
C8H10NO
C2H9NPO4
C10H11N2
C4H9NPO4
C5H15NPO4
C8H19NPO4
glycine
lipid
tyrosine
glycine
NIPAM
arginine
L-serine
methionine
proline
threonine
*
arginine, proline, leucine
L-serine
glycine
valine
arginine
threonine
histidine
valine
glutamine
glycine
*
*
*
TCPS
histidine
asparagine
arginine
tyrosine
*
glycine
NIPAM
glycine
phenylalanine
phenylalanine
histidine
arginine
tryptophan
phenylalanine
tyrosine
lipid
tryptophan
lipid
lipid
lipid
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Figure 3. Results from PCA of positive ion ToF-SIMS data
comparing control ppNIPAM surfaces to surfaces where cells
were removed via mechanical dissociation. In the scores plot (top),
spectra from ppNIPAM control surfaces (samples 1–15) are
distinctly separated from surfaces from which cells were removed
via mechanical dissociation (samples 16–30). The loadings plot
(bottom) indicates that this separation is primarily due to the
presence of NIPAM monomer fragments (m/z 58, 114) present in
the ppNIPAM controls, and the presence of lipid fragments (m/z
86, 184, 224) present in the post-mechanical dissociation surfaces.
Results and Discussion
Previously, we compared the release of cell sheets via lowtemperature liftoff from ppNIPAM treated surfaces as a
non-destructive alternative for cell removal to enzymatic
digestion and mechanical dissociation[14] (see Figure 2). In
this study, we combine PCA with positive ion ToF-SIMS
data to thoroughly examine the effect that mechanical
dissociation has on the ECM. Post-mechanical dissociation
surfaces were compared to control ppNIPAM surfaces that
were not used for cell culture. In contrast to the previous
study, where a ‘‘complete’’ peak set was constructed by
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using all of the major peaks in the 0–200 m/z region from
each sample type (i.e., surfaces after cell removal by lowtemperature liftoff, enzymatic digestion, and mechanical
dissociation), the current analysis used a ‘‘limited’’ peak
set: only fragments previously identified as arising from
amino acids,[27,32] lipid moieties,[30,31] and those indicative
of NIPAM monomer[25] (see Table 1).
Figure 3a shows a scores plot of principal component 1
(PC 1), which captures 81% of the variance in the data
comparing post-mechanical dissociation surfaces to control
ppNIPAM surfaces. Examination of Figure 3a shows the
surfaces after cell removal by mechanical dissociation
(samples 16–30) are clearly distinguished from control
ppNIPAM surfaces (samples 1–15). The dashes drawn
around each group indicate the 95% confidence interval of
each grouping.[27] Examination of Figure 3 indicates that
PCA is clearly able to distinguish between the control
ppNIPAM surfaces and the post-mechanical dissociation
surfaces. This suggests that mechanical digestion does not
completely remove the ECM and fully expose the underlying ppNIPAM surface.
Figure 3b represents the loadings for PC 1. Each of the
peaks that load negatively in the PC 1 loadings plot
corresponds to samples with negative scores in the scores
plot; each of the peaks that loads positively in the PC 1
loadings plot corresponds to samples with positive scores in
the PC 1 scores plot. Examination of Figure 3a and 3b
indicates that fragments originating from the NIPAM
monomer (m/z 58: C3H8N, and m/z 114: C6H12NO) both
load negatively, and correspond to the blank ppNIPAM
surface. In comparison, there are high mass fragments that
load positively (m/z 166: C4H9NPO4, m/z 184: C5H15NPO4,
and m/z 224: C8H19NPO4), that correspond to the postmechanical dissociation surfaces. In previous studies by
Winograd et al., these fragments have been correlated to
the phosphocholine headgroup of phosphatidylcholine, a
component of the plasma membrane of cells.[30,31] As noted
previously, m/z 86 (C5H12N) fragment associated with the
Figure 4. Plots showing normalized ToF-SIMS positive ion data for m/z 114 (a), 184 (b), 70 (c), and 61 (d). m/z 114,
which corresponds to the C6H12NO moiety of the NIPAM monomer, is present in the ppNIPAM control (blank) and
trypsinized surfaces (a). m/z 184, which corresponds to the C5H15NPO4 moiety of lipids, is present in the post-scrape
surfaces (b). m/z 70 and 61 – which correspond to the C4H8N and C2H5S moieties of the amino acids arginine and
methionine, respectively – are present in the post-liftoff and post-trypsinized surfaces (c,d).
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post-mechanical dissociation surfaces could arise from
either the headgroup of phospholipids or from the amino
acids leucine and isoleucine.[27]
Figure 4a–d represents plots showing normalized
ToF-SIMS positive ion data for the masses that contributed
the largest amount of variance when comparing blank
ppNIPAM control surfaces to surfaces from which cells had
been removed either by enzymatic digestion, mechanical
dissociation, or low-temperature liftoff.
Figure 4a presents the raw data for m/z 114, which
corresponds to the C6H12NO moiety of the NIPAM
monomer. Examination of Figure 4a indicates that this part
of the NIPAM monomer is associated with both the
ppNIPAM control (blank) surface and the post-enzymatic
digestion surfaces. It is interesting to note that, of the three
removal methods, enzymatic digestion leaves behind a
surface most similar to that of blank ppNIPAM, but that
there is a great deal of variance in this data. Previously the
blank ppNIPAM surface was easily distinguished from the
post-enzymatic digestion surfaces;[14] however, that result
was primarily based on the presence of salt ions (e.g., Naþ,
Kþ) on the surface after cell removal by enzymatic
dissociation. It was proposed that the salt ions most likely
arose from salts associated with the trypsin/EDTA solution
used to remove the cells from the surface. Their presence
persisted despite repeated and extended rinsing procedures
used to remove salts from the surfaces. The current analysis
used a peak set limited to only those fragments identified
as arising from amino acids,[27] lipid moieties,[30,31] and the
NIPAM monomer;[25] therefore the presence of these salt
ions does not directly influence the current analysis.
Furthermore, we previously noted that the variation within
this group was extremely large, and postulated that it
reflected the fact that treatment with trypsin does not affect
the surfaces in a regular, predictable manner, even when
those surfaces are prepared at the same time, in the same
TCPS culture plate. This may, in turn, be a reflection of a
heterogeneous distribution of ECM proteins being produced by the cells. The fact that fragments indicative of the
ppNIPAM substrate are observed on the post-enzymatic
digestion surface seems to confirm this idea.
In contrast, Figure 4b presents the raw data for m/z 184,
which corresponds to the C5H15NPO4 moiety of lipids. As
with the PCA results presented in Figure 3, the raw data
indicates that the lipid fragments correspond most strongly
with the post-mechanical dissociation surfaces. In contrast,
the raw data indicates that neither the ppNIPAM control nor
the post-enzymatic digestion surfaces are characterized by
lipid fragments. The low-temperature liftoff surfaces are
associated with lipid fragments, though to a lesser extent than
mechanical dissociation. Previous studies have demonstrated
by XPS that low-temperature liftoff surfaces have less
hydrocarbon character than post-mechanical dissociation
surfaces.[14] However, in that data, as in the current data, there
is a high degree of variability. This suggests that the
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detachment of cell sheets from ppNIPAM is not a smooth
fracture of the ECM interlayer, but may occasionally rupture
cell/ECM junctions, along with protein/protein or protein/
surface interfaces. The sensitivity of the ToF-SIMS method
coupled with PCA provides a unique tool to pursue this
mechanism, which will be done in future publications.
Finally, Figure 4c and 4d present the raw data for m/z 70 and
61, which correspond to the C4H8N and C2H5S moieties of the
amino acids arginine and methionine, respectively. These
fragments – which are indicative of protein being left at the
surface – are associated with both the low-temperature liftoff
and post-enzymatic digestion surfaces. However, the postmechanical dissociation surfaces are not associated with
protein. These results are consistent with the previous XPS
findings that the amount of hydrocarbon present after
mechanical dissociation is second only to blank ppNIPAM.[14]
From these results, it is concluded that cell removal by
mechanical dissociation ruptures cell walls during the
removal process. This hypothesis is consistent with the
previous observation that mechanical dissociation affects
the morphological appearance of the cells being harvested,
yielding a few, isolated cells surrounded by a crystalline
matrix,[1–5] possibly due to the disruption of the cellular
membranes and glycocalyx.[6,7] That these high-mass lipid
fragments are not found after cells are removed by enzymatic digestion is consistent with the action of trypsin as a
proteinase (i.e., ECM-protein disruptive rather than cell/
cell junction disruptive).[33] Furthermore, this indicates that
it is the presence of these lipids from the plasma membrane
(blebs), and not the presence of ECM proteins, that make
surfaces from which cells were mechanically dissociated
supportive of cell growth (as previously demonstrated).[14]
Conclusion
In a previous study comparing the effect that different cell
removal methods (low-temperature liftoff, enzymatic digestion, and mechanical dissociation) have on cell viability,
it was found that mechanical dissociation of cells from
surfaces left behind a surface that clearly promoted new cell
growth. However, the composition of those surfaces differed
from the post-enzymatic digestion and low-temperature
liftoff surfaces. In this study, PCA analysis of ToF-SIMS data
showed a clear separation of the post-mechanical dissociation surfaces from controls, due primarily to the presence of
lipid fragments and the absence of NIPAM monomer
fragments on post-mechanical dissociation surfaces. These
observations are consistent with the hypothesis that the
process of mechanical disruption breaks cell walls, leaving
lipids on the ppNIPAM surface.
An additional implication of this work relates to mechanisms of cell death. In contrast to the apoptotic mechanism,
the obvious disruption of the lipid membranes seen here with
mechanical harvesting will release intracellular contents
ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A Plasma-Deposited Surface for Cell Sheet Engineering: Advantages over Mechanical Dissociation of Cells
leading to inflammation. Thus, cells harvested by mechanical
means will always be suspect when used in follow-up studies
since inflammatory mediators may be present. Cells harvested
by enzymatic or thermal lift-off methods should be more
suitable for use in subsequent research. However, the damage
to the ECM from enzymatic harvesting may also impact
subsequent studies with harvested cells.
Acknowledgements: This research was supported by NIBIB
grant EB-002027 to the National ESCA and Surface Analysis
Center for Biomedical Problems (NESAC/BIO), and NSF ERC
grant EEC-9529161 to University of Washington Engineered
Biomaterials (UWEB). The authors thank Winston Ciridon, Max
Greenfeld, Roger Michel, and Jamie Reed for supplies, helpful
discussions, and expertise.
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