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Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser
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Biomaterials 28 (2007) 1141–1151 www.elsevier.com/locate/biomaterials
Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser Per Gustavssona,, Fredrik Johanssona, Martin Kanjea, Lars Wallmanb,c, Cecilia Eriksson Linsmeiera a
Department of Cell and Organism Biology, Lund University, Helgonava¨gen 3B, SE 223 62, Lund, Sweden Department of Electrical Measurements, Lund Institute of Technology, Box 118, SE 221 00, Lund, Sweden c Neural Interfaces, Department of Experimental Medical Science, Lund University, BMC F10, SE 221 84, Lund, Sweden b
Received 25 August 2006; accepted 21 October 2006 Available online 15 November 2006
Abstract In this study, we developed a microdispenser technique in order to create protein patterns for guidance of neurites from cultured adult mouse dorsal root ganglia (DRG). The microdispenser is a micromachined silicon device that ejects 100 picolitre droplets and has the ability to position the droplets with a precision of 6–8 mm. Laminin and bovine serum albumin (BSA) was used to create adhesive and non-adhesive protein lines on polystyrene surfaces (cell culture dishes). Whole-mounted DRGs were then positioned close to the patterns and neurite outgrowth was monitored. The neurites preferred to grow on laminin lines as compared to the unpatterned plastic. When patterns were made from BSA the neurites preferred to grow in between the lines on the unpatterned plastic surface. We conclude that microdispensing can be used for guidance of sensory neurites. The advantages of microdispensing is that it is fast, flexible, allows deposition of different protein concentrations and enables patterning on delicate surfaces due to its non-contact mode of operation. It is conceivable that microdispensing can be utilized for the creation of protein patterns for guiding neurites to obtain in vitro neural networks, in tissue engineering or rapid screening for guiding proteins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Micropatterning; Nerve tissue engineering; Nerve regeneration; Protein; Cell adhesion
1. Introduction It has previously been demonstrated that micropatterns of proteins and other molecules can be used for the guidance of nerve cell processes such as neurites but also to create patterns of whole neurons and glial cells [1–8]. Techniques for creating these patterns include soft lithography methods such as microcontact printing [5–8] and microfluidics [2,9–11], microlithography [1–3] and UVirradition protein inactivation [4]. Commonly patterned molecules are general adhesive molecules such as poly-Llysine or extracellular matrix (ECM) compontents such as laminin, which has long been known for its ability to guide neurites and promote neuronal adhesion [12,13]. Protein and chemical micropatterns can be used for studies of cell Corresponding author. Tel.: +46 46 222 93 54; fax: +46 46 222 45 39.
E-mail address: [email protected] (P. Gustavsson). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.10.028
adhesion, guidance and chemotaxia, but also for the specific guidance of neurites to microelectrodes capable of generating and measuring neural electrical signals [14]. Such neuro-electrical interfaces could have a number of applications, e.g. connecting prosthetic devices to the nervous system for transduction of sensory and motor information, creating cell-based biosensors [15] or creating neural networks in vitro [16]. An alternative method for micropattern formation is microdispensing or inkjet printing, which has total flexibility for pattern generation and does not require contact with the substrate as opposed to other techniques for protein patterning [17–21]. Previous studies using microdispensing have shown printing of cell adhesion molecules for controlled patterning of dissociated cells on surfaces. In this study we used microdispensing to dispense droplets of adhesive laminin and non-adhesive bovine serum albumin (BSA) solutions to create protein micropatterns on polystyrene tissue culture dishes.
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We examined the effects of these patterns on guidance of neurites from organcultured adult dorsal root ganglia (DRG).
2. Materials and methods 2.1. Proteins The proteins used for micropatterning were laminin from EngelbrethHolm-Swarm murine sarcoma (0.5 and 0.05 mg/ml, Sigma, Sweden), BSA fraction V (10, 1 and 0.1 mg/ml, ICN Biomedicals, USA) and fatty-acidfree BSA (10, 1 and 0.1 mg/ml, Sigma, Sweden). For measurements of the spatial resolution and size of the protein micropatterns, 0.1 mg/ml of AlexaFluor 488 labelled BSA was used (Molecular Probes, USA). All proteins were diluted in sterile 0.1 mM phosphate buffered saline (PBS), pH 7.2.
2.2. Micropattern generation Micropatterns were generated using the microdispenser system developed at the Department of Electrical Measurements at Lund Institute of Technology and previously described by Laurell et al. [22]. The experimental setup is shown in Figs. 1 and 2. In short, the microdispenser is composed of a piezoelectric element connected to a push-bar situated over a channel etched in a silicon. The channel has one inlet at each end and a nozzle in the middle (Fig. 2). When a voltage is applied across the piezoelectric element it will expand and via the silicon membrane it will expel the liquid in the nozzle. The dispenser is loaded with a protein solution and the target is moved using a computer-controlled x–y table in order to generate protein patterns. The CCD-camera shown in Fig. 1 allows real-time visual inspection of the microdispensing process and enables positioning of patterns on the substrate. The substrates were heated to 30 1C by a heater element on the x–y table in order to increase the spatial resolution by increased droplet evaporation, thus preventing the dispersion of the drops on the surface. The patterns generated consisted of lines with a drop pitch of 40 mm and a line spacing of 300 mm. We used 35 mm NunclonTM polysterene cell culture dishes (Nunc, Denmark) as substrates.
Fig. 2. Close-up of the microdispenser. Top panel: the dispenser is fixed in position by a metal arm, protein solution is fed into the dispenser head through two silicone tubes functioning as inlets at each end of the channel. Bottom panel: schematic section drawing of the microdispenser. A piezoelectric element is situated above a channel etched in silicon. The element is connected to a push-bar that forms the roof of the channel. When a voltage is applied to the element it will expand and the membrane will push the liquid through the nozzle in the middle of the channel.
2.3. Organotypic culture of DRG All procedures involving animals were conducted according to the Swedish experimental animal ethical guidelines and approved by the local Ethical Committee on Animal Experiments. Adult female NMRI mice (B&K Universal, Sweden) were killed by an intraperitoneal injection of sodium pentobarbital (Apoteksbolaget, Sweden) and the DRG from lumbar 6 to lumbar 4 were dissected and mounted in the ECM-substitute Matri-gel onto culture dishes with microdispensed protein patterns. In each culture dish two ganglia were mounted diagonally on opposite sides of the pattern and placed as closely as possible to the edge of the patterns. The DRGs were cultured for three days at 37 1C in an atmosphere of 6.5% CO2 and 93.5% O2 in RPMI 1640 media (Sigma, Sweden) supplemented with 20 ng/ml NGF (PeproTech, USA) to promote neurite outgrowth, L-glutamine (Gibco, USA) and penicillin/ streptomycin (Gibco).
2.4. Immunocytochemistry
Fig. 1. The experimental setup with the microdispenser is located in the centre of the picture (1). The dispenser is held in position by a metal arm above a computer-controlled x–y table (2). The cell culture dishes are placed on the x–y table and during dispensing the table moves the dish while the dispenser stays fixed. The frequency of droplet ejection and the pulse formation, is controlled by a signal generator (3). A CCD-camera with magnifying optics allows real-time inspection of the dispensing process in order to determine if to modify the parameters (4).
After 3 days in culture the DRG cultures were fixed in Stefanini’s fixative (4% paraformaldehyde, 0.03% saturated picric acid in 0.1 M PBS, pH 7.2) over night at 4 1C. Following rinses in PBS, the cultures were incubated over night at 4 1C with primary antibodies diluted in PBS containing 0.25% Triton X-100. The antibodies used were mouse monoclonal antibodies recognizing b-tubulin isotype III (1:400; Sigma, USA) to visualize neurites; rabbit polyclonal antibodies recognizing BSA (1:50, DAKO, Denmark) or rabbit polyclonal antibodies recognizing laminin (1:25, Sigma, USA) to visualize the microdispensed protein patterns. After repeated rinses in PBS, the preparations were further incubated for 1 h at room temperature with the polyclonal secondary antibodies AlexaFluor 594 goat anti-mouse and AlexaFluor 488 goat
ARTICLE IN PRESS P. Gustavsson et al. / Biomaterials 28 (2007) 1141–1151 anti-rabbit (1:500, Molecular Probes, USA), repeatedly rinsed in PBS, mounted in 1:1 PBS/glycerol and coverslipped.
2.5. Photography and image analysis The preparations were photographed using an Olympus AX70 fluorescence microscope equipped with a DP50 CCD camera and images were acquired using the software StudioLite (Olympus, Japan). Images were converted to 8-bit grayscale TIFF using Adobe Photoshop 7.0 (Adobe, USA) and imported into NIH Image 1.63 (a public domain image analysis programme developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). The Plot Profile function was used to measure the intensity values of the AlexaFluor488 BSA gradients in order to confirm that gradients had formed. The distances and width of protein lines and arays was determined using the distance measurment function. Unpaired Students t-test (Microsoft Excel, Microsoft Corp. USA) was used to test for significant differences in distance and width. A p-value p0.05 was considered as statistically significant. To quantify neurite outgrowth on the patterns, the immunoreactivity to bIII-tubulin was determined on the microdispensed protein lines and on the unpatterned surface. The immunoreactivity to neuronal tubulin corresponds to the cytoskeleton inside the neurites and could thus be regarded to be a measure of neurite outgrowth. The intensity cut-off, above which the tubulin immunoreactivity was considered positive, was determined separately for each image in order to keep the background fluorescence of the substrate surface below the detection level. The number of immunopositive pixels was measured on the protein lines and between the lines, using NIH Image, and then the ratio between immunopositive pixels and total number of pixels in the region of interest was calculated. Unpaired Students t-test (Microsoft Excel) was used to test for significant differences in pixel ratios between protein patterns and non-patterned surface. This procedure thus gave a measure of the amount of axons growing on the patterns and between the patterns. A p-value p0.05 was considered as statistically significant.
3. Results 3.1. Protein micropattern generation Fluorescent BSA was used to investigate the precision of the microdispenser system (the system is shown in Figs. 1 and 2), but not used for cell culture since the influence of the fluorophores on neurite outgrowth was not known. Figs. 3 and 4 show arrays and lines, respectively, generated by microdispensing 0.1 mg/ml fluorescent BSA on plasmatreated polystyrene. In Fig. 3, three different types of arrays are shown. The arrays were generated by modulating the drop pitch in both x- and y-direction to 100, 200 and 300 mm, respectively. Fig. 3A shows an array with a pitch of 100 mm, where the array became a square due to
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merging of the droplets on the substrate. Image analysis showed that the array had a mean dimension of 1033721 mm 1027710 mm (n ¼ 10). In Fig. 3B, an array with a pitch of 200 mm is shown. The resulting array had discrete points, as opposed to the array in Fig. 3A. The mean distance between neighbouring points was 19976 mm (n ¼ 180) and the mean diameter of the points was 15674 mm (n ¼ 100). In Fig. 3C, the drop pitch was set to 300 mm, which also resulted in an array with discrete points. The mean distance between neighbouring points was measured to 29978 mm (n ¼ 134). The average diameter of the points in an array with a 300 mm pitch was 158710 mm (n ¼ 70). In Figs. 3B and C, one can see satellite droplets that formed around the dispensed drops. These satellites appeared when the liquid used for dispensing contained contaminating dust particles or when salt and protein crystals formed in the nozzle, thus disturbing the trajectory of the drops. The formation of satellite droplets could be reduced by modulating the pulse applied to the piezo ceramic element of the dispenser and by cleaning the nozzle with a moist tissue between rounds of dispensing. Figs. 4A–D shows linear patterns generated by microdispensing. The drop pitch in the y-direction was set to 30 mm in Figs. 4A and B and 50 mm in Figs. 4C and D, while the x-direction was kept constant at 300 mm. The mean width of the lines with 30 mm pitch was 23974 mm (n ¼ 29) at when the substrate was kept at room temperature during dispensing and 240715 mm (n ¼ 40) when the substrate was heated to 30 1C. The mean width of the lines with 50 mm pitch was 19973 mm (n ¼ 36) at room temperature and 19871 mm (n ¼ 36) when substrates were heated to 30 1C. There was no statistical difference between substrates made at room temperature or heated substrates but by visual inspection of patterns (Figs. 4A–D), it could be seen that the patterns were more irregular on nonheated substrates while they were more regular and the protein lines more straight when the substrates were heated. The width of the lines at a pitch of 30 and 50 mm was however significantly different, irrespective of the temperature during dispensing (po0:001). To further investigate the possibilities to generate patterns, we created gradients of fluorescent BSA. The gradients were created by changing the number of droplets dispensed in each point. The array show in Fig. 5A consists
Fig. 3. Arrays of microdispensed AlexaFluor 488 labelled BSA (0.1 mg/ml). The droplet pitch in A was 100 mm, in B 200 mm and in C 300 mm. The numbers 1–3 in the figures indicate how the pattern resolution was measured. Scale bar ¼ 500 mm.
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Fig. 4. Lines of microdispensed AlexaFluor 488 labelled BSA (0.1 mg/ml). The x-direction droplet pitch was changed from the top panel to the lower panel as follows: A–B: 30 mm pitch, C–D: 50 mm pitch. In A and C the substrate was kept at room temperature during dispensing, while in B and D the substrate was heated to 30 1C to increase evaporation to prevent the spreading of dispensed droplets over the surface. Scale bar ¼ 1000 mm.
Fig. 5. (A–D) Gradients of AlexaFluor 488 labelled BSA (0.1 mg/ml). (A) Gradient consisting of 10 spots, the first two spots contained one droplet and the last two contained five droplets, the pitch was 50 mm. (B) Intensity plot for the gradient shown in A. (C) Gradient consisting of 20 spots, the first two spots contained one droplet each and the last two spots contained 10 droplets each, the pitch was 50 mm. (D) Intensity plot for the gradient shown in C. Scale bar ¼ 200 mm.
of 10 points with the two first points each containing 1 droplet, the next two points contains 2 droplets each and so forth with the last two point containing 5 droplets each. An intentsity profile of the gradients is shown in Fig. 5B. Fig. 5C shows a gradient created in a similar way, but consisting of 20 points, with the first two points containing 1 droplet each and the last two points containing 10 droplets each. An intensity plot for this array is shown in Fig. 5D. From these images it is possible to see that
gradients can be created with microdispensing, however they are not very smooth. We tested glass cover slips as substrates for fluorescent BSA. However, glass cover slips yielded poorer patterns due to rapid merging of adjacent droplets, causing dispersion of the pattern (results not shown). Better patterns on glass could be achieved by decreasing the delay between droplets as to allow the previous drop to evaporate before the next. Still, this did not result in as
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uniform patterns as those formed on plastic cell culture dishes. 3.2. Neurite outgrowth on laminin patterns Fig. 6A shows neurite outgrowth (visualised by tubulin immunostaining, red) on patterns containing 0.5 mg/ml laminin and Fig. 6B shows the corresponding laminin patterns (visualised by laminin immunostaining, green). From Fig. 6A it can be seen that there was a higher proportion of neurites growing on the microdispensed lines compared to the number growing between the lines. The guidance was not perfect, and some neurites followed the lines for a short distance but then left to grow in between the lines. A certain amount of satellite droplets had also been generated during microdispensing, which could have provided a certain increased attachment between the patterns. When the images were analysed for the ratio of tubulin immunopositive pixels (those pixels that corresponded to neurites) to total number of pixels in the analysed area, there was a significantly higher percentage of positive pixels on the laminin containing lines than between the lines, corroborating the ocular observations (Fig. 7). Fig. 6C shows neurite outgrowth (red staining) on protein patterns containing 0.05 mg/ml laminin and Fig. 6D shows the corresponding laminin patterns (green staining). Here the neurites did not follow the laminin lines, instead the neurites grew randomly across the pattern. Image analysis confirmed these observations, and there was no significant difference between the percentage of tubulin
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immunopositive pixels on the lines and in between the lines (Fig. 7). 3.3. Neurite outgrowth on 98% pure BSA patterns Fig. 8A shows neurite outgrowth (visualised by tubulin immunostaining, red) on protein patterns made from 10 mg/ml BSA and Fig. 8B shows the corresponding protein patterns (visualised by BSA immunostaining, green). From Fig. 8A, it can be seen that most of the
Fig. 7. Ratio between tubulin positive pixels and total pixel area on laminin lines and between laminin lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 4 for all experiments).
Fig. 6. (A–D) Neurite outgrowth on laminin patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by laminin immunostaining (green). (A) Neurite outgrowth on 0.5 mg/ml laminin. (B) 0.5 mg/ml laminin patterns. (C) Neurite outgrowth on 0.05 mg/ml laminin. (D) 0.05 mg/ml laminin patterns. Scale bars ¼ 200 mm.
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Fig. 8. (A–F) Neurite outgrowth on BSA patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by BSA immunostaining (green). (A) Neurite outgrowth on 10 mg/ml BSA. (B) 10 mg/ml BSA patterns. (C) Neurite outgrowth on 1 mg/ml BSA. (D) 1 mg/ml BSA patterns. (E) Neurite outgrowth on 0.1 mg/ml BSA. (F): 0.1 mg/ml BSA patterns. Scale bars ¼ 200 mm.
neurites avoided the protein lines and grew in between the lines. Some neurites started growing on the unpatterned surface, then grew on to the patterns but then grew back on the unpatterned surface. Image analysis revealed that there was a significant difference between the relative area of tubulin positive pixels on and between protein lines (Fig. 9). In Fig. 8C the neurite outgrowth (red staining) on patterns made of 1 mg/ml BSA is shown and in Fig. 8D the corresponding protein patterns (green staining) are shown. The 1 mg/ml BSA patterns showed a similar guidance as the 10 mg/ml patterns, but an increasing number of neurites grew on the lines in these experiments. Image analysis once again revealed that there was a significant difference between the relative area of tubulin positive pixels on the lines and the positive pixels between the lines (Fig. 9). Fig. 8E shows the neurite outgrowth (red staining) on patterns made from 0.1 mg/ml BSA and in Fig. 8F the corresponding protein patterns (green staining) are shown. When the patterns were made from a 0.1 mg/ml BSA solution, the neurites did not prefer to grow between
Fig. 9. Ratio between tubulin positive pixels and total pixel area on BSA lines and between BSA lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 5 for 10 mg/ml, n ¼ 8 for 1 mg/ml and n ¼ 4 for 0.1 mg/ml).
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the patterns, but instead grew in a random fashion over the surface, both on the lines and in between the lines. Image analysis showed that there was no significant difference between the percentage of tubulin immunopositive pixels occupying the lines or the unpatterned surface (Fig. 9). A general observation for the BSA lines was that a higher concentration of BSA (10 mg/ml, shown in Fig. 8B) resulted in finer patterns due to increased viscosity as compared to a lower concentration (e.g. 0.1 mg/ml, shown in Fig. 8F). 3.4. Growth on 96% pure fatty-acid-free BSA patterns As BSA is known for its non-permissive properties we also sought to investigate if fatty-acids bound to BSA could be involved in this property. To this end we created micropatterns of fatty-acid-free BSA and cultured DRGs on these patterns. Fig. 10A shows neurite outgrowth (visulised by tubulin immunostaining, red) on protein
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patterns made from 10 mg/ml fatty-acid-free BSA and Fig. 10B shows the corresponding protein patterns (visulised by fatty-acid-free BSA immunostaining, green). It can be seen that the neurites preferred to grow on the unpatterned surface between the lines containing BSA and avoided the patterned lines. Image analysis revealed that there was a significantly higher percentage of tubulin immunopositive pixels between the lines than on the lines, showing that the bulk of the neurites grew between lines (Fig. 11). Fig. 10C shows neurite outgrowth (red staining) on patterns of 1 mg/ml fatty-acid-free BSA and Fig. 10D shows the corresponding protein patterns (green staining). Most of the neurites avoided the protein lines and grew in between on the unpatterned surface. Some neurites could be found growing on the protein lines. Image analysis showed a significantly higher proportion of tubulin positive pixels between the lines (Fig. 11) compared to the proportion on the lines. Fig. 10E shows neurite outgrowth (red staining) on patterns made of 0.1 mg/ml fatty-acid-free
Fig. 10. (A–F) Neurite outgrowth on fatty-acid-free BSA patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by fatty-acid-free BSA immunostaining (green). (A) Neurite outgrowth on 10 mg/ml fatty-acid-free BSA. (B) 10 mg/ml fatty-acid-free BSA patterns. (C) Neurite outgrowth on 1 mg/ml fatty-acid-free BSA. (D) 1 mg/ml fatty-acid-free BSA patterns. (E) Neurite outgrowth on 0.1 mg/ml fatty-acid-free BSA. (F) 0.1 mg/ml fatty-acid-free BSA patterns. Scale bars ¼ 200 mm.
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Fig. 11. Ratio between tubulin positive pixels and total pixel area on fatty-acid-free BSA lines and between fatty-acid-free lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 3 for 10 mg/ml, n ¼ 4 for 1 mg/ml and n ¼ 3 for 0.1 mg/ml).
BSA and Fig. 10F shows the corresponding protein pattern (green staining). It can be seen in Fig. 10E that the outgrowing neurites grew randomly over the surface and did not show any preference for protein lines or the uncoated surface. Image analysis corroborated these observations as there was no significant difference in the percentage of tubulin immunopositive pixels on or between the 0.1 mg/ml BSA patterns (Fig. 11). A general observation for these patterns made from fatty-acid-free BSA, is that a higher concentration of BSA (10 mg/ml, shown in Fig. 10B) resulted in finer patterns as compared to lower concentrations (e.g. 0.1 mg/ml, shown in Fig. 10F), reflecting the influence of the higher viscosity of 10 mg/ml. 4. Discussion In this investigation the major finding was that protein micropatterns generated by a piezoelectric microdispenser can be used for guidance of normal regenerating nerve cell processes in this case, sensory neurites from the DRG. The protein patterns generated were made of laminin and BSA. The resolution of the micropatterns generated by microdispensing depends on a number of factors including the viscosity of the liquid being dispensed, the drop pitch (the distance between the centre of two adjacent droplets in a line or array) and on the substrate. Increased viscosity gives thinner protein lines compared to a lower viscosity. This can be seen for the BSA lines in Figs. 8 and 10, where the 10 mg/ml lines were thinner as compared to the 1 and 0.1 mg/ml lines. However, if the viscosity is increased too much the liquid becomes impossible to expel from the nozzle. The critical level of BSA was at 10 mg/ml. The effect of drop pitch can be seen in Fig. 4, where a lower drop pitch resulted in wider patterns. The effect of a low drop pitch on pattern wideness is probably due to merging
of adjacent droplets and reduced evaporation from the surface. Heating did not influence the width of lines significantly but it generally provided more uniform protein lines. As the temperature was kept below 37 1C, the heating should not affect the state of the proteins more negatively than if the substrates were at room temperature, although denaturing and reduced biological effects cannot be ruled out. The resolution of microdispensed protein spots on substrates with various hydrophilictiy–hydrophobicity has been described by Ressine et al. [23] who used the same microdispensing system as we did. They showed that hydrophilic substrates such as glass exhibited a lower contact angle and gave wider spots while highly hydrophobic macro/nanoporous silicon exhibited a higher contact angle and resulted in smaller spots. We used plastic cell culture dishes, which are rather hydrophilic, since these dishes are primed for cell culture. These dishes are also sterile and easy to handle and mount a DRG on. For rapid screening purposes there is no disadvantage of using these dishes as substrates, but in order to make finer patterns and increasing spatial resolution a hydrophobic substrate could be an advantage. During the development of the system we also tried glass coverslips as substrates. However these substrates yielded poorer patterns due to rapid merging of adjacent droplets, causing dispersion of the pattern. Better patterns on glass could be achieved by decreasing the droplet frequency, as to allow the previous drop to evaporate before the next. Still, this did not result in as uniform patterns as those formed on plastic cell culture dishes. We were also able to make surface bound protein gradient using AlexaFluor 488 labelled BSA. This was achieved by increasing the number of droplets dispensed at each point along the gradient. The gradients are however not smooth, due to overlap between droplets and uneven droplet evaporation. Droplet over lap is inevitable, since it is necessary in order to create continuous lines. The uneven droplet evaporation leads to a local increase of protein concentration, which is reflected in the peaks seen in the surface plots. Smoother droplet evaporation can be achieved by modification of the properties of the solution dispensed. Addition of glycerol for instance, increases the viscosity of the liquid and yields spots with smaller diameter and reduces evaporation artifacts [24]. Modifications of the substrate can also be used to create finer patterns and more uniform droplets [25]. The use of gradients like the ones described here, could be to screen molecules for possible chemotactic properties or to pattern cells at various densities by creating gradients of adhesive molecules. It is conceivable that such gradients could be made from laminin or similar adhesive proteins. Certain dispensed liquids generated satellite droplets, which could be reduced by modulating the waveform of the pulse applied to the dispensers piezoceramic element in order to improve pattern resolution. A total reduction in satellites could not always be achieved, which was especially true for the highest concentration of laminin.
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Laminin is a well-known ECM protein with cell adhesive and neurite guiding properties [12,13,26] and here we show that patterns of laminin for sensory neurite guidance can be made by microdispensing. At a concentration of 0.5 mg/ml laminin, the neurite guiding properties of laminin was clear and most axons grew and followed the lines, whereas at 0.05 mg/ml the axons grew more or less randomly on the surface. The reason why some neurites did not prefer to grow on the tracks made from 0.5 mg/ml laminin, could be due to the fact that the substrate used for dispensing are cell culture dishes which from the start are primed to enhance attachment of cells and cellular processes. To improve guidance, the laminin could have been patterned on a non-permissive substrate, such as BSA or polyethylene glycol (PEG). For instance, Sanjana and Fuller [20] printed permissive collagen/poly-D-lysine on a nonpermissive surface of PEG to control cell spreading. Another option would be to treat the polystyrene surface with e.g. BSA after creation of the microdispensed patterns, thus blocking available binding sites on the surface. Occasional satellite droplets of laminin between the patterns could also have influenced the guidance, promoting some outgrowth between the lines. When doing organotypic culture of DRGs, fibroblasts and Schwann cells migrate from the ganglia to a certain extent, especially from DRGs of newborn animals. These cells can interact with the outgrowing neurites and influence their growth. In our experiments we observed very few migrating cells, and the ones that had migrated stayed close to the Matri-gel border, while the axons extended much further. In this respect, DRGs from adult animals are superior to those from newborns, which exhibit extensive migration of Schwann cells. Neurites avoided the lines made of BSA providing the concentration of the solution was 10 and 1 mg/ml BSA. The avoidance was concentration dependant and at 0.1 mg/ml BSA the neurites were able to grow on the protein lines. BSA is a common molecule to use for making substrates nonpermissive. In the context of neuroscience, Thompson and Buettner [2] for instance took advantage of this property of BSA and made substrates with alternating stripes of laminin and BSA in order to create patterns of Schwann cells. It could in this context be noted that serum albumin is confined to the blood vessles and neurites do not normally come in contact with serum albumins. During nerve injury however, the blood nerve barrier is breached and serum factors make contact with axons and Schwann cells. In addition, the blood-nerve barrier of DRG is virtually absent and it has been found that horse radish peroxidase and fluorescent BSA enters intact sensory ganglia and peripheral nerves [27]. Interestingly Dyer et al. [28,29] showed that an isoform of serum albumin, called active serum albumin, which contained certain lipids could induce growth cone collapse of PC12 cells. We therefore tested if fatty-acid-free BSA exhibited the same properties as regular BSA. Lines created with fatty-acid-free BSA, were as nonpermissive for neurite as those created with BSA containing
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fatty acids. Thus fatty acids bound to BSA are probably not the reason for the non-permissive nature of BSA. Microdispensing was first introduced by Klebe [17] who used a commercial inkjet printer to pattern fibronectin and anti-EGF receptor antibodies for selective cell attachment. Others including Turcu et al. [18] have used a microdispensing system similar to ours to create protein patterns on various surfaces including glass, pure silicon, gold-coated silicon and glassy carbon. Watanabe et al. [19] and Sanjana and Fuller [20] used a customized commercial piezo electric inkjet printer. Roth et al. [21] on the other hand used a modified commercial bubble-jet printer to print collagen and pattern dissociated DRG neurons. Additional techniques to chemically pattern a substrate include microcontact printing, microfluidics and microlithography. They all have in common that they require special process laboratories and are rather work intensive, especially the soft lithograpic methods of microcontact printing and microfluidics. As compared to microdispensing their advantage is that they offer a higher spatial resolution. For microcontact printing, a master stamp is first made by etching the desired pattern into a silicon wafer or a quartz crystal. A solution of polymer such as polydimethylsiloxane (PDMS) is poured on the etched master and cured in an oven. The PDMS is removed and can then be coated with a reactive linker solution or a protein solution. By pressing the PDMS stamp on a surface, a linker or protein imprint is created onto which cells can be seeded and thus patterned. This method has been used for neuronal cell patterning and is described in [5–7]. Microfluidics shares the manufacture steps involved in microcontact printing, has a high resolution and can be used to create protein gradients. It has been used for studies of neuronal cells by Romanova et al. [30], Martinoia et al. [31], Ryan et al. [32] and Patel et al. [10]. Microlithography, used by Thompson and Buettner [2] and Kleinfeld et al. [3] among others, works by covering a substrate with photoresist and areas where proteins are supposed to be deposited are exposed to UV light to remove the resist. The substrate is then treated with a solution of reactive linker molecules, the remaining photoresist is removed and the surface is treated with protein. The advantages of using a microdispensing technique, compared to those described above, is the flexibility of the microdispensing. Due to the microdispensers non-contact mode of operation it further allows controlled patterning of molecules on substrates that are unsuitable for methods involving physical contact with the substrate. Such substrates where microdispensing could be advantageous include surfaces with delicate micro or nanotopology, which could be destroyed by contact with a stamp or mask. Patterning of proteins or other molecules on invasive microelectrodes is one area where microdispensing could be a method of choice. Foley et al. [33] did show the possibility to pattern proteins in a microfluidics device using a special kind of microcontact printing. This enabled
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patterning on a surface with a high aspect ratio, but it is conceivable that non-contact microdispensing could also be used for this purpose. With microdispensing it is possible to vary the amount of protein deposited at one position by dispensing fewer or more droplets at that position, thus creating gradient patterns. The dispensing technique could be used for delivery of substances to cells in culture or patterning of molecules during cell culture. An additional advantage of microdispensing is the speed of making patterned substrates. Creation of a micropattern such as those described here, takes roughly 5–10 min. The piezo-electric microdispenser technique used in this investigation does not require heating of the liquid during dispensing as commercial bubble-jet printers. Hence, piezoelectric microdispensing allows dispensing of heat-sensitive proteins and fluids as well as living cells. It should however be added that the cell printing done by Xu et al. [34] was done using thermal inkjets and they found that there was little adverse effects on cell survival due to increased temperature. 5. Conclusions Here we describe a piezo electric microdispensing method to create protein micropatterns for neurite guidance. The method is fast and flexible and can also be used for creation of protein gradients. Using this method we could successfully guide neurites from dorsal root ganglia on protein micropatterns of laminin and BSA. Acknowledgements We wish to express our gratitude to Inger Antonsson for her expert technical assistance. The BSA antibody was a generous gift from Professor Bjo¨rn Westro¨m, The Department of Cell and Organism Biology, Lund University. This project was sponsored by the Research School in Pharmaceutical Science in Lund, The Royal Physiographic Society in Lund, The Swedish Research Council and VINNOVA. References [1] Clark P, Britland S, Connolly P. Growth cone guidance and neuron morphology on micropatterned laminin surfaces. J Cell Sci 1993; 105(Pt 1):203–12. [2] Thompson DM, Buettner HM. Schwann cell response to micropatterned laminin surfaces. Tissue Eng 2001;7(3):247–65. [3] Kleinfeld D, Kahler KH, Hockberger PE. Controlled outgrowth of dissociated neurons on patterned substrates. J Neurosci 1988; 8(11):4098–120. [4] Hammarback JA, McCarthy JB, Palm SL, Furcht LT, Letourneau PC. Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity. Dev Biol 1988;126(1): 29–39. [5] Kam LC, St John PM, Craighead HG, Isaacson M, Turner JN, Shain W, et al. Astrocyte adhesion to peptide-modified substrates. Abst Papers Am Chem Soc 1997;213:360-Coll. [6] St John PM, Kam L, Turner SW, Craighead HG, Issacson M, Turner JN, et al. Preferential glial cell attachment to microcontact printed surfaces. J Neurosci Methods 1997;75(2):171–7.
[7] Kam L, Shain W, Turner JN, Bizios R. Axonal outgrowth of hippocampal neurons on micro-scale networks of polylysine-conjugated laminin. Biomaterials 2001;22(10):1049–54. [8] Oliva Jr AA, James CD, Kingman CE, Craighead HG, Banker GA. Patterning axonal guidance molecules using a novel strategy for microcontact printing. Neurochem Res 2003;28(11): 1639–48. [9] Folch A, Toner M. Cellular micropatterns on biocompatible materials. Biotechnol Prog 1998;14(3):388–92. [10] Patel N, Padera R, Sanders GH, Cannizzaro SM, Davies MC, Langer R, et al. Spatially controlled cell engineering on biodegradable polymer surfaces. Faseb J 1998;12(14):1447–54. [11] Kane RS, Takayama S, Ostuni E, Ingber DE, Whitesides GM. Patterning proteins and cells using soft lithography. Biomaterials 1999;20(23–24):2363–76. [12] Rogers SL, Letourneau PC, Palm SL, McCarthy J, Furcht LT. Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev Biol 1983;98(1):212–20. [13] Hammarback JA, Palm SL, Furcht LT, Letourneau PC. Guidance of neurite outgrowth by pathways of substratum-adsorbed laminin. J Neurosci Res 1985;13(1-2):213–20. [14] James CD, Davis R, Meyer M, Turner A, Turner S, Withers G, et al. Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays. IEEE Trans Biomed Eng 2000;47(1):17–21. [15] O’Shaughnessy TJ, Gray SA, Pancrazio JJ. Cultured neuronal networks as environmental biosensors. J Appl Toxicol 2004;24(5): 379–85. [16] Jenkner M, Muller B, Fromherz P. Interfacing a silicon chip to pairs of snail neurons connected by electrical synapses. Biol Cybern 2001;84(4):239–49. [17] Klebe RJ. Cytoscribing: a method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues. Exp Cell Res 1988;179(2):362–73. [18] Turcu F, Tratsk-Nitz K, Thanos S, Schuhmann W, Heiduschka P. Ink-jet printing for micropattern generation of laminin for neuronal adhesion. J Neurosci Methods 2003;131(1-2):141–8. [19] Watanabe K, Miyazaki T, Matsuda R. Growth factor array fabrication using a color ink jet printer. Zool Sci 2003;20(4):429–34. [20] Sanjana NE, Fuller SB. A fast flexible ink-jet printing method for patterning dissociated neurons in culture. J Neurosci Methods 2004; 136(2):151–63. [21] Roth EA, Xu T, Das M, Gregory C, Hickman JJ, Boland T. Inkjet printing for high-throughput cell patterning. Biomaterials 2004; 25(17):3707–15. [22] Laurell T, Wallman L, Nilsson J. Design and development of a silicon microfabricated flow-through dispenser for on-line picolitre sample handling. J Micromech Microeng 1999;9(4):369–76. [23] Ressine A, Finnskog D, Malm J, Becker C, Lilja H, Marko Varga G, et al. Macro/nano-structured silicon as solid support for antibody design. NanoBiotechnology 2005;1(1):93–104. [24] Olle EW, Messamore J, Deogracias MP, McClintock SD, Anderson TD, Johnson KJ. Comparison of antibody array substrates and the use of glycerol to normalize spot morphology. Exp Mol Pathol 2005;79(3):206–9. [25] Ressine A, Ekstrom S, Marko-Varga G, Laurell T. Macro-/ nanoporous silicon as a support for high-performance protein microarrays. Anal Chem 2003;75(24):6968–74. [26] Lander AD, Fujii DK, Reichardt LF. Laminin is associated with the ‘‘neurite outgrowth-promoting factors’’ found in conditioned media. Proc Natl Acad Sci USA 1985;82(7):2183–7. [27] Allen DT, Kiernan JA. Permeation of proteins from the blood into peripheral nerves and ganglia. Neuroscience 1994; 59(3):755–64. [28] Dyer D, Tigyi G, Miledi R. The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their
ARTICLE IN PRESS P. Gustavsson et al. / Biomaterials 28 (2007) 1141–1151 role in neurite retraction. Brain Res Mol Brain Res 1992;14(4): 302–9. [29] Dyer D, Tigyi G, Miledi R. The effect of active serum albumin on PC12 cells: I. Neurite retraction and activation of the phosphoinositide second messenger system. Brain Res Mol Brain Res 1992;14(4): 293–301. [30] Romanova EV, Fosser KA, Rubakhin SS, Nuzzo RG, Sweedler JV. Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning. Faseb J 2004;18(11):1267–9.
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[31] Martinoia S, Bove M, Tedesco M, Margesin B, Grattarola M. A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates. J Neurosci Methods 1999;87(1): 35–44. [32] Ryan AF, Wittig J, Evans A, Dazert S, Mullen L. Environmental micro-patterning for the study of spiral ganglion neurite guidance. Audiol Neurootol 2006;11(2):134–43. [33] Foley J, Schmid H, Stutz R, Delamarche E. Microcontact printing of proteins inside microstructures. Langmuir 2005;21(24):11296–303. [34] Xu T, Jin J, Gregory C, Hickman JJ, Boland T. Inkjet printing of viable mammalian cells. Biomaterials 2005;26(1):93–9.
Biomaterials 28 (2007) 1141–1151 www.elsevier.com/locate/biomaterials
Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser Per Gustavssona,, Fredrik Johanssona, Martin Kanjea, Lars Wallmanb,c, Cecilia Eriksson Linsmeiera a
Department of Cell and Organism Biology, Lund University, Helgonava¨gen 3B, SE 223 62, Lund, Sweden Department of Electrical Measurements, Lund Institute of Technology, Box 118, SE 221 00, Lund, Sweden c Neural Interfaces, Department of Experimental Medical Science, Lund University, BMC F10, SE 221 84, Lund, Sweden b
Received 25 August 2006; accepted 21 October 2006 Available online 15 November 2006
Abstract In this study, we developed a microdispenser technique in order to create protein patterns for guidance of neurites from cultured adult mouse dorsal root ganglia (DRG). The microdispenser is a micromachined silicon device that ejects 100 picolitre droplets and has the ability to position the droplets with a precision of 6–8 mm. Laminin and bovine serum albumin (BSA) was used to create adhesive and non-adhesive protein lines on polystyrene surfaces (cell culture dishes). Whole-mounted DRGs were then positioned close to the patterns and neurite outgrowth was monitored. The neurites preferred to grow on laminin lines as compared to the unpatterned plastic. When patterns were made from BSA the neurites preferred to grow in between the lines on the unpatterned plastic surface. We conclude that microdispensing can be used for guidance of sensory neurites. The advantages of microdispensing is that it is fast, flexible, allows deposition of different protein concentrations and enables patterning on delicate surfaces due to its non-contact mode of operation. It is conceivable that microdispensing can be utilized for the creation of protein patterns for guiding neurites to obtain in vitro neural networks, in tissue engineering or rapid screening for guiding proteins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Micropatterning; Nerve tissue engineering; Nerve regeneration; Protein; Cell adhesion
1. Introduction It has previously been demonstrated that micropatterns of proteins and other molecules can be used for the guidance of nerve cell processes such as neurites but also to create patterns of whole neurons and glial cells [1–8]. Techniques for creating these patterns include soft lithography methods such as microcontact printing [5–8] and microfluidics [2,9–11], microlithography [1–3] and UVirradition protein inactivation [4]. Commonly patterned molecules are general adhesive molecules such as poly-Llysine or extracellular matrix (ECM) compontents such as laminin, which has long been known for its ability to guide neurites and promote neuronal adhesion [12,13]. Protein and chemical micropatterns can be used for studies of cell Corresponding author. Tel.: +46 46 222 93 54; fax: +46 46 222 45 39.
E-mail address: [email protected] (P. Gustavsson). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.10.028
adhesion, guidance and chemotaxia, but also for the specific guidance of neurites to microelectrodes capable of generating and measuring neural electrical signals [14]. Such neuro-electrical interfaces could have a number of applications, e.g. connecting prosthetic devices to the nervous system for transduction of sensory and motor information, creating cell-based biosensors [15] or creating neural networks in vitro [16]. An alternative method for micropattern formation is microdispensing or inkjet printing, which has total flexibility for pattern generation and does not require contact with the substrate as opposed to other techniques for protein patterning [17–21]. Previous studies using microdispensing have shown printing of cell adhesion molecules for controlled patterning of dissociated cells on surfaces. In this study we used microdispensing to dispense droplets of adhesive laminin and non-adhesive bovine serum albumin (BSA) solutions to create protein micropatterns on polystyrene tissue culture dishes.
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We examined the effects of these patterns on guidance of neurites from organcultured adult dorsal root ganglia (DRG).
2. Materials and methods 2.1. Proteins The proteins used for micropatterning were laminin from EngelbrethHolm-Swarm murine sarcoma (0.5 and 0.05 mg/ml, Sigma, Sweden), BSA fraction V (10, 1 and 0.1 mg/ml, ICN Biomedicals, USA) and fatty-acidfree BSA (10, 1 and 0.1 mg/ml, Sigma, Sweden). For measurements of the spatial resolution and size of the protein micropatterns, 0.1 mg/ml of AlexaFluor 488 labelled BSA was used (Molecular Probes, USA). All proteins were diluted in sterile 0.1 mM phosphate buffered saline (PBS), pH 7.2.
2.2. Micropattern generation Micropatterns were generated using the microdispenser system developed at the Department of Electrical Measurements at Lund Institute of Technology and previously described by Laurell et al. [22]. The experimental setup is shown in Figs. 1 and 2. In short, the microdispenser is composed of a piezoelectric element connected to a push-bar situated over a channel etched in a silicon. The channel has one inlet at each end and a nozzle in the middle (Fig. 2). When a voltage is applied across the piezoelectric element it will expand and via the silicon membrane it will expel the liquid in the nozzle. The dispenser is loaded with a protein solution and the target is moved using a computer-controlled x–y table in order to generate protein patterns. The CCD-camera shown in Fig. 1 allows real-time visual inspection of the microdispensing process and enables positioning of patterns on the substrate. The substrates were heated to 30 1C by a heater element on the x–y table in order to increase the spatial resolution by increased droplet evaporation, thus preventing the dispersion of the drops on the surface. The patterns generated consisted of lines with a drop pitch of 40 mm and a line spacing of 300 mm. We used 35 mm NunclonTM polysterene cell culture dishes (Nunc, Denmark) as substrates.
Fig. 2. Close-up of the microdispenser. Top panel: the dispenser is fixed in position by a metal arm, protein solution is fed into the dispenser head through two silicone tubes functioning as inlets at each end of the channel. Bottom panel: schematic section drawing of the microdispenser. A piezoelectric element is situated above a channel etched in silicon. The element is connected to a push-bar that forms the roof of the channel. When a voltage is applied to the element it will expand and the membrane will push the liquid through the nozzle in the middle of the channel.
2.3. Organotypic culture of DRG All procedures involving animals were conducted according to the Swedish experimental animal ethical guidelines and approved by the local Ethical Committee on Animal Experiments. Adult female NMRI mice (B&K Universal, Sweden) were killed by an intraperitoneal injection of sodium pentobarbital (Apoteksbolaget, Sweden) and the DRG from lumbar 6 to lumbar 4 were dissected and mounted in the ECM-substitute Matri-gel onto culture dishes with microdispensed protein patterns. In each culture dish two ganglia were mounted diagonally on opposite sides of the pattern and placed as closely as possible to the edge of the patterns. The DRGs were cultured for three days at 37 1C in an atmosphere of 6.5% CO2 and 93.5% O2 in RPMI 1640 media (Sigma, Sweden) supplemented with 20 ng/ml NGF (PeproTech, USA) to promote neurite outgrowth, L-glutamine (Gibco, USA) and penicillin/ streptomycin (Gibco).
2.4. Immunocytochemistry
Fig. 1. The experimental setup with the microdispenser is located in the centre of the picture (1). The dispenser is held in position by a metal arm above a computer-controlled x–y table (2). The cell culture dishes are placed on the x–y table and during dispensing the table moves the dish while the dispenser stays fixed. The frequency of droplet ejection and the pulse formation, is controlled by a signal generator (3). A CCD-camera with magnifying optics allows real-time inspection of the dispensing process in order to determine if to modify the parameters (4).
After 3 days in culture the DRG cultures were fixed in Stefanini’s fixative (4% paraformaldehyde, 0.03% saturated picric acid in 0.1 M PBS, pH 7.2) over night at 4 1C. Following rinses in PBS, the cultures were incubated over night at 4 1C with primary antibodies diluted in PBS containing 0.25% Triton X-100. The antibodies used were mouse monoclonal antibodies recognizing b-tubulin isotype III (1:400; Sigma, USA) to visualize neurites; rabbit polyclonal antibodies recognizing BSA (1:50, DAKO, Denmark) or rabbit polyclonal antibodies recognizing laminin (1:25, Sigma, USA) to visualize the microdispensed protein patterns. After repeated rinses in PBS, the preparations were further incubated for 1 h at room temperature with the polyclonal secondary antibodies AlexaFluor 594 goat anti-mouse and AlexaFluor 488 goat
ARTICLE IN PRESS P. Gustavsson et al. / Biomaterials 28 (2007) 1141–1151 anti-rabbit (1:500, Molecular Probes, USA), repeatedly rinsed in PBS, mounted in 1:1 PBS/glycerol and coverslipped.
2.5. Photography and image analysis The preparations were photographed using an Olympus AX70 fluorescence microscope equipped with a DP50 CCD camera and images were acquired using the software StudioLite (Olympus, Japan). Images were converted to 8-bit grayscale TIFF using Adobe Photoshop 7.0 (Adobe, USA) and imported into NIH Image 1.63 (a public domain image analysis programme developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). The Plot Profile function was used to measure the intensity values of the AlexaFluor488 BSA gradients in order to confirm that gradients had formed. The distances and width of protein lines and arays was determined using the distance measurment function. Unpaired Students t-test (Microsoft Excel, Microsoft Corp. USA) was used to test for significant differences in distance and width. A p-value p0.05 was considered as statistically significant. To quantify neurite outgrowth on the patterns, the immunoreactivity to bIII-tubulin was determined on the microdispensed protein lines and on the unpatterned surface. The immunoreactivity to neuronal tubulin corresponds to the cytoskeleton inside the neurites and could thus be regarded to be a measure of neurite outgrowth. The intensity cut-off, above which the tubulin immunoreactivity was considered positive, was determined separately for each image in order to keep the background fluorescence of the substrate surface below the detection level. The number of immunopositive pixels was measured on the protein lines and between the lines, using NIH Image, and then the ratio between immunopositive pixels and total number of pixels in the region of interest was calculated. Unpaired Students t-test (Microsoft Excel) was used to test for significant differences in pixel ratios between protein patterns and non-patterned surface. This procedure thus gave a measure of the amount of axons growing on the patterns and between the patterns. A p-value p0.05 was considered as statistically significant.
3. Results 3.1. Protein micropattern generation Fluorescent BSA was used to investigate the precision of the microdispenser system (the system is shown in Figs. 1 and 2), but not used for cell culture since the influence of the fluorophores on neurite outgrowth was not known. Figs. 3 and 4 show arrays and lines, respectively, generated by microdispensing 0.1 mg/ml fluorescent BSA on plasmatreated polystyrene. In Fig. 3, three different types of arrays are shown. The arrays were generated by modulating the drop pitch in both x- and y-direction to 100, 200 and 300 mm, respectively. Fig. 3A shows an array with a pitch of 100 mm, where the array became a square due to
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merging of the droplets on the substrate. Image analysis showed that the array had a mean dimension of 1033721 mm 1027710 mm (n ¼ 10). In Fig. 3B, an array with a pitch of 200 mm is shown. The resulting array had discrete points, as opposed to the array in Fig. 3A. The mean distance between neighbouring points was 19976 mm (n ¼ 180) and the mean diameter of the points was 15674 mm (n ¼ 100). In Fig. 3C, the drop pitch was set to 300 mm, which also resulted in an array with discrete points. The mean distance between neighbouring points was measured to 29978 mm (n ¼ 134). The average diameter of the points in an array with a 300 mm pitch was 158710 mm (n ¼ 70). In Figs. 3B and C, one can see satellite droplets that formed around the dispensed drops. These satellites appeared when the liquid used for dispensing contained contaminating dust particles or when salt and protein crystals formed in the nozzle, thus disturbing the trajectory of the drops. The formation of satellite droplets could be reduced by modulating the pulse applied to the piezo ceramic element of the dispenser and by cleaning the nozzle with a moist tissue between rounds of dispensing. Figs. 4A–D shows linear patterns generated by microdispensing. The drop pitch in the y-direction was set to 30 mm in Figs. 4A and B and 50 mm in Figs. 4C and D, while the x-direction was kept constant at 300 mm. The mean width of the lines with 30 mm pitch was 23974 mm (n ¼ 29) at when the substrate was kept at room temperature during dispensing and 240715 mm (n ¼ 40) when the substrate was heated to 30 1C. The mean width of the lines with 50 mm pitch was 19973 mm (n ¼ 36) at room temperature and 19871 mm (n ¼ 36) when substrates were heated to 30 1C. There was no statistical difference between substrates made at room temperature or heated substrates but by visual inspection of patterns (Figs. 4A–D), it could be seen that the patterns were more irregular on nonheated substrates while they were more regular and the protein lines more straight when the substrates were heated. The width of the lines at a pitch of 30 and 50 mm was however significantly different, irrespective of the temperature during dispensing (po0:001). To further investigate the possibilities to generate patterns, we created gradients of fluorescent BSA. The gradients were created by changing the number of droplets dispensed in each point. The array show in Fig. 5A consists
Fig. 3. Arrays of microdispensed AlexaFluor 488 labelled BSA (0.1 mg/ml). The droplet pitch in A was 100 mm, in B 200 mm and in C 300 mm. The numbers 1–3 in the figures indicate how the pattern resolution was measured. Scale bar ¼ 500 mm.
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Fig. 4. Lines of microdispensed AlexaFluor 488 labelled BSA (0.1 mg/ml). The x-direction droplet pitch was changed from the top panel to the lower panel as follows: A–B: 30 mm pitch, C–D: 50 mm pitch. In A and C the substrate was kept at room temperature during dispensing, while in B and D the substrate was heated to 30 1C to increase evaporation to prevent the spreading of dispensed droplets over the surface. Scale bar ¼ 1000 mm.
Fig. 5. (A–D) Gradients of AlexaFluor 488 labelled BSA (0.1 mg/ml). (A) Gradient consisting of 10 spots, the first two spots contained one droplet and the last two contained five droplets, the pitch was 50 mm. (B) Intensity plot for the gradient shown in A. (C) Gradient consisting of 20 spots, the first two spots contained one droplet each and the last two spots contained 10 droplets each, the pitch was 50 mm. (D) Intensity plot for the gradient shown in C. Scale bar ¼ 200 mm.
of 10 points with the two first points each containing 1 droplet, the next two points contains 2 droplets each and so forth with the last two point containing 5 droplets each. An intentsity profile of the gradients is shown in Fig. 5B. Fig. 5C shows a gradient created in a similar way, but consisting of 20 points, with the first two points containing 1 droplet each and the last two points containing 10 droplets each. An intensity plot for this array is shown in Fig. 5D. From these images it is possible to see that
gradients can be created with microdispensing, however they are not very smooth. We tested glass cover slips as substrates for fluorescent BSA. However, glass cover slips yielded poorer patterns due to rapid merging of adjacent droplets, causing dispersion of the pattern (results not shown). Better patterns on glass could be achieved by decreasing the delay between droplets as to allow the previous drop to evaporate before the next. Still, this did not result in as
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uniform patterns as those formed on plastic cell culture dishes. 3.2. Neurite outgrowth on laminin patterns Fig. 6A shows neurite outgrowth (visualised by tubulin immunostaining, red) on patterns containing 0.5 mg/ml laminin and Fig. 6B shows the corresponding laminin patterns (visualised by laminin immunostaining, green). From Fig. 6A it can be seen that there was a higher proportion of neurites growing on the microdispensed lines compared to the number growing between the lines. The guidance was not perfect, and some neurites followed the lines for a short distance but then left to grow in between the lines. A certain amount of satellite droplets had also been generated during microdispensing, which could have provided a certain increased attachment between the patterns. When the images were analysed for the ratio of tubulin immunopositive pixels (those pixels that corresponded to neurites) to total number of pixels in the analysed area, there was a significantly higher percentage of positive pixels on the laminin containing lines than between the lines, corroborating the ocular observations (Fig. 7). Fig. 6C shows neurite outgrowth (red staining) on protein patterns containing 0.05 mg/ml laminin and Fig. 6D shows the corresponding laminin patterns (green staining). Here the neurites did not follow the laminin lines, instead the neurites grew randomly across the pattern. Image analysis confirmed these observations, and there was no significant difference between the percentage of tubulin
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immunopositive pixels on the lines and in between the lines (Fig. 7). 3.3. Neurite outgrowth on 98% pure BSA patterns Fig. 8A shows neurite outgrowth (visualised by tubulin immunostaining, red) on protein patterns made from 10 mg/ml BSA and Fig. 8B shows the corresponding protein patterns (visualised by BSA immunostaining, green). From Fig. 8A, it can be seen that most of the
Fig. 7. Ratio between tubulin positive pixels and total pixel area on laminin lines and between laminin lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 4 for all experiments).
Fig. 6. (A–D) Neurite outgrowth on laminin patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by laminin immunostaining (green). (A) Neurite outgrowth on 0.5 mg/ml laminin. (B) 0.5 mg/ml laminin patterns. (C) Neurite outgrowth on 0.05 mg/ml laminin. (D) 0.05 mg/ml laminin patterns. Scale bars ¼ 200 mm.
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Fig. 8. (A–F) Neurite outgrowth on BSA patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by BSA immunostaining (green). (A) Neurite outgrowth on 10 mg/ml BSA. (B) 10 mg/ml BSA patterns. (C) Neurite outgrowth on 1 mg/ml BSA. (D) 1 mg/ml BSA patterns. (E) Neurite outgrowth on 0.1 mg/ml BSA. (F): 0.1 mg/ml BSA patterns. Scale bars ¼ 200 mm.
neurites avoided the protein lines and grew in between the lines. Some neurites started growing on the unpatterned surface, then grew on to the patterns but then grew back on the unpatterned surface. Image analysis revealed that there was a significant difference between the relative area of tubulin positive pixels on and between protein lines (Fig. 9). In Fig. 8C the neurite outgrowth (red staining) on patterns made of 1 mg/ml BSA is shown and in Fig. 8D the corresponding protein patterns (green staining) are shown. The 1 mg/ml BSA patterns showed a similar guidance as the 10 mg/ml patterns, but an increasing number of neurites grew on the lines in these experiments. Image analysis once again revealed that there was a significant difference between the relative area of tubulin positive pixels on the lines and the positive pixels between the lines (Fig. 9). Fig. 8E shows the neurite outgrowth (red staining) on patterns made from 0.1 mg/ml BSA and in Fig. 8F the corresponding protein patterns (green staining) are shown. When the patterns were made from a 0.1 mg/ml BSA solution, the neurites did not prefer to grow between
Fig. 9. Ratio between tubulin positive pixels and total pixel area on BSA lines and between BSA lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 5 for 10 mg/ml, n ¼ 8 for 1 mg/ml and n ¼ 4 for 0.1 mg/ml).
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the patterns, but instead grew in a random fashion over the surface, both on the lines and in between the lines. Image analysis showed that there was no significant difference between the percentage of tubulin immunopositive pixels occupying the lines or the unpatterned surface (Fig. 9). A general observation for the BSA lines was that a higher concentration of BSA (10 mg/ml, shown in Fig. 8B) resulted in finer patterns due to increased viscosity as compared to a lower concentration (e.g. 0.1 mg/ml, shown in Fig. 8F). 3.4. Growth on 96% pure fatty-acid-free BSA patterns As BSA is known for its non-permissive properties we also sought to investigate if fatty-acids bound to BSA could be involved in this property. To this end we created micropatterns of fatty-acid-free BSA and cultured DRGs on these patterns. Fig. 10A shows neurite outgrowth (visulised by tubulin immunostaining, red) on protein
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patterns made from 10 mg/ml fatty-acid-free BSA and Fig. 10B shows the corresponding protein patterns (visulised by fatty-acid-free BSA immunostaining, green). It can be seen that the neurites preferred to grow on the unpatterned surface between the lines containing BSA and avoided the patterned lines. Image analysis revealed that there was a significantly higher percentage of tubulin immunopositive pixels between the lines than on the lines, showing that the bulk of the neurites grew between lines (Fig. 11). Fig. 10C shows neurite outgrowth (red staining) on patterns of 1 mg/ml fatty-acid-free BSA and Fig. 10D shows the corresponding protein patterns (green staining). Most of the neurites avoided the protein lines and grew in between on the unpatterned surface. Some neurites could be found growing on the protein lines. Image analysis showed a significantly higher proportion of tubulin positive pixels between the lines (Fig. 11) compared to the proportion on the lines. Fig. 10E shows neurite outgrowth (red staining) on patterns made of 0.1 mg/ml fatty-acid-free
Fig. 10. (A–F) Neurite outgrowth on fatty-acid-free BSA patterns. Neurites were visualized by tubulin immunostaining (red) and protein patterns by fatty-acid-free BSA immunostaining (green). (A) Neurite outgrowth on 10 mg/ml fatty-acid-free BSA. (B) 10 mg/ml fatty-acid-free BSA patterns. (C) Neurite outgrowth on 1 mg/ml fatty-acid-free BSA. (D) 1 mg/ml fatty-acid-free BSA patterns. (E) Neurite outgrowth on 0.1 mg/ml fatty-acid-free BSA. (F) 0.1 mg/ml fatty-acid-free BSA patterns. Scale bars ¼ 200 mm.
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Fig. 11. Ratio between tubulin positive pixels and total pixel area on fatty-acid-free BSA lines and between fatty-acid-free lines. Black bars indicate outgrowth on protein lines, white bars indicate outgrowth between protein lines. Error bars ¼ S.E.M. (n ¼ 3 for 10 mg/ml, n ¼ 4 for 1 mg/ml and n ¼ 3 for 0.1 mg/ml).
BSA and Fig. 10F shows the corresponding protein pattern (green staining). It can be seen in Fig. 10E that the outgrowing neurites grew randomly over the surface and did not show any preference for protein lines or the uncoated surface. Image analysis corroborated these observations as there was no significant difference in the percentage of tubulin immunopositive pixels on or between the 0.1 mg/ml BSA patterns (Fig. 11). A general observation for these patterns made from fatty-acid-free BSA, is that a higher concentration of BSA (10 mg/ml, shown in Fig. 10B) resulted in finer patterns as compared to lower concentrations (e.g. 0.1 mg/ml, shown in Fig. 10F), reflecting the influence of the higher viscosity of 10 mg/ml. 4. Discussion In this investigation the major finding was that protein micropatterns generated by a piezoelectric microdispenser can be used for guidance of normal regenerating nerve cell processes in this case, sensory neurites from the DRG. The protein patterns generated were made of laminin and BSA. The resolution of the micropatterns generated by microdispensing depends on a number of factors including the viscosity of the liquid being dispensed, the drop pitch (the distance between the centre of two adjacent droplets in a line or array) and on the substrate. Increased viscosity gives thinner protein lines compared to a lower viscosity. This can be seen for the BSA lines in Figs. 8 and 10, where the 10 mg/ml lines were thinner as compared to the 1 and 0.1 mg/ml lines. However, if the viscosity is increased too much the liquid becomes impossible to expel from the nozzle. The critical level of BSA was at 10 mg/ml. The effect of drop pitch can be seen in Fig. 4, where a lower drop pitch resulted in wider patterns. The effect of a low drop pitch on pattern wideness is probably due to merging
of adjacent droplets and reduced evaporation from the surface. Heating did not influence the width of lines significantly but it generally provided more uniform protein lines. As the temperature was kept below 37 1C, the heating should not affect the state of the proteins more negatively than if the substrates were at room temperature, although denaturing and reduced biological effects cannot be ruled out. The resolution of microdispensed protein spots on substrates with various hydrophilictiy–hydrophobicity has been described by Ressine et al. [23] who used the same microdispensing system as we did. They showed that hydrophilic substrates such as glass exhibited a lower contact angle and gave wider spots while highly hydrophobic macro/nanoporous silicon exhibited a higher contact angle and resulted in smaller spots. We used plastic cell culture dishes, which are rather hydrophilic, since these dishes are primed for cell culture. These dishes are also sterile and easy to handle and mount a DRG on. For rapid screening purposes there is no disadvantage of using these dishes as substrates, but in order to make finer patterns and increasing spatial resolution a hydrophobic substrate could be an advantage. During the development of the system we also tried glass coverslips as substrates. However these substrates yielded poorer patterns due to rapid merging of adjacent droplets, causing dispersion of the pattern. Better patterns on glass could be achieved by decreasing the droplet frequency, as to allow the previous drop to evaporate before the next. Still, this did not result in as uniform patterns as those formed on plastic cell culture dishes. We were also able to make surface bound protein gradient using AlexaFluor 488 labelled BSA. This was achieved by increasing the number of droplets dispensed at each point along the gradient. The gradients are however not smooth, due to overlap between droplets and uneven droplet evaporation. Droplet over lap is inevitable, since it is necessary in order to create continuous lines. The uneven droplet evaporation leads to a local increase of protein concentration, which is reflected in the peaks seen in the surface plots. Smoother droplet evaporation can be achieved by modification of the properties of the solution dispensed. Addition of glycerol for instance, increases the viscosity of the liquid and yields spots with smaller diameter and reduces evaporation artifacts [24]. Modifications of the substrate can also be used to create finer patterns and more uniform droplets [25]. The use of gradients like the ones described here, could be to screen molecules for possible chemotactic properties or to pattern cells at various densities by creating gradients of adhesive molecules. It is conceivable that such gradients could be made from laminin or similar adhesive proteins. Certain dispensed liquids generated satellite droplets, which could be reduced by modulating the waveform of the pulse applied to the dispensers piezoceramic element in order to improve pattern resolution. A total reduction in satellites could not always be achieved, which was especially true for the highest concentration of laminin.
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Laminin is a well-known ECM protein with cell adhesive and neurite guiding properties [12,13,26] and here we show that patterns of laminin for sensory neurite guidance can be made by microdispensing. At a concentration of 0.5 mg/ml laminin, the neurite guiding properties of laminin was clear and most axons grew and followed the lines, whereas at 0.05 mg/ml the axons grew more or less randomly on the surface. The reason why some neurites did not prefer to grow on the tracks made from 0.5 mg/ml laminin, could be due to the fact that the substrate used for dispensing are cell culture dishes which from the start are primed to enhance attachment of cells and cellular processes. To improve guidance, the laminin could have been patterned on a non-permissive substrate, such as BSA or polyethylene glycol (PEG). For instance, Sanjana and Fuller [20] printed permissive collagen/poly-D-lysine on a nonpermissive surface of PEG to control cell spreading. Another option would be to treat the polystyrene surface with e.g. BSA after creation of the microdispensed patterns, thus blocking available binding sites on the surface. Occasional satellite droplets of laminin between the patterns could also have influenced the guidance, promoting some outgrowth between the lines. When doing organotypic culture of DRGs, fibroblasts and Schwann cells migrate from the ganglia to a certain extent, especially from DRGs of newborn animals. These cells can interact with the outgrowing neurites and influence their growth. In our experiments we observed very few migrating cells, and the ones that had migrated stayed close to the Matri-gel border, while the axons extended much further. In this respect, DRGs from adult animals are superior to those from newborns, which exhibit extensive migration of Schwann cells. Neurites avoided the lines made of BSA providing the concentration of the solution was 10 and 1 mg/ml BSA. The avoidance was concentration dependant and at 0.1 mg/ml BSA the neurites were able to grow on the protein lines. BSA is a common molecule to use for making substrates nonpermissive. In the context of neuroscience, Thompson and Buettner [2] for instance took advantage of this property of BSA and made substrates with alternating stripes of laminin and BSA in order to create patterns of Schwann cells. It could in this context be noted that serum albumin is confined to the blood vessles and neurites do not normally come in contact with serum albumins. During nerve injury however, the blood nerve barrier is breached and serum factors make contact with axons and Schwann cells. In addition, the blood-nerve barrier of DRG is virtually absent and it has been found that horse radish peroxidase and fluorescent BSA enters intact sensory ganglia and peripheral nerves [27]. Interestingly Dyer et al. [28,29] showed that an isoform of serum albumin, called active serum albumin, which contained certain lipids could induce growth cone collapse of PC12 cells. We therefore tested if fatty-acid-free BSA exhibited the same properties as regular BSA. Lines created with fatty-acid-free BSA, were as nonpermissive for neurite as those created with BSA containing
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fatty acids. Thus fatty acids bound to BSA are probably not the reason for the non-permissive nature of BSA. Microdispensing was first introduced by Klebe [17] who used a commercial inkjet printer to pattern fibronectin and anti-EGF receptor antibodies for selective cell attachment. Others including Turcu et al. [18] have used a microdispensing system similar to ours to create protein patterns on various surfaces including glass, pure silicon, gold-coated silicon and glassy carbon. Watanabe et al. [19] and Sanjana and Fuller [20] used a customized commercial piezo electric inkjet printer. Roth et al. [21] on the other hand used a modified commercial bubble-jet printer to print collagen and pattern dissociated DRG neurons. Additional techniques to chemically pattern a substrate include microcontact printing, microfluidics and microlithography. They all have in common that they require special process laboratories and are rather work intensive, especially the soft lithograpic methods of microcontact printing and microfluidics. As compared to microdispensing their advantage is that they offer a higher spatial resolution. For microcontact printing, a master stamp is first made by etching the desired pattern into a silicon wafer or a quartz crystal. A solution of polymer such as polydimethylsiloxane (PDMS) is poured on the etched master and cured in an oven. The PDMS is removed and can then be coated with a reactive linker solution or a protein solution. By pressing the PDMS stamp on a surface, a linker or protein imprint is created onto which cells can be seeded and thus patterned. This method has been used for neuronal cell patterning and is described in [5–7]. Microfluidics shares the manufacture steps involved in microcontact printing, has a high resolution and can be used to create protein gradients. It has been used for studies of neuronal cells by Romanova et al. [30], Martinoia et al. [31], Ryan et al. [32] and Patel et al. [10]. Microlithography, used by Thompson and Buettner [2] and Kleinfeld et al. [3] among others, works by covering a substrate with photoresist and areas where proteins are supposed to be deposited are exposed to UV light to remove the resist. The substrate is then treated with a solution of reactive linker molecules, the remaining photoresist is removed and the surface is treated with protein. The advantages of using a microdispensing technique, compared to those described above, is the flexibility of the microdispensing. Due to the microdispensers non-contact mode of operation it further allows controlled patterning of molecules on substrates that are unsuitable for methods involving physical contact with the substrate. Such substrates where microdispensing could be advantageous include surfaces with delicate micro or nanotopology, which could be destroyed by contact with a stamp or mask. Patterning of proteins or other molecules on invasive microelectrodes is one area where microdispensing could be a method of choice. Foley et al. [33] did show the possibility to pattern proteins in a microfluidics device using a special kind of microcontact printing. This enabled
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patterning on a surface with a high aspect ratio, but it is conceivable that non-contact microdispensing could also be used for this purpose. With microdispensing it is possible to vary the amount of protein deposited at one position by dispensing fewer or more droplets at that position, thus creating gradient patterns. The dispensing technique could be used for delivery of substances to cells in culture or patterning of molecules during cell culture. An additional advantage of microdispensing is the speed of making patterned substrates. Creation of a micropattern such as those described here, takes roughly 5–10 min. The piezo-electric microdispenser technique used in this investigation does not require heating of the liquid during dispensing as commercial bubble-jet printers. Hence, piezoelectric microdispensing allows dispensing of heat-sensitive proteins and fluids as well as living cells. It should however be added that the cell printing done by Xu et al. [34] was done using thermal inkjets and they found that there was little adverse effects on cell survival due to increased temperature. 5. Conclusions Here we describe a piezo electric microdispensing method to create protein micropatterns for neurite guidance. The method is fast and flexible and can also be used for creation of protein gradients. Using this method we could successfully guide neurites from dorsal root ganglia on protein micropatterns of laminin and BSA. Acknowledgements We wish to express our gratitude to Inger Antonsson for her expert technical assistance. The BSA antibody was a generous gift from Professor Bjo¨rn Westro¨m, The Department of Cell and Organism Biology, Lund University. This project was sponsored by the Research School in Pharmaceutical Science in Lund, The Royal Physiographic Society in Lund, The Swedish Research Council and VINNOVA. References [1] Clark P, Britland S, Connolly P. Growth cone guidance and neuron morphology on micropatterned laminin surfaces. J Cell Sci 1993; 105(Pt 1):203–12. [2] Thompson DM, Buettner HM. Schwann cell response to micropatterned laminin surfaces. Tissue Eng 2001;7(3):247–65. [3] Kleinfeld D, Kahler KH, Hockberger PE. Controlled outgrowth of dissociated neurons on patterned substrates. J Neurosci 1988; 8(11):4098–120. [4] Hammarback JA, McCarthy JB, Palm SL, Furcht LT, Letourneau PC. Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity. Dev Biol 1988;126(1): 29–39. [5] Kam LC, St John PM, Craighead HG, Isaacson M, Turner JN, Shain W, et al. Astrocyte adhesion to peptide-modified substrates. Abst Papers Am Chem Soc 1997;213:360-Coll. [6] St John PM, Kam L, Turner SW, Craighead HG, Issacson M, Turner JN, et al. Preferential glial cell attachment to microcontact printed surfaces. J Neurosci Methods 1997;75(2):171–7.
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