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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 ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A Plasma-Deposited Surface for Cell Sheet Engineering: Advantages over Mechanical Dissociation of Cells 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 Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org 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. ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 517 518 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. Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org 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 ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 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). Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 519 520 H. E. Canavan, X. Cheng, D. J. Graham, B. D. Ratner, D. G. Castner 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 Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org 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 ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A Plasma-Deposited Surface for Cell Sheet Engineering: Advantages over Mechanical Dissociation of Cells 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). Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 521 522 H. E. Canavan, X. Cheng, D. J. Graham, B. D. Ratner, D. G. Castner 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 Plasma Process. Polym. 2006, 3, 516–523 www.plasma-polymers.org 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. [1] L. J. Anghileri, Oncology 1976, 33, 17. [2] J. Bayad, N. Sabolovic, D. Bagrel, J. Magdalou, G. Siest, J. Pharmacol. Meth. 1991, 25, 85. [3] B. P. Chelobanov, P. P. Laktionov, M. V. Khar’kova, E. Y. Rykova, D. V. Pyshnyi, I. A. Pyshnaya, V. N. 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