Micropatterned Thermoresponsive Cell Culture Substrates for

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Cite This: ACS Appl. Bio Mater. 2019, 2, 2853−2861

Micropatterned Thermoresponsive Cell Culture Substrates for Dynamically Controlling Neurite Outgrowth and Neuronal Connectivity in Vitro Laura V. J. Behm,† Susanna Gerike,† M. Katharina Grauel,‡ Katja Uhlig,† Felix Pfisterer,† Werner Baumann,§ Frank F. Bier,† Claus Duschl,† and Michael Kirschbaum*,† †

Fraunhofer Institute for Cell Therapy and Immunology, Branch Potsdam IZI-BB, Am Muehlenberg 13, 14476 Potsdam, Germany Institute of Neurophysiology, Charité-Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany § Chair for Biophysics, University of Rostock, Gertrudenstr. 11a, 18057 Rostock, Germany

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‡

S Supporting Information *

ABSTRACT: In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2Dmicropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks. KEYWORDS: neuronal networks, micropatterns, neurite outgrowth, thermoresponsive polymers, growth control, cell polarization, smart coatings, lab-on-a-chip

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chambers with microchannels for axon growth,8−11 or 2D patterns of cell-repellent and cell-attractive surface properties. Some of the 2D pattern approaches have also been successfully used for single-cell patterning and the control of the polarization of neuronal cells.12−15 However, the formation of directed neuronal cell connections (i.e., with clearly defined pre- and postsynaptic compartments) remains difficult with permanent 2D surface patterns. This is mainly because such approaches are not suitable for solving two initially contradictory requirements for this demand in the course of network formation: On the one hand, the microenvironment of any given cell must be accessible for axonal projections of a neighboring cell. On the other hand, the outgrowth of its own axonal projections toward this neighboring cell must be blocked if both cells are to connect with predefined directionality via axon and dendrite. To tackle this problem, Vogt et al. employed interrupted pathways of microcontact-printed extracellular matrix proteins for both forcing neuronal cells grow into a preferential

INTRODUCTION

Neurons in cell culture are commonly used for detailed studies of neuronal function, as they are less complex than in vivo studies. In addition, such approaches may provide detailed control over essential parameters and readily allow cell analysis by various methods like high-resolution microscopy, immunocytochemistry, or different -omics techniques. With emerging possibilities to produce diverse types of human neuronal cells from induced pluripotent stem cells (iPSCs),1−3 there are also promising alternatives to animal experiments for the study of neurological and neurodegenerative diseases.4 However, even though neurons in cell culture show many in vivo characteristics and form functional synaptic connections, when grown on standard cell culture substrates, they do not form defined architectures as they do in vivo. Defined connections with directional signal transmission, however, are underlying many neuronal key functions. Approaches to pattern neuronal cells and to guide axon growth in vitro started already in the 70s and 80s pioneered by Letourneau, Campenot, and Kleinfeld.5−7 Today, there are many different techniques to pattern neuronal cells and to create neuronal networks, such as microfluidic cell culture © 2019 American Chemical Society

Received: March 22, 2019 Accepted: May 27, 2019 Published: May 27, 2019 2853

DOI: 10.1021/acsabm.9b00246 ACS Appl. Bio Mater. 2019, 2, 2853−2861

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ACS Applied Bio Materials

(granules 99.99%, MaTeck, Germany) using a BOC Edwards Auto 500 electron beam evaporation system (Edwards, UK). Note that a 2−5 mm wide edge area of the glass slides was left uncoated. The metal coating was then microstructured by laser ablation or photolithography according to design 1 or design 2 (see Figure 1A), respectively. Photolithographically structured slides underwent a

direction and at the same time keeping them accessible for projections of neighboring cells from the opposing side.13 Others proposed switchable microstructures for changing the microenvironmental conditions like surface properties or topological cues during cultivation of the cells, thereby achieving a dynamic (i.e., time-gated) control of surface accessibility and, as a consequence, axon outgrowth.15−20 Dynamic in situ guidance of axons has been achieved with different methods such as photoreactive self-assembled monolayers,16 laser-written microgrooves in an agarose matrix,17,18 AFM tip-mediated modification of surface coatings,19 or AC electrokinetic forces in microfluidic channels.20 However, the approaches described here seem very tedious, require expertise with sophisticated techniques, or are too blurred, either in their effect on the polarization behavior of the cells or in their local resolution to robustly control directionality of cellular interactions in complex neuronal networks. An elegant approach to control cell adhesion and -growth dynamically is using stimuli-responsive substrates, as they enable straightforward control over cell adhesion by a simple switch of external cues like temperature or pH during cell cultivation.21,22 Among them, substrates coated with thermoresponsive polymers (TRP) are most common and are widely used in biomedical fields.23 They can be switched from a protein- and cell-repellent state to a protein- and cell-adhesive state by shifting the substrate temperature above a certain transition temperature (lower critical solution temperature, LCST) and vice versa.24 Surface coatings of TRP can be easily microstructured and have been technically implemented in diverse and fascinating applications.25−27 For example, patterned thermoresponsive substrates allow the spatiotemporal control of (non-neuronal) cell growth28 or the creation of unidirectional neuron bundles.29 However, to our knowledge, they have never been used before for the dynamic guidance of neurite outgrowth. In this paper, we present a proof-of-concept for the use of micropatterned thermoresponsive cell culture substrates to spatially and temporally control the outgrowth of neurites and the time-gated induction of neuronal interconnections.

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Figure 1. Schematic illustration of the micropatterned thermoresponsive substrates for temperature-dependent neurite outgrowth. (A) Geometry of micropatterns with designs 1 and 2. Patterns were produced on 20 × 20 mm2 gold-coated glass slides with an uncoated edge area (not shown) for feeder cell layers. Design 1 consists of 50 μm sized square adhesion spots for somal adhesion, separated by 200 μm and surrounded by TRP-coated gold. The unit cell of design 2 comprises triplets of three spots for somal adhesion two of which extend a 150 μm long and 7−9 μm narrow neurite pathway toward the neighboring spot. (B) TRP is immobilized on gold, while the glass patterns stay TRP-free. At 33 °C, the TRP coating is cell-repellent, that is, cells are restricted to the spots for somal adhesion, and neurite pathways are accessible for neurites. At 37 °C, the TRP coating is celladhesive, which provides a high chance for the neurites (i.e., axons) to cross the gap between neurite pathway and neighboring spot toward the neighboring cell. Illustrations are not to scale.

EXPERIMENTAL SECTION

Thermoresponsive Polymers. In this work, we used a polyethylene glycol (PEG)-based TRP (synthesized by Erik Wischerhoff, Fraunhofer IAP, Potsdam, Germany), which is a copolymer of the monomers 2-(2-methoxyethoxy) ethylmethacrylat (MEO2MA) and oligo(ethylenglycol) methacrylat (OEGMA). The ratio of the amount of these two monomers (here 89 units and 11 units, respectively) together with the length of the oligo-ethylenglycol (OEG)-chains (here 7.5 units) define the lower critical solution temperature (LCST) of the polymer,30 which is 35.2 °C (measured with 3 g/L in PBS by cloud point curve as described by Wischerhoff et al.,31 data not shown). We chose this LCST because cell cultivation is possible both slightly below (i.e., at 33 °C) and slightly above (i.e., at 37 °C) this temperature. We immobilized the TRP onto gold surfaces through its disulfide group.31 At temperatures below the LCST, the polymer brushes are in a fully hydrated and extended confirmation, which inhibits proteins and consequently cells to adhere. Above the LCST, the polymer has a compact chainconformation and water is excluded, which then allows proteins and cells to adhere.30,32,33 Fabrication of Micropatterned Thermoresponsive Cell Culture Substrates. Cleaned glass slides (20 × 20 mm2, 0.23− 0.32 mm thickness, Menzel, Germany) were coated with 3 nm chromium (granules, 99.98%, ChemPur, Germany) and 47 nm gold

further cleaning step with acetone and hot NaOH (8%, 70 °C) to dissolve any resin residuals. Directly before immobilization of the polymer (TRP or, as a control, PEG) the structured gold surfaces were cleaned in a solution of 50 mM KOH and 25% H2O2 for 10 min at room temperature34 and then rinsed with 1 l of deionized water. Five micromolar TRP or 0.56 mM PEG (CH3O-PEG-SH, MW 2 kDa, Rapp Polymere GmbH, Germany) was dissolved in pure ethanol (LiChrosolv, Merck, Germany) in glass dishes. The cleaned substrates were immersed in this solution for 3 h at room temperature. Uncoupled polymer was removed by rinsing and immersing the substrates in ethanol three times and 30 min, respectively. Finally, the substrates were dried with nitrogen and directly used for cell cultivation experiments. 2854


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ACS Applied Bio Materials Cell Cultivation on Substrates. The human neuroblastoma cell line SH-SY5Y (ACC 209, DSMZ, Germany) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, Germany) supplemented with 15% (v/v) fetal bovine serum (FBS), 4 mM L-glutamine, and 0.1 mg/mL penicillin/streptomycin (all Biochrom, Germany) at 37 °C and 5% CO2. Prior to the experiments, cells were differentiated for 10 days (i.e., differentiation started 1 day after seeding with 10 μM retinoic acid (Sigma-Aldrich, Germany) in full medium, which was applied for 4−5 days, and continued in serum-free medium with 50 ng/mL BDNF (Sigma-Aldrich, Germany) for the next 4−5 days). Differentiated cells were harvested using 0.05% Trypsin (w/v; Biochrom, Germany). For removal of large cell clusters, the cell suspension was let sediment in medium containing 10 μM RA for 10 min before only the upper fraction of the suspension was used for counting and plating. Then 3 ×105−6 × 105 cells (in max. 4 mL of RA-containing medium) were plated onto the microstructured substrates, which were placed in nontreated cell culture dishes (3 cm diameter, Greiner Bio-one, Germany). The dishes were agitated for 30 min at 33 °C and 5% CO2 and afterward kept for 1 day at 33 °C and 5% CO2 without agitation to let cells adhere to the polymer-free glass areas (including the uncoated edge area of the glass slides, which served for a feeder cell layer and for control of cell viability). The substrates were then rinsed thoroughly from all sides and transferred to fresh medium to remove nonadherent cells. The substrates were further cultivated at 33 °C and medium was refreshed after 4−6 days. In some cases (especially when incubated for more than 5 days at 33 °C), the medium was exchanged to serumfree, BDNF-containing medium 3 days after plating. At different time points (4 or 5 days after plating), the temperature was increased to 37 °C for further cultivation above the LCST of the TRP. For astrocyte feeder cultures, cortices of newborn mice (postnatal day zero, P0) were digested with trypsin. Cells were grown in T75 flasks in DMEM (ThermoFisher Scientific, Germany) supplemented with 10% FBS (PanBiotech, Germany), 0.2% penicillin−streptomycin (Sigma-Aldrich, Germany) for 1 week. 1.5 × 105 astrocytes were plated onto the microstructured TRP-coated substrates, which were placed in nontreated cell culture dishes (3 cm diameter, Greiner Bioone, Germany) and cultivated at 32 °C for 1 day. The slides were rinsed with neuronal medium (Neurobasal A, supplemented with B27, glutamax (all ThermoFisher Scientific), 2 μg/mL doxicycline (SigmaAldrich, Germany), and 2.5% fetal bovine serum) before human induced neurons (iNs) were plated. Excitatory human iNs had been produced essentially as described elsewhere3,35 and expressed an inducible eGFP for better distinction. Approximately 3 × 105 cells were plated onto the rinsed microstructured TRP-coated substrates with mouse feeder astrocytes. After 1 day at 32 °C the substrates were again rinsed with neuronal medium to remove nonadherent cells and then substrates were transferred to fresh six-well plates preplated with astrocyte feeder cultures (1 week before). The iNs were cultivated for another 4 days at 32 °C before the temperature was increased to 37 °C. Imaging and Analysis. Time points for imaging were chosen to be 1 day after plating (i.e., 1 day in vitro, DIV), and usually every day after the fourth day (4 DIV). At each of these time points, cells were imaged at five random positions on the microstructured substrates. Images were captured at room temperature with an optical microscope (Leica DM IL, Germany) using the 5× and 10× objective and a digital camera (Nikon, Germany). To enable imaging of cells on gold- (i.e., polymer-) coated areas by transmission microscopy, we employed strong illumination settings. In contrast, imaging of cells on gold- (i.e., polymer-) free areas was done with standard illumination settings. For illustration, differently exposed images of representative positions were combined using ImageJ. For time lapse imaging, the substrates were placed onto a fully automated microscope system (cell^R, Olympus, Germany) equipped with a CCD-camera (F-view II, Olympus, Germany). For controlling the ambient temperature (33 or 37 °C), CO2 (5%), and the humidity (ca. 60%), a climate chamber (Evotec Technologies GmbH, Germany) was attached to the microscope. Four to six random positions were captured in a 10 min interval with a 10× objective (UPLFLN, Olympus, Germany).

Images of the iNs on TRP-substrates were captured using a color CCD camera (DP70, Olympus, Germany) mounted to an optical microscope (CKX53 inverted microscope, Olympus, Germany) equipped with a 10× objective (CACHN10XIPC, Olympus, Germany), an integrated LED illumination and phase contrast system, a 100 W mercury lamp (U-LH100HG with power supply unit U-RFL-T, Olympus, Germany), and an eGFP filter set. If not otherwise stated, the cell density in polymer-coated areas was evaluated by dividing the number of all counted cells by the total of the polymer-coated area of all analyzed images. In the polymer-free adhesion spots, cells were growing in clusters. For evaluating the cell density there, the number of occupied spots was analyzed and multiplied with the expected cell number per occupied spot (i.e., 6.0), which was determined in advance by a staining with NucBlue (Thermofisher, supplementary data, Figure S1). Alternatively, for estimating the confluency on the polymer-coated area in time-lapse images or at the end of an experiment, we employed the PHANTAST Plugin of ImageJ36 (using values for sigma and epsilon of 4.5 and 0.09, respectively, and computational halo correction). In experiments with substrates of design 2, we estimated the amount of gaps crossed by neurites by relating the number of such events to the overall number of gaps between neurite pathway and spot in all captured images of these experiments. The statistical differences between results obtained on TRP-coated substrates that were transferred to 37 °C or kept at 33 °C and between those obtained on TRP-coated substrates and PEG-coated substrates, both transferred to 37 °C were tested with the 2-tail Fisher’s exact test. Immunocytochemistry. The cells on the substrates were fixed for 10 min with 4% PFA, permeabilized for 10 min with 0.5% Triton X-100 in PBS, and blocked for 20 min with 1% BSA in PBS. Incubation with the primary antibody ß3-Tubulin (monoclonal, 1:300 in PBS, TUJ-I, Santa Cruz) and, after rinsing three times with PBS, with the secondary antibody (1:300 in PBS, Cy3, Jackson Immunoresearch), was done for 2 h. The substrates were then rinsed twice with PBS, incubated for 10 min with DAPI (0.5 μg/mL) in PBS, and rinsed again three times with PBS before they were microscopically analyzed. Note that all steps in this protocol were performed using prewarmed solutions and executing careful rinsing and incubation steps at 37 °C to avoid switching of the TRP and detachment of cells. Fluorescence images were recorded using an epifluorescence microscope (IX81, Olympus, Germany) equipped with a CCD-camera (F-view II, Olympus, Germany), a 10× objective (UPLFLN, Olympus, Germany), a Xenon short arc lamp (Ushio, Japan), and an appropriate filter set (Ex, 540−550 nm; Em, 575−625 nm).

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RESULTS AND DISCUSSION Micropatterned Thermoresponsive Cell Culture Substrates. To spatiotemporally control the outgrowth of neuronal cells, we structured the thermoresponsive surfaces into temperature-dependent and temperature-independent areas. Since our TRP is immobilized by covalent binding of a central disulfide group to the gold surface,30 we accomplished these patterns by micropatterning a gold-coating on glass slides. After the immobilization of TRP, only gold-coated areas were covered by TRP, while gold-free glass patterns remained TRP-free and consequently allowed for temperature-independent cell adhesion (Figure 1B). We developed two different micropattern designs for the TRP coating (Figure 1A). Design 1 comprises 50 μm wide spots of TRP-free glass for somal adhesion (i.e., adhesion of cell bodies), which are separated from each other by 200 μm and surrounded by TRP. Their size should allow only a few cells or even a single cell to adhere. As long as the surrounding TRP is cell-repellent, the distance of 200 μm cannot be crossed by cells or neurites. Consequently, the cells will stay in the adhesion spots. In contrast, when the TRP is switched to the cell-adhesive state, neurites or cells can 2855


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ACS Applied Bio Materials grow or migrate toward the 200 μm distant neighboring cells. Design 2 extends Design 1 by small, TRP-free pathways that serve as guidance structures for neurites between the spots for somal adhesion. More specifically, the key element of the pattern is formed by three spots for somal adhesion with two of them extending a 150 μm long and 7−9 μm wide TRP-free pathway toward the neighboring spot. The neurite pathways should optimally allow only neurites to enter and exclude cell bodies. The 50 μm wide gap of TRP between the end of the neurite pathway and the next adhesion spot allows us to control neuronal cell growth and neurite outgrowth between the spots in a temperature-dependent manner (Figure 1B). Confinement to the Pattern. We established a network formation protocol on the basis of the patterned thermoresponsive cell culture substrates. For that, we used neuronal cells of the neuroblastoma cell line SH-SY5Y, which can easily be propagated and differentiated into cells with morphology and biochemistry similar to mature human neurons.37,38 After plating SH-SY5Y cells onto the thermoresponsive substrates, cell growth and migration could be restricted for at least 7 days to the TRP-free adhesion spots when the substrates were kept below the LCST (i.e., at 33 °C, TRP is cell-repellent). Nearly all cells adhering to the chip surface were located at the TRP-free adhesion spots, although their area made up less than 3% of the whole microstructured area. Consequently, the analyzed cell density on the adhesion spots was roughly thousand-fold higher than on the cell-repellent TRP-coated area. In the adhesion spots, it was 1800 cells/mm2 after 7 days in vitro (DIV, with 6 cells/spot on average), whereas it was only 3.7 cells/mm2 on the TRP-coated area (Figure 2). This distribution did not change significantly during the 7 days of cultivation at 33 °C, even though we observed improved pattern conformity of the cells over the first

5 days (i.e., 1500 cells/mm2 after 1 DIV and 1900 cells/mm2 after 5 DIV). Improvement of conformity over time was also reported elsewhere and could be due to cell migration toward the adhesive regions or washing off the cells from the repellent regions.12 Judged by morphological analysis on the uncoated edge area, we observed no difference in the viability of cells that had been cultivated at temperatures of 37 or 33 °C, respectively (data not shown). On design 2, approximately 1−3 days after plating the SHSY5Y cells, they started to grow into the narrow, TRP-free pathways, which led off from the adhesion spots. After 4 days of incubation at 33 °C, 96% of the patterns were occupied by neurites or cells. Within these occupied patterns, 89% of the pathways were occupied, without major migration of cells or neurites to outside the patterns. Sometimes it was difficult to decide on the basis of the microscope images whether a pathway was occupied by a cell body or a neurite. However, since the pathways could not be made thinner than 7−9 μm due to technical limitations of our fabrication process, we had to accept that cell bodies may have migrated into the pathways. More narrow pathways (e.g., 3 μm) would probably improve the selectivity for neurites and exclusion of cells.8 Induced Outgrowth of Cells from Adhesion Spots. We further employed design 1 of the thermoresponsive cell culture substrates to initiate the outgrowth of neurites and cells from the TRP-free adhesion spots at defined time points. For that, we cultivated the cells for 4−5 days at 33 °C as described above. After this time, we switched the TRP to its cell-adhesive state by simply increasing the ambient temperature from 33 to 37 °C (i.e., above the LCST, Figure 3A,B). Approximately 5 h after the temperature change, the cells started to extend neurites onto the TRP-coated area to grow completely out of the spots (another 1 h later) and to migrate across the TRPcoated area toward other cells and finally connect with them (Figure 3B and Supporting Information, Video 1 and Video 2). To quantify this behavior, we analyzed the confluency on the TRP-coated area (see Figure 3E). The confluency increased for approximately 10 h and reached a plateau, when all cells were migrating over the surface. In repeated tests, the cells showed consistent behavior (Figure 3F). We observed variability in the increase of confluency due to retarded outgrowth of the cells or reduced initial numbers of cells in the adhesion spots. This variability of confluency is the main cause of the error bars in Figure 3F being so prominent. In control experiments, we did not change the temperature but kept the substrates at 33 °C for 6 days. In this case, the cells stayed within the TRP-free adhesion spots over the entire duration of the experiment, which indicated that the observed outgrowth in the previously described experiment was not due to a time-dependent loss of TRP functionality (Figure 3C). To further exclude other temperature-dependent effects than TRP switching as an explanation for the observed neurite outgrowth (e.g., an increased cell activity), we performed the same experiment on substrates that were coated with PEG (i.e., a temperature-independent, cell-repellent polymer) instead of TRP. On these substrates, the cells stayed within the adhesion spots for both the first 4 days at 33 °C and for the next 1 or 2 days at 37 °C (Figure 3D−F). Spatiotemporal Control of Neuronal Connectivity. As shown above, thermoresponsive substrates of design 1 allowed us to control the timing of neurite outgrowth and migration of neuronal cells. In a next step, we aimed for directional control on neurite outgrowth and the formation of small neuronal

Figure 2. Confinement of the SH-SY5Y cells to the TRP-free adhesion spots below the LCST (i.e., TRP is cell-repellent). Cells were plated onto TRP-coated substrates (design 1 + 2) and cultivated at 33 °C for up to 7 days. Phase contrast images were captured for each experiment at 1 and 4 DIV (n = 13) and for up to 7 DIV (4 ≤ n ≤ 6). The cell density in the adhesion spots (≤4% of the microstructured area) and on the TRP-coated region (≥96% of the microstructured area) was analyzed. Nearly all cells were attached to the adhesion spots. Error bars, SD. 2856


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Figure 3. Confinement and induced outgrowth of neuronal cells on TRP- or PEG-coated substrates (design 1). Cells were plated onto the substrate and restricted to the polymer-free adhesion spots by cultivation at 33 °C for 4 days. After that time, the ambient temperature was increased to 37 °C or left unchanged for one or two additional days. (A) After 4 days of cultivation at 33 °C, most of the cells were confined to the uncoated adhesion spots (arrowheads). (B) Increasing the temperature to 37 °C switched the TRP to its cell-adhesive state and consequently enabled outgrowth and migration of the cells and the formation of cell−cell connections (arrows point to migrating cells). (C) Keeping the temperature at 33 °C instead restricted the cells further to the uncoated adhesion spots. (D) On substrates that were coated with PEG instead of TRP, the cells were restricted to the pattern independent of the temperature. Note the white halo around the adhesion spots is an artifact from strong illumination. Scale bar is 100 μm. (E−F) Change of confluency on TRP- or PEG-coated areas during cultivation at different temperatures. (E) Temporal course of the confluency on TRP- or PEG-coated areas 1 day before and 1 day after the switching time point. (F) End-point analysis after 1−2 additional days of cultivation at 37 °C on TRP- and PEG-coated areas (n = 6 and n = 2, respectively). Error bars, SD.

4A). Neurites were growing into the neurite pathways toward the neighboring adhesion spot but did not cross the gap of (cell-repellent) TRP. Upon switching the TRP into the celladhesive state by a temperature increase to 37 °C, neurites started to cross the TRP-coated gaps between the neurite pathways and adhesion spots and connected with the

circuits. For this purpose, we used substrates of design 2, which comprised 150 μm long TRP-free neurite pathways as well as a 50 μm wide, TRP-coated gap between the adhesion spots. The cells were plated onto the substrates as described above. In accordance with the results described above, SH-SY5Y cells were confined to the pattern for 4−5 days at 33 °C (Figure 2857


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Figure 4. Dynamic control of outgrowth of neurites and formation of small neuronal circuits on substrates of design 2. SH-SY5Y cells were plated onto TRP- or PEG-coated substrates and restricted to the polymer-free area for 4 days by cultivation at 33 °C. After that time, the ambient temperature was increased to 37 °C for one or two additional days or left unchanged. (A) After 4 days of cultivation at 33 °C, most of the cells were restricted to the uncoated glass areas (arrows point to cell- and neurite-free gaps). (B, C) Increasing the temperature to 37 °C switched the TRP to its cell-adhesive state and consequently enabled outgrowth of neurites and cells across the TRP-coated gaps (arrowheads). (E, F) On substrates that were coated with PEG, the cells stayed mostly in the pattern even after a temperature increase to 37 °C. (D) Same behavior was observed for cells on TRP-coated substrates, when cultivated at 33 °C for two additional days. Scale bar is 100 μm. (G) Quantification of the number of crossed gaps at different time points of analysis. The amount of crossed gaps was significantly higher after 2 days at 37 °C on TRP-coated substrates than on PEG-coated substrates or than on TRP-coated substrates that were cultivated at 33 °C for additional 2 days (∗: p < 0.05, 2-tail Fishers Exact Test); 23 ≤ n ≤ 80 number of analyzed gaps (in 2−6 experiments).

Figure 5. Expression of the neuronal marker ß3-tubulin in cells that had been cultivated on the microstructured thermoresponsive cell culture substrates. Differentiated SH-SY5Y cells were cultivated for 4 days at 33 °C and for two additional days at 37 °C on TRP-coated substrates. Afterward, the cells were immunocytochemically stained against ß3-tubulin and analyzed by fluorescence microscopy. Scale bar is 100 μm.

neighboring cells (Figure 4B). After 2 days at 37 °C, we analyzed the percentage of crossed gaps. The values were significantly higher for TRP-coated substrates than for substrates that were coated with temperature-independent PEG and for TRP-coated substrates that were kept at 33 °C (i.e., TRP in the cell-repellent state, Figure 4G). It should be mentioned that, after temperature increase to 37 °C, not only neurites crossed the gaps but also cells were migrating out of the adhesion spots, just as they did on substrates of design 1. This is comprehensible since we did not specifically switch the TRP-coated gap but heated up the whole substrate and thereby switched all the surrounding TRP into the cell-adhesive state. This unintended outgrowth and

migration of cells was even more pronounced the longer the substrates were kept at 37 °C (Figure 4C). In the future, integrated microheaters for local heating of the surface could help to specifically switch distinct areas into the cell-adhesive state, while the rest of the substrate can be continuously cultivated at 33 °C to keep the other regions cell-repellent. In this way, outgrowth of the longest neurites (i.e., axons) could be triggered only between the neurite pathways and the neighboring spots, while the outgrowth at all other regions is not induced. We then immunocytochemically stained for the neuronal marker ß3-tubulin in differentiated SH-SY5Y cells after cultivation on the thermoresponsive substrates. We showed 2858


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Figure 6. Dynamic control of outgrowth of astrocytes and human induced neurons (iNs) on TRP-coated microstructured thermoresponsive cell culture substrates. Astrocytes were plated 1 day prior to the iNs onto substrates of design 2. (A) Astrocytes were confined to the polymer-free area for 5 days and iNs for 4 days at a cultivation temperature of 32 °C (arrows). (B) Switching the TRP to its cell-adhesive state by increasing the temperature to 37 °C permitted the outgrowth of neurites and cells across the TRP-coated gaps (arrowheads). For better distinction, human iNs were transduced with eGFP. Fluorescence images (left) are presented side-by-side to the corresponding phase-contrast images (right). Scale bar is 100 μm.

patterned cells was also mentioned for other structured surfaces.7,14 After rinsing the substrates, their transfer into dishes with cell layers (e.g., of glial cells), which did not previously undergo the procedure of splitting and rinsing of the substrates, was assumed to be helpful for the cultivation of more delicate cells on these substrates. Finally, as stated before, narrower neurite pathways would help to inhibit migration of cell bodies along the TRP-free neurite pathways and allow only neurites or axons to grow along the pathways, which is a prerequisite for unidirectional connections. We plan to optimize our fabrication process such that a minimal structure width of 3−5 μm can be employed within the gold layer.

that the differentiated SH-SY5Y cells expressed the neuronal marker ß3-tubulin also in the networks created. This experiment also demonstrates that immunocyctochemistry can easily be performed on these substrates like on standard coverslips without further steps (Figure 5). To demonstrate the applicability of our approach to other neurobiologically relevant cell models, we plated human iPSCderived neuronal cells (iNs) onto the TRP-coated substrates of design 2. The employment of iNs requires feeder mouse astrocytes to be seeded onto the substrates on the preceding day. To facilitate their distinction, the neuronal cells were transduced with enhanced green fluorescent protein (eGFP). We succeeded to confine both astrocytes and iNs to the TRPfree patterns by cultivation at 32 °C (Figure 6A). After cultivation of astrocytes for 5 and iNs for 4 days at 32 °C, we switched the TRP to the cell-adhesive state by increasing the ambient temperature to 37 °C. Analog to our experiments with the SH-SY5Y cell line, this induced an outgrowth of the cells from the patterns over the TRP-coated area (Figure 6B). As expected, the iNs followed the astrocytes in their growth patterns. For long-term applications, the repellent properties of the TRP may need to be further optimized. This could be achieved, for example, by varying the polymer concentration or by optimizing cell culture conditions. In particular, the use of serum-free medium from the earliest date practicable might minimize fading of cell-repellent properties due to unwanted adsorption of serum proteins to the TRP-coating. Moreover, the attraction of the TRP-free patterns could be increased by selectively coating the glass spots using adhesion mediating polymers or proteins. This might result in a longer retention time of the cells within the patterns. Prolonged confinement of the cells to the adhesion spots will allow cells to have more time for maturation. This is highly beneficial, as most neuronal cells (rodent primary neurons and human induced neurons) take at least 10 days in culture to develop electrical excitability.7,13,15 For longer cultivation at temperatures below 37 °C, the neuronal development and viability of the desired cell type should be analyzed beforehand. In this regard, the uncoated edge areas on the glass slides adjacent to the microstructured polymer-coated gold surfaces are assumed to be important, as they allow for the formation of a feeder cell layer. The importance of feeder layers for supporting the

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CONCLUSIONS

We developed microstructured thermoresponsive cell culture substrates for dynamically controlling growth of neuronal cells in vitro. The substrate surface is equipped with a microstructured coating of thermoresponsive polymers, which can be switched between a cell-repellent and a cell-adhesive state. We used the substrates to pattern neuronal cells as well as to restrict neurite outgrowth to predefined areas of the substrate surface. By changing the temperature at defined time points of cultivation, we induced neurite outgrowth between the patterned cells. For the first time, we showed that thermoresponsive polymer coatings can be used to guide neurite outgrowth in time and space and help to control connectivity in neuronal cell cultures. We applied our protocol to both a human neuronal cell line and human iPSC-derived neuronal cells, which are of high interest for studying, for example, patient-specific neurological diseases. The use of the substrates in cell culture experiments did not require any sophisticated techniques. In contrast to microfluidic-based approaches, such relatively simple micropatterned cell culture substrates can be used in an open format, making the cells on the substrates directly accessible to electrophysiological recordings by, for example, the patch clamp technique. In the future, we will combine the substrates with microheaters to heat-up the surface locally and, thus, to further improve spatial control over cell migration and neurite outgrowth. 2859


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00246.

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Confinement of neuronal cells on TRP-coated substrates of Design 1 (MP4) Confinement and induced outgrowth of neuronal cells on TRP- or PEG-coated substrates of Design 1 (MP4) Quantification of the number of cells in adhesion spots (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: + 49/331/581 87−303. Fax: + 49/331/581 87−399. ORCID

Michael Kirschbaum: 0000-0003-3686-8349 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support by the German Research Foundation DFG (DU 167/4-1) and the Berlin-Brandenburg School for Regenerative Therapies GSC 203. We gratefully thank Philipp Wysotzki (University of Rostock, Germany) for enabling the laser ablation of gold surfaces, Beate Morgenstern (Fraunhofer Institute for Cell Therapy and Immunology, Branch Potsdam, Germany) for cell culture assistance, and Roland Lauster (Technical University of Berlin, Germany) for helpful discussions. We also thank Pascal Fenske (Charité Universitätsmedizin Berlin, Germany) for providing human induced neurons.

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REFERENCES

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