Invited Feature Article pubs.acs.org/Langmuir
Easy to Apply Polyoxazoline-Based Coating for Precise and Long-Term Control of Neural Patterns Serge Weydert,† Stefan Zürcher,‡ Stefanie Tanner,† Ning Zhang,†,§ Rebecca Ritter,† Thomas Peter,† Mathias J. Aebersold,† Greta Thompson-Steckel,† Csaba Forró,† Markus Rottmar,∥ Flurin Stauffer,† Irene A. Valassina,‡ Giulia Morgese,⊥ Edmondo M. Benetti,⊥ Samuele Tosatti,‡ and János Vörös*,† †
Laboratory of Biosensors and Bioelectronics, ETH Zurich, Gloriastrasse 35, 8092 Zurich, Switzerland SuSoS AG, Lagerstrasse 14, 8600 Dübendorf, Switzerland § State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, 210096 Nanjing, China ∥ Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, 9014 St. Gallen, Switzerland ⊥ Laboratory for Surface Science and Technology, ETH Zürich, Vladimir-Prelog-Weg 5, 8093 Zurich, Switzerland ‡
S Supporting Information *
ABSTRACT: Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottomup neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building welldefined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer’s ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.
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studying cytoskeleton development10 and morphogenesis;11 and for controlling tissue organization.12 While a porous surface topography, combined with lubricants and hydrophobic coatings,13,14 or oleophobic coatings,15 can be used to render surfaces antifouling, the most popular approaches
INTRODUCTION Anti-, non-, and low-fouling coatings are surface treatments which prevent the adhesion of proteins, cells, and microorganisms and are relevant in diverse fields, among them the medtech industry where they are used for implants,1 catheters,2 contact lenses,3 and drug-delivery systems.4 They also play an important role in reducing nonspecific binding in biosensing.5 In biological research, antifouling coatings have been extensively used in vitro for studies where the geometry of cell patterns is of importance,6 e.g., for controlling cell−cell contacts,7 the microenvironment around stem cells,8 and apoptosis;9 for © XXXX American Chemical Society
Special Issue: Surfaces and Interfaces for Molecular Monitoring Received: April 27, 2017 Revised: July 4, 2017
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connect to neighboring cells over unattractive substrates.54 Undesired connections and uncontrolled network outgrowth after 2 weeks is common throughout the whole field. The most promising results for stability were shown with PLL-g-PEG (>95% pattern occupancy after DIV 940 and many studies presenting promising images but no pattern failure statistics for more than 1 week in vitro55−60) and reasonable results were also reported with microcontact printing on agarose,61 but quantitative statistics for pattern compliance are missing in the field. Several issues related to PLL-g-PEG have been identified. It is known that PEG is subject to oxidation under long-term cell culture conditions.17,19,62 To this end, poly(2-methyl-2-oxazoline) (PMOXA) is a promising alternative, as it has similar molecular properties such as PEG (hydrogen-bond acceptor, polar, no hydrogen-bond donors, no net charge) and is equally excellent in rendering surfaces nonfouling63,64 but is more stable in oxidative environments.33,65 PLL-g-PEG has been shown to lose 40−50% of the coating thickness in representative tests with 10 mM hydrogen peroxide solutions, whereas for PLL-gPMOXA the reduction was below 20%.65 Also, a better pattern stability for PLL-g-PMOXA than for PLL-g-PEG was reported for endothelial cells and fibroblasts cultivated on 2D micropatterns.62,66 Another potential problem for PLL-based coatings is potential detachment at high ionic strength or with changing pH as well as exchange processes with biomolecules in solution.67,68 By adding catechol side chains to PLL next to PEG, an idea first suggested in 2005,69 increased stability was demonstrated on titanium, whereas the nonfouling performance was still excellent.67 Just last year, this idea was amended by using dimethylsilanol instead of catechol for covalent bonding to tissue culture surfaces such as glass or plasma-treated polystyrene, and increased stability in high-ionic-concentration buffers and detergents was demonstrated.68 For example, after overnight exposure to a physiological NaCl concentration of 150 mM, the purely electrostatically bound PLL-g-PEG analogue, PAcrAm-g-(PEG, NH2), lost 40% of its original thickness, and after incubation in 2 M NaCl, it was not protein-resistant anymore. In the same study,68 it was shown that additional dimethylsilanol side chains made the coating and also its antifouling property stable in salt water. For the synthesis, instead of PLL, Serrano et al. used reactive poly(pentafluorophenyl acrylate) as a backbone and N-boc-hexanediamine as an electrostatic component, equivalent to the amine in PLL. (See Figure 2 and Scheme S3 for the synthesis.) Despite the reported improvements in stability, the PMOXA approach and the idea of adding dimethylsilanol side chains have not been tested for cell patterning and also have never been combined. The presented work merges oxidation-resistant PMOXA polymer with positively charged amines for spontaneous adhesion and dimethylsilanol side chains for covalent bonding to culture substrates. When poly(pentafluorophenyl acrylate) was used as a backbone, poly(acrylamide)-g-(PMOXA, 1,6hexanediamine, 3-aminopropyldimethylsilanol) was synthesized as a promising new candidate for a flexible and stable antifouling modification of glass and polymer cell culture substrates (Figure 1). Its protein resistance was characterized, and the performance in maintaining adhesion inhibition in patterned long-term cultures was compared to the gold standard, PLL-g-PEG. An assay with primary embryonic rat neurons on isolated adhesion spots was selected, and as a property meaningful to bottom-up neuroscience, the formation of unwanted connections between neuronal nodes was quantified. The versatility of the presented coating is demonstrated with long-term cultures of fibroblasts
use hydrophilic polymers (such as the widespread poly(ethylene glycol) (PEG), polysaccharides (e.g., agarose), and polyamides) and zwitterionic polymers.16−19 They all share, with a few exceptions, the existence of hydrogen-bond acceptors and polarity as well as the absence of hydrogen-bond donors or net charge. Their nonfouling functionality is based on preventing the adhesion of proteins and other biomolecules, which normally is the first step in the biofouling cascade. Simple steric hindrance and the formation of a thick hydration layer around the coating are responsible for this effect, which makes the surface behave almost like water when interacting with close-by biomolecules. In this context, there has lately been increased interest in the scientific community in zwitterionic polymers because their hydration shell has been found to be especially strong and dense20,21 and their performance in protein resistance is very good.22 They can be classified in polybetaines (positively and negatively charged groups on the same monomer) and polyampholytes (positively and negatively charged groups on different monomers). In a recent study, poly(sulfobetainemethacrylate) has been reported to yield a higher signal-to-noise ratio than PEG when used in biosensing for the selective capturing of disease-related proteins.23 Nevertheless, PEG is currently still the best-studied and the most-used and -referenced antifouling polymer and stands out for very good protein resistance as well as for the inhibition of cell and bacterial adhesion.19,24−26 A widely used means to functionalize surfaces with PEG is copolymer poly(L-lysine)-graf t-poly(ethylene glycol) (PLL-g-PEG) with excellent nonfouling properties,27−30 and for geometrically controlling cell adhesion, Lussi et al. reported good stability of up to 2 weeks31 in a cell culture. The major advantage of PLL-g-PEG is its ability to spontaneously adhere from buffered solutions onto various negatively charged surfaces such as glass, metal oxides, and tissue culture polymers and silicon when plasma-activated. This reduces the coating procedure to a simple dipping/pipetting step; therefore, PLL-g-PEG evolved to become the gold standard for quick and versatile nonfouling surface modification.32,33 For cell adhesion studies, it is often used for backfilling, e.g., after microcontact printing of an adhesion promoter molecule such as poly(L-lysine) or extracellular matrix proteins, which is a fast and convenient method for surface patterning.34 In the past decade, surface patterning techniques using antifouling coatings also started to play a role in neuroscience. An emerging subfield in the 21st century is bottom-up neuroscience,35 which promotes the approach of understanding the brain step-by-step via engineering and analyzing small, well-defined neural networks in vitro as a representation of elementary brain functions. One of the first attempts to control the geometry of neural networks was made in 1996,36 and many have followed.15,37−42 Unique activity features have been characterized for patterned networks,43,44 and studies have demonstrated, to some degree, control over in vitro axonal outgrowth,45,46 guidance,47,48 cell placement,49 and directionality between nodes50,51 as well as in situ modifications of the network topography.52,53 But the field is in its infancy, and to date there is no report on networks with controlled architecture (well-defined neural nodes and connections) reliably maintained over weeks in culture. In particular, the long-term maintenance of defect-free network topologies would be of importance because primary neurons start to be electrically active only after substantial time in culture, e.g., roughly 2 weeks for primary rat neurons. A major limitation for surface-coating-based approaches is the persistent drive of neurons to spread their axons and dendrites and also B
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was obtained from SuSoS AG. SU-8 photoresists were purchased at Gersteltec (Gersteltec, GM 1070), and polyester masks were ordered at Microlitho (Microlitho UK, high-resolution film, dark field). Developers were from Micro Resist Technology GmbH (mr-DEV600). PDMS elastomer and the corresponding cross-linker were purchased from Dow Corning (Sylgard 184). Phosphate-buffered saline (PBS, Gibco, 10010015) and fibrinogen Oregon Green 488 (Molecular Probes, F7496) were purchased from Thermo-Fisher and Hellmanex III from Hellma Analytics. Ultrapure water (18 MΩ/cm) is produced with a Milli-Q gradient A 10 from Merck-MilliPore. The following abbreviations are used for different buffers: HEPES 0 buffer (1 mM HEPES, pH 7.4); HEPES I buffer (10 mM HEPES, pH 7.4), and physiologic HEPES II buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). For culture media and dissociation, see the corresponding sections. Polymer Synthesis. Poly(pentafluorophenyl acrylate) (pPFPAc) (Mw = 238.18 g/mol monomer, DP = 100) was synthesized as previously published.68 α-Methyl-ω-amine poly-2-methyl-2-oxazoline (PMOXA-NH2) was synthesized as recently published70−72 from MeOTf (0.49 mL, 4.4 mmol, 1 equiv) as a crop initiator and MOXA (18.6 mL, 0.22 mol, 50 equiv). The polymerization was terminated with a 3-fold excess of ammonia in dry THF. The polymer was dialyzed using a regenerated cellulose membrane (Spectra Por, MWCO 1000) and finally freezedried. Yield: 5.01 g (26.8%). 1H NMR (500 MHz, D2O): δ[ppm] 3.5 (−CH2CH2− in a polymer chain, 210 H), 3.15−2.9 (CH3−N end group, 3H), 2.1 (CH3−CO, 157 H). DP(1H NMR) = 52 corresponding to a Mn of 4420 g/mol. PDI (GPC) Mw/Mn = 1.12. The synthesis (see also the synthesis in scheme S3) of graft copolymer poly(acrylamide)-g-(PMOXA, 1,6-hexanediamine, 3-aminopropyldimethylsilanol) (7000:4425:116.2:161.3 Mr; 0.15:0.425:0.425 d) (PAcrAm-g-(PMOXA, NH2, Si) was carried out as follows. A solution of PMOXA-NH2 (500 mg, 0.113 mmol) in 5 mL of DMF together with NEt3 (31 μL, 0.226 mmol, 2-fold excess) was prepared under stirring and added dropwise to another solution of pPFPAc (179 mg, 0.753 mmol of monomer) in 2 mL of DMF. The mixture was stirred overnight at 50 °C. N-boc-1,6-hexanediamine hydrochloride (80 mg, 0.32 mmol) was dissolved in 1 mL of DMF together with NEt3 (88.5 μL, 0.64 mmol, 2-fold excess with respect to the amine) and added dropwise to the resulting PAcrAm-g-(PMOXA, PFPAc) solution from above. The resulting clear solution was left to react overnight under stirring at 50 °C. A solution of 3-aminopropyldimethylethoxysilane (125 μL, 0.64 mmol, 2-fold excess with respect to the remaining nonreacted active ester groups) and NEt3 (177 μL, 1.28 mmol, 2-fold excess with respect to the silane) was added to the above solution and left to react overnight under stirring at 50 °C. DMF was removed under reduced pressure, and the crude product was redissolved in 10 mL of DCM to obtain a yellow solution. TFA (2.5 mL) was added, and the mixture was stirred overnight at room temperature. The solvents were evaporated under reduced pressure, and the crude oily material was dispersed in 10 mL of DI water. NaOH solution (15%, m/v) was added until a clear solution was obtained, which was dialyzed (3.5 kDa MWCO membrane) for 2 days against ultrapure water. The polymer was isolated by freezedrying as an off-white fluffy powder. Yield: 412 mg (66% with respect to pPFPAc). 1H NMR (400 MHz, D2O): δ[ppm] 3.7−3.2 (−CH2CH2− in a PMOXA polymer chain), 3.2−2.6 (CH3−N end group, overlapped with broad signals from the PAcrAm backbone, −CH2−NH3+ and −NH−CH2−), 2.2−1.8 (CH3−CO in the PMOXA polymer chain) 1.6, 1.4, and 1.3 (broad signals from −CH2− of hexylamine and silane side chains), 0.52 (−CH2−Si(CH3)2OH), and 0.07 (−CH2−Si(CH3)2OH). Variable-Angle Spectroscopic Ellipsometry (VASE). The thickness of the silicon oxide on silicon wafers, as well as the adsorbed polymer layers before and after HEPES incubation and finally after serum exposure, was measured with an M-2000F variable-angle spectroscopic ellipsometer (J. A. Woollam Co., Inc.). The measurements were made using focal probes at 70° relative to the surface normal under ambient conditions in the spectral range of 370−1000 nm. Ellipsometry data were fitted with multilayer models using custom analysis software (WVASE 32) and a Cauchy model (A = 1.45, Bn = 0.01, Cn = 0) to obtain a best estimate for the refractive index and the dry thickness over the entire spectrum for the adsorbed polymer and the
Figure 1. Scheme of new copolymer poly(acrylamide)-g-(poly (2-methyl-2-oxazoline), 1,6-hexanediamine, 3-aminopropyldimethylsilanol), short PAcrAm-g-(PMOXA, NH2, Si) (a) vs poly(L-lysine)-gpoly(ethylene glycol), short PLL-g-PEG (b), the current gold standard for versatile antifouling surface modification. Grafting densities and polymer lengths for PLL-g-PEG: a = 0.29, b = 0.71, m = 100 (DP for PLL is 100, 20 kDa), and n = 42−43 (1.9 kDa). For PAcrAm-g-(PMOXA, NH2, Si): c = 0.425, a = 0.15, b = 0.425, m = 100 (DP for PAcrAm is 100), and n = 52 (4.4 kDa).
and C2C12 myoblasts on poly(dimethylsiloxane) (PDMS) and polystyrene. The study is supplemented by two tests for cell adhesion and protein adsorption under long-term culture conditions, providing complementary characterizations to standard coating tests. This coating is expected to be relevant to scientists working with geometrically arranged cell cultures on any substrate, e.g., in well plates, on electrode arrays, or in microfluidic systems. In addition, we predict that PAcrAm-g-(PMOXA, NH2, Si) will bring neuroscientists a step further toward engineering artificial brains on chips.
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EXPERIMENTAL SECTION
Unless otherwise stated, all chemicals listed were used as received. Methyl trifluoromethanesulfonate (≥98%, MeOTf), 2-methyl-2-oxazoline (98%, MOXA), extra dry ammonia (0.5 M in THF), deuterium oxide (99.9% atom % D), N-boc-1,6-hexandiamine hydrochloride (≥98%), 3-aminopropyl-dimethylethoxysilane, sodium chloride (NaCl, ≥99.5%), trichloro(1H,1H,2H,2H-perfluorooctyl)silane (448931), poly(D-lysine) (PDL, P6407), fibronectin (F1141), Atto-633-labeled anti-mouse IgG (78102, produced in goat), hexamethyldisilazane (440191), paraformaldehyde (158127), glutaraldehyde (G7651), and extra-dry-grade acetonitrile (ACN, Fluka) were obtained from SigmaAldrich. MeOTf was distilled under an inert nitrogen atmosphere, right before use, with Ca(OH)2 used as a drying agent, MOXA monomer was dried with KOH and distilled under inert nitrogen atmosphere, and the obtained distillate was stored under an argon atmosphere. N,NDimethylformamide (DMF, ≥99,8%), triethylamine (NEt3), trifluoroacetic acid (TFA) toluene (analytical grade), 2-propanol (analytical grade), and dichloromethane (DCM) were purchased from Merck. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, ≥99%) was acquired from BDH Biochemical. Full human serum (PrecinormU) was purchased from Roche Diagnostics. Poly(L-lysine)-graft-(poly(ethylene glycol)) (20 000:2000 Mr; 0.29d) (PLL(20)-g[3.5]-PEG(2)) C
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serum layer. Dry aerial mass densities were calculated via dry densities of 1.3 g/cm3 for proteins,73,74 1.17 g/cm3 for PEG polymers,67 and 1.01 g/cm3 for PMOXA33 polymers. The results reported represent an average of at least three measurements on three different samples. X-ray Photoelectron Spectroscopy (XPS). Surface characterization was carried out by X-ray photoelectron spectroscopy (XPS). Measuring conditions for XPS were as previously reported by Rodenstein et al.75 All spectra were recorded using a PHI5000 Versa probe (ULVAC-PHI, INC., Chigasaki, Japan). The spectrometer is equipped with a 180° spherical-capacitor energy analyzer and a multichannel detection system with 16 channels. Spectra were acquired at a base pressure of 5 × 10−8 Pa using a focused, scanning, monochromatic Al Kα source (1486.6 eV) with a spot size of 200 μm and 47.6 W power. The instrument was run in FAT analyzer mode, with electrons emitted at 45° to the surface normal. The pass energies used for survey scans were 187.85 and 46.95 eV for detailed spectra. The full width at half-maximum (fwhm) of this setup is 95% for PLL-g-PEG. The detection limit of VASE measurements is approximately 10 ng/cm2. The uptake of human serum albumin in the standard incubation test (30−60 min) for PEG and PMOXA coatings on glass and metal oxides is
usually low28 (around the OWLS and QCM detection limit of 1 ng/cm2 84), similar to that for full serum.27,30,32,63 For the latter, although, published measurements vary for different antifouling coatings, being up to 80 ng/cm2 wet mass for poly(vinyl alcohol),25 22 ng/cm2 for a zwitterionic poly(sulfobetaine methacrylate) (interestingly only 10.5 ng/cm2 if 10% serum is used),85 65 ng/cm2 for dextran,25 177 ng/cm2 for poly(vinylpyrrolidone),25 and in one study 25 ng/cm2 dry mass for PEG.28 Good results in protein repulsion tests contribute to the popularity of PLL-g-PEG but are in partial contrast to its longterm performance under culture conditions, as later described in this study. The above-mentioned results from short-term serum incubation tests with many coatings, which successfully prevent adhesion in cell culture studies, are an indication that the standard serum test is too simplified and does not represent realistic long-term conditions in a tissue culture environment. The role of long-term stability in protein resistance (also under
Figure 2. C 1s detailed spectra for PLL-g-PEG (left) and PAcrAm-g(PMOXA, NH2, Si) (center) on silicon wafers with reference spectra (on the right, uncoated wafer). (Top) After aging overnight in HEPES II. (Bottom) After immersion in full human serum for 30 min. Aliphatic carbon C−C (285 eV), C−O/C−N (286.5 eV), and CON (288.3 eV).
Figure 3. Percentage of nonfouling polymer covered by protein clumps after 4 weeks of incubation in medium supplemented with 10 μg/mL fibrinogen Oregon Green 488. The significance level is 0.5%, calculated via the Welch unequal variance t test. White areas on the small images reflect printed PDL. F
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solutions and aging overnight in HEPES II buffer, for both polymers the C 1s signal was increasing and the different carbon contributions typical of PEG and PMOXA were present (Figure 2). Also, N in the form of amides (399.5 eV) and ammonium groups (401.5 eV) was present. Immersing these samples in human serum leads to only a minor change in surface composition, demonstrating the low uptake of proteins. Comparing the overlayer/substrate ratio, which is proportional to the film thickness as well as the change in N before and after immersion in human serum, PAcrAm-g-(PMOXA, NH2, Si) seemed slightly less protein-resistant than PLL-g-PEG under these experimental conditions, consistent with ellipsometry. When evaluating surface modifications for their fouling resistance, usually protein adhesion assays are performed with ellipsometry, quartz crystal microbalance, surface plasmon resonance, or X-ray photoelectron spectroscopy after immersion in a protein solution in aqueous buffer. Unfortunately, such measurements come with limitations, e.g., not taking into account exposure to the culture medium at 37 °C, long-term replacement and oxidation phenomena, resistance against specific proteins involved in adhesion, and protein aggregation effects. As an alternative method, fluorescence microscopy was used in this study to quantify the number of protein clumps on the antifouling layer after keeping coated substrates under culture conditions for 4 weeks. As can be seen in Figure 3, more clump formation is observed on PLL-g-PEG. This result is reflecting
harsh conditions) seems to be generally underestimated when testing the antifouling performance. For instance, for the presented approach with the PAcrAm backbone, we reported in an earlier publication with Serrano et al.68 a lower coating thickness and a lower resistance against human serum when dimethylsilanol side chains were added to PAcrAm-g-(PEG, NH2) but a much better performance after exposure to harsh conditions (e.g., high ionic strength). One rather obvious critique regarding the serum incubation test is that serum is dominated by albumins and globulins, whereas it would be more meaningful to quantify the adsorption of fibrinogen (see the discussion for Figure 3) or adhesion-relevant proteins such as laminin or NCAM. XPS was measured on samples coated and aged in HEPES II buffer overnight as well as after immersion for 30 min in full human serum. Normalized atomic concentrations for all detected elements are given in Table 2. As reference, an uncoated sample after O2 plasma cleaning and after immersion in human serum was measured for comparison. After O2 plasma cleaning, a small amount of adventitious C, O from hydroxyl groups, and trace amounts of N can be assigned to the overlayer contribution. Mainly, substrate elements Si and O originating from the natural SiO2 layer on top of the Si substrate were present. After the immersion of such a clean substrate in HEPES II overnight and subsequently in serum, strong increases in C and N were found. After the immersion of the clean substrates in the polymer
Figure 4. Comparison of cell resistance of PLL-g-PEG or PAcrAm-g-(PMOXA, NH2, Si) in patterned neuronal cultures. Small confocal microscopy images show examples for the outgrowing neurites over the first 4 weeks (cyan, microcontact-printed PDL adhesive layer; yellow, GFP-expressing neurons). On the right, SEM images show patterned neurons and the microcontact-printed PDL squares (slightly brighter) of the same test culture after 6 weeks in vitro. Data: Percentage of antifouling polymer area overgrown by neurites. Excluded from the analysis are the squares containing PDL. The box plots show the median with the two inner quartiles around and the most extreme values as whiskers. Five percent significance is reached for two stars, and 0.5% is reached for four stars, calculated via the Whitney-Mann U test. G
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known stability issues with PLL-g-PEG in culture medium such as potential detachment, exchange processes with biomolecules,67,68 and the oxidation of PEG.17,19,62 Branch et al.86 measured the biofilm thickness on PEG in neural cultures and found a significant increase after 2 weeks. On PAcrAm-g(PMOXA, NH2, Si), only very low protein adhesion is observed, which is in agreement with the results from the cell culture assays in the presented study and might serve as a direct explanation for the excellent cell resistance. The two polymers were compared in multiple tests for their performance when applied in long-term cell cultures. To assess the applicability for neural connectivity control, adhesive squares with different distances were microcontact printed on glass slides and backfilled with either PAcrAm-g-(PMOXA, NH2, Si) or PLL-g-PEG (three pattern versions in total: 100, 150, and 200 μm distances with corresponding square widths of 150, 200, and 300 μm). Hippocampal rat neurons were seeded, and the percentage of nonfouling polymer area with unwanted cell overgrowth was compared for up to 6 weeks in vitro (Figure 4). While primary neurons covered over 5% of the PLL-g-PEGcoated areas after 2 weeks, the PAcrAm-g-(PMOXA, NH2, Si) polymer maintained its cell resistance throughout the 6 weeks of the experiment. Interestingly, a small difference between the two coatings can already by seen after half a week, which can potentially be explained by exchange processes between biomolecules and the electrostatically attached PLL-g-PEG in the culture medium.67,68 For bottom-up neuroscience studies, where specific connectivity patterns between nodes have to be reliably maintained over time, the ability of an antifouling coating to prevent uncontrolled connections is highly relevant. To quantify this, the percentage of nodes with undesired connections was counted for hippocampus cultures (Figure 5). To be able to successfully create a neural network with 10 nodes with defined connections,
it is suggested by the authors that a false connection rate of
