Peptide nanofiber substrates for long-term culturing of primary neurons

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Biological and Medical Applications of Materials and Interfaces

Peptide nanofiber substrates for long-term culturing of primary neurons Adam D Martin, Sook Wern Chua, Carol G Au, Holly Stefen, Magda Przybyla, Yijun Lin, Josefine Bertz, Pall Thordarson, Thomas Fath, Yazi D Ke, and Lars Matthias Ittner ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07560 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Applied Materials & Interfaces

Peptide nanofiber substrates for long-term culturing of primary neurons Adam D. Martin,*a,c Sook Wern Chua,a Carol G. Au,a Holly Stefen,b Magdalena Przybyla,a Yijun Lin,a Josefine Bertz,a Pall Thordarson,c Thomas Fath,b,d Yazi D. Ke,a Lars M. Ittner*a,d,e a

Dementia Research Unit, School of Medical Sciences, Faculty of Medicine, University of New

South Wales, Sydney, NSW 2052, Australia. b

Neurodegeneration and Repair Unit, School of Medical Sciences and Neuronal Culture Core

Facility, University of New South Wales, Sydney, NSW 2052, Australia. c

Department of Chemistry, The Australian Centre for Nanomedicine and the ARC Centre of

Excellence in Convergent Bio-Nano Science, University of New South Wales, Sydney, NSW 2052, Australia. d

Dementia Research Centre, Faculty of Medicine and Health Sciences, Macquarie University,

Sydney, NSW 2109, Australia e

Neuroscience Research Australia, Sydney, NSW 2031, Australia.

ABSTRACT: The culturing of primary neurons represents a central pillar of neuroscience research. Primary neurons are derived directly from brain tissue and recapitulate key aspects of neuronal development in an in vitro setting. Unlike neural stem cells, primary neurons do not

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divide, thus initial attachment of cells to a suitable substrate is critical. Commonly used polylysine substrates can suffer from batch variability owing to their polymeric nature. Herein we report the use of chemically well-defined, self-assembling tetrapeptides as substrates for primary neuronal culture. These water soluble peptides assemble into fibres which facilitate adhesion and development of primary neurons, their long-term survival (>40 days), synaptic maturation and electrical activity. Furthermore, these substrates are permissive towards neuronal transfection and transduction, which coupled with their uniformity and reproducible nature, make them suitable for a wide variety of applications in neuroscience.

KEYWORDS: Neuroscience, self-assembling peptides, cell culture, tissue engineering

INTRODUCTION Neuronal cultures are a powerful and highly versatile tool for dissecting molecular and cellular mechanisms in neuroscience. Primary neurons have been established from different species, such as rat, mouse and chicken, using different developing organs as source for cells.1 This includes tissues from different brain areas, as well as dorsal root ganglia and retina.2 Furthermore, neurons can be obtained by differentiation of precursor or stem cells, including embryonic3,4 or neural crest stem cells5,6 from mice, as well as human embryonic7,8 or iPSCs,9,10 or by directly transdifferentiating fibroblasts.11,12 Primary neuronal cultures are often used in order to provide a more realistic representation of the in vivo environment, as compared to immortalized neuronal cell lines such as HEK, PC12 and NT2 cells,13-16 or chemically programmed stem cells or progenitor cells.17,18 As primary neurons are notoriously sensitive and do not proliferate, initial adhesion of neurons to the culture


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substrate is of paramount importance. Most commonly, primary neurons are seeded onto glass coverslips coated with a thin film of poly-lysine.19 Whilst effective, variability between batches of poly-lysine owing to its polymeric nature, are known to occur. Such variation can adversely affect neuronal viability, resulting in lower reproducibility of results. Many alternative materials have been used to support the development of primary neurons, with varying degrees of success. Polymer-based materials functionalized with adhesive ligands such as fibronectin and laminin have shown ability to support primary neurons,20,21 however significant costs and potential toxicity are associated with coupling proteins from the native ECM onto polymer scaffolds. Notably, the use of Matrigel® has been unsuccessful for culture of primary neurons, including in our hands. It has however been used to culture iPSC-derived or immortalized neuronal cell lines.22 Pioneering work by Zhang showed that the self-assembling peptide RADA16 could be used to support neural stem cells, and primary neurons.23-27 Based upon this principle of ionic selfassociation, a number of other self-assembling peptides have been developed for neural cell culture, including the KLD system of Gelain,28-30 the MAX8 peptides of Pochan,31 and the peptide amphiphiles of Stupp17,32 and Guler33,34 among others. However, the majority of these examples employ neural stem cells (NSCs), which typically require expansion of cultures and chemical treatment in order to maintain stemness. NSCs are capable of proliferation, which, while useful for co-cultures and cell matrix interactions, does not necessarily represent the in vivo environment of the brain. Distinct from the aforementioned complementary ionic peptides are short peptide hydrogels (2-8 amino acids), which are capped at their N-terminus with an aromatic moiety. Here, selfassembly into nanofibres is driven through hydrophobic interactions.35-37 The ease of synthesis


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and tunability make these materials attractive prospects in tissue engineering.37,38 These hydrogels have been used to culture a variety of different cell types, including fibroblasts,39,40 mesenchymal stem cells,41 neural and cortical progenitor cells42,43 and neuronal type cells,44 however to our knowledge, this is the first report of the culture of primary neurons on short peptide nanofiber substrates. Owing to their water solubility, these self-assembling nanofiber substrates significantly simplify the current procedures for preparing neuronal long-term cultures. We show that these chemically well-defined, reproducible substrates facilitate the attachment and initial development of primary hippocampal neurons. Furthermore, we demonstrate the feasibility of long-term neuronal cultures, complete with synapse formation and electrical activity. RESULTS AND DISCUSSION Peptide design We and others have previously described short peptides which self-assemble into fibrous substrates and support culturing of cancer cell lines.45-47 Fmoc-diphenylalanine (Fmoc-FF) and related derivatives are known to self-assemble into nanofibers, however the majority of these peptides require dissolution under basic pH conditions, followed by a switch to acidic pH to induce self-assembly.48 These pH changes make most self-assembled short peptides incompatible with cell culture methods. Incorporation of charged amino acids is known to affect the pKa of peptides, altering the pH at which they self-assemble. This has been shown for the hydrophobic Ile-Lys-Val-Ala-Val (IKVAV) sequence, where aspartic acid groups were added, resulting in self-assembly at neutral pH.49,50 Based upon the most frequently used substrate for primary neurons, poly-D-lysine (PDL),1 we designed new Fmoc-peptides containing multiple positively charged

D-lysine

(k) groups, incorporated into the well-known Fmoc-FF scaffold.


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Here, D-lysines were selected to avoid matrix degradation at longer culture time periods through the action of proteolytic enzymes.51

Figure 1. Lysine containing tetrapeptides (a) Fmoc-Phe-Phe-Lys-Lys and (b) Fmoc-Phe-LysPhe-Lys, (c, d) atomic force microscopy images of their self-assembly into fibers and (e) the method employed to create peptide nanofiber coatings on glass coverslips, compared to


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traditionally used 2D cultures. AFM samples were prepared at a concentration of 0.5% (w/v) peptide and spread coated onto a freshly cleaved mica substrate. Scale bar represents 1 µm. Analogues bearing only one lysine residue were synthesized, however were too insoluble to be investigated further. Peptides Fmoc-Phe-Phe-D-Lys-D-Lys (Fmoc-FFkk, where the lower-case k denotes the presence of a D-lysine) and Fmoc-Phe- D-Lys-Phe- D-Lys (Fmoc-FkFk), bearing two lysine groups (Figure 1a, b) were dissolved in water and spontaneously self-assemble into fibers with β-sheet secondary structures, according to circular dichroism, viscosity and atomic force microscopy measurements (Figure S1 and 2, ESI† and Figure 1c, d). Fiber diameters for FmocFFkk and Fmoc-FkFk were measured as (7.7 ± 0.3) and (5.1 ± 0.8) nm, respectively. The small standard deviation is indicative of a very uniform fiber size, and the observed AFM fiber sizes were supported through small angle neutron scattering (SANS) measurements (Figure S3, ESI†). Some lateral association of fibers is observed in Fmoc-FFkk, however this may be due to drying effects.48 Due to the presence of lysine residues, these fibers are positively charged. When initially dissolved, the fibers have a pH of 4-5 (Figure S4, ESI†), which is then neutralized upon the addition of Neurobasal media when neurons are seeded atop the nanofibers. Similar charge and pH neutralization has been reported for other peptide nanofibers.49-52 Development and maturation of primary neurons on nanofiber substrates We used the tetrapeptides Fmoc-FFkk and Fmoc-FkFk to create nanofiber layers on glass coverslips and seeded freshly prepared primary neurons from E16.5 C57Bl/6 mouse embryos onto these substrates. A schematic of the coating procedure is outlined in Figure 1e. The selfassembled peptide nanofibers are incubated with the glass coverslip overnight, followed by aspiration of excess solution. It is important to note that no washing step is required here. PDL is generally prepared in borate buffer,1 which is incompatible with cell culture conditions. Thus,


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each coverslip must be washed multiple times, which is a time consuming and laborious process. Thus the coating procedure for these tetrapeptide nanofiber substrates represents a considerable time saving.

Figure 2. (a) Development of primary neurons seeded atop PDL or peptide nanofibers from 1-5 days in vitro (DIV). Development is as according to Banker staging, defined by axonal extension


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at DIV3 followed by dendrite development. Representative overlays for all channels are shown, scale bars 10 µm. Long-term neuronal cultures on nanofiber substrates, showing (b) cell viability of primary neurons seeded on peptide nanofibers as determined using an Alamar Blue colorimetric assay, with PDL used as the positive control (100% viability), (c) Primary neurons fixed after DIV40 cultured on PDL, (d) Fmoc-FFkk and (e) Fmoc-FkFk. Representative overlays for all channels are shown, scale bars 100 µm. In all cases neurons are stained for β3-tubulin (green), MAP2 (red) and nucleus (blue). Primary murine neurons undergo well-defined developmental stages during their maturation in culture.53 To examine whether our nanofiber substrates promoted neuronal adhesion, we seeded primary hippocampal neurons derived from E16.5 mouse embryos on coverslips, which had been coated with solutions of PDL, or nanofibers of Fmoc-FFkk or Fmoc-FkFk as illustrated in Fig. 1e. Cultures were fixed and stained every 24 hours for 1-5 days in vitro (DIV) (Figure 2a). No differences were observed in the early stages of neuronal development between Fmoc-FFkk, Fmoc-FkFk and poly-D-lysine substrates (Figure S5, ESI†). In all cultures, lamellipodia were present at day 1, minor processes extended after day 2, axonal polarization and extension occurred at day 3, followed by the development of dendrites on day 4 and marked branching of processes at day 5. This suggests that the nanofiber substrates of Fmoc-FFkk and Fmoc-FkFk support the early stages of neuronal development. Since both peptide nanofiber substrates supported the initial development of primary neurons, we next determined their ability to maintain long-term cultures and allow maturation of synaptic connections. Therefore, primary hippocampal neurons were cultured on coverslips coated with Fmoc-FFkk, Fmoc-FkFk or PDL for 10, 20, 30 or 40 days. Cell viability was determined using an Alamar Blue assay, before fixing and staining. No significant differences in viability were


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observed between the different substrates. Coverslips coated with PDL were used as a positive (100% viability) control. At DIV10 and DIV20, the viability of neurons cultured on peptide nanofibers showed a trend towards reduction. However at longer time points, no significant differences in viability relative to PDL were observed (Figure 2b). Staining of cultures fixed at DIV40 revealed that these long-term cultures form dense networks on peptide nanofiber substrates (Figure 2d, e and Figure S6, ESI†), comparable to PDL (Figure 2c). This clearly indicates that Fmoc-FFkk and Fmoc-FkFk substrates support the long-term culture of primary neurons. Purity and synaptic development of neuronal cultures on nanofiber substrates


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Figure 3. (a) Primary neurons were cultured on PDL or peptide nanofibers and fixed at the times indicated above. Staining was performed with β3-tubulin (red) as a marker for neurons, GFAP (green) to stain astrocytes and IBA-1 (cyan) to identify glial cells. The cell nucleus was also stained (blue). Primary neurons cultured on peptide nanofibers are virtually devoid of any glia, and only few astrocytes are observed. Scale bars, 20 µm. (b) Primary neurons were cultured on


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PDL or peptide nanofibers and fixed at the indicated time points. Staining was performed with synaptophysin (green) marking the pre-synapse, PSD-95 (red) to stain the post-synapse and the cell nucleus (blue). The density of synapses along dendrites was observed over days 10 to 30, (c) with evident marker co-localization. Scale bars, (b) 50 µm and (c) 10 µm. (d) Quantification of synaptic density reveals similar results for neurons cultured on PDL or peptide nanofibers, suggesting the nanofiber network supports the synaptic development of neurons. Since primary neurons cannot divide, it is important to ensure that dividing astrocytes do not overrun the cultures. Therefore, we next established the purity of our neuronal cultures. Primary cultures at different time points (7, 10, 14 and 17 days in culture) were fixed and stained for the presence of neurons (β3-tubulin), astrocytes (GFAP) and microglia (IBA-1) as the major cell types found at different numbers in mixed cultures, Figure 3a. The vast majority of cells in all cultures were neurons; however, a limited number of astrocytes were present. No microglia were observed in any of the cultures, consistent with special requirements needed for this cell type.1 It should be noted that the number of astrocytes does not significantly differ between PDL coated coverslips and peptide nanofibers, and were overall rare. This indicates that Fmoc-FFkk and Fmoc-FkFk substrates are permissive for neuronal growth, which do not invoke neuronal cell death or promote growth of non-neuronal cell types. With long-term culturing of primary neurons on our peptide substrates established, we next assessed their maturation compared to cultures on PDL. It has previously been reported that synapses begin to form in cultured neurons as early as 3 days;54 however development of dendritic spines requires culturing times of at least two weeks.55 The formation of synapses in neurons cultured on coverslips coated with PDL or peptide nanofibers was visualized by immunostaining for pre-synaptic synaptophysin and post-synaptic PSD-95 at time points ranging


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from DIV10 to DIV30 (Figure 3b, Figure S7 and S8, ESI†). Proximity of synaptophysin and PSD-95 staining along dendrites was evident in neurons beyond 14 days in culture, for both PDL and nanofiber coated coverslips (Figure 3c). Synaptic density was quantified, and no significant differences were found between neurons cultured on PDL versus those cultured on peptide nanofiber substrates (Figure 3d). This suggested that neurons seeded on Fmoc-FFkk and FmocFkFk substrates undergo normal maturation. Electrical activity of primary neurons on peptide nanofiber substrates

Figure 4. Electrical activity of neurons cultured on peptide nanofibers. Primary neurons were cultured on PDL, Fmoc-FFkk or Fmoc-FkFk and transfected with GCaMP6s at DIV11, before imaging was undertaken four days later. (a) Still images from videos show synchronized firing at DIV15 for neurons cultured on PDL as well as Fmoc-FFkk and Fmoc-FkFk substrates. Scale bar, 100 µm. Quantification of firing events over time shows (b) random firing at DIV8 and (c) synchronous firing at DIV15.


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To further assess the maturation of primary neurons cultured on peptide nanofibers, we performed adeno-associated virus (AAV)-mediated transductions with GFP at DIV7, before fixing cells at DIV11 and 14, in order to visualize the development of dendritic spines (Figure S9, ESI†). In all cases, the development of spines was evident at DIV14, and was comparable between PDL and peptide nanofiber substrates. Importantly, these experiments also showed that primary neurons cultured on our substrates are suitable for AAV-mediated gene expression. To test this further, a transfection was performed with Lipofectamine, resulting in typically low transfection efficiencies (~2%, Figure S10, ESI†) which have previously been reported for cultures using PDL coating.56 Notably, across primary neurons cultured on PDL or peptide nanofibers, similar transduction efficiencies were observed for AAV-mediated transduction or liposome-mediated transfections, implying that the presence of the cationic peptide does not interfere with the transduction process. This suggests the lysine residues do not interfere with the cationic liposomes of the Lipofectamine reagent, or the AAV. Finally, primary neurons were transduced with a GCaMP construct (AAV-hSyn1-GCaMP6snls-dTomato) in order to visualize electrical activity. Upon binding of calcium, GCaMP undergoes a conformational change which results in fluorescence emission.57,58 Primary neurons were transduced at both DIV4 and DIV11, while imaging was undertaken at DIV8 and DIV15, respectively. In all cases, spontaneous synaptic activity for neurons cultured on peptide nanofibers was observed (Supplementary videos S1-S4). As expected, primary neurons imaged at DIV8 undergo random firing events (Figure S11, ESI†), whereas primary neurons recorded at DIV15 fired in tandem (Figure 4a). Firing events for multiple neurons over time were quantified (Figure S12, ESI†, and averaged, showing random firing events occurring on all substrates at DIV8 (Figure 4b), and highly synchronous firing at DIV15 (Figure 4c). Similar network activity


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was observed for both PDL and both peptide substrates, confirming the development of electrically active neuronal networks. The substrates reported herein provide a platform for the future development of hydrogel cultures to mimic the in vivo environment of the brain. Currently, commonly used culture ware coatings such as poly-lysine, laminin and fibronectin can only form 2D films on coverslips. Production of these extracellular proteins can be expensive and low yielding, whereas PDL polymers may suffer from batch to batch variability. Short peptides offer an alternative where the physical and chemical properties of the peptide can be tuned using a facile, scalable solid phase synthetic method. This includes charge, self-assembly kinetics and fiber morphology. Furthermore, self-assembled peptides are chemically uniform, reducing any batch-to-batch variability between substrates. We have performed proof of concept experiments showing that the peptide nanofibers of Fmoc-FFkk and Fmoc-FkFk can be extended to give 3D cultures that support primary neurons, however optimization of this 3D culture system is ongoing (Figure S13, ESI†). CONCLUSION In conclusion, we have developed two novel tetrapeptides Fmoc-FFkk and Fmoc-FkFk which self-assemble into fibers when dissolved in water. These nanofibers can be coated atop glass coverslips, which have been used to successfully culture primary neurons. These tetrapeptides offer significant time savings over the widely used PDL, as no washing steps are required before seeding of primary neurons onto coverslips. Nanofiber coatings of Fmoc-FFkk and Fmoc-FkFk can be used to culture neurons long-term in vitro. Initially, neurons undergo development as described by Banker,53 before synapse generation occurs. This is followed by the appearance of dendritic spines and electrical activity of the cultured neurons, as confirmed through GCaMP


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calcium imaging. Preliminary results show that these peptides may be used to create 3D primary neuronal cultures for studying drug delivery, neurogenesis or disease models in a setting more relevant to the in vivo environment. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge: Supporting information pdf file and supporting movie files showing electrical activity of neurons cultured on nanofiber substrates. AUTHOR INFORMATION Corresponding Author *Dr Adam. D. Martin, [email protected]. *Professor Lars M. Ittner, [email protected] Author Contributions ADM, PT, TF, YDK and LMI all equally contributed to the design of this work. ADM synthesized and characterized the self-assembling peptides, performed immunostaining, viability measurements and electrical imaging. SWC, CGA and HS prepared the primary neurons. YL cloned GCaMP6s into viral vectors for use in this work. JB supplied AAV-GFP and performed these transfections. MP performed the quantification of neuronal firing at different time points. ACKNOWLEDGMENT We would like to thank the Mark Wainwright Analytical Centre (UNSW) for access to instruments and the Australian Nuclear Science and Technology Organisation (ANSTO) for access to the QUOKKA SANS beamline (proposal 6020). The authors received funding from the


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National Health and Medical Research Council (NHMRC) (1081916, 1132524) to L.I and (1143848, 1143978) to Y.K., the Australian Research Council (ARC) (DP150104321) to Y.K., (CE140100036) to P.T. and (DP170100781, DP170100843). A.D.M. is an ARC-NHMRC Dementia Development Research Fellow (APP1106751), Y.K. is a NHMRC Career Development Fellow (1123564) and L.I. is a NHMRC Principal Research Fellow (1136241). REFERENCES 1. Fath, T.; Ke, Y. D.; Gunning, P.; Gotz, J.; Ittner, L. M. Primary support cultures of hippocampal and substantia nigra neurons. Nat. Protoc., 2009, 4, 78-85. 2. Nakai, J. Dissociated Dorsal Root Ganglia in Tissue Culture. Am. J. Anat., 1956, 99, 81129. 3. Reynolds, B. A.; Tetzlaff, W.; Weiss, S. A Multipotent EGF-responsive Striatal Embryogenic Progenitor Cell Produces Neurons and Astrocytes. J. Neurosci., 1992, 12, 4565-4572. 4. Keller, G. Embryonic Stem Cell Differentiation: Emergence of a New Era in Biology and Medicine. Gene. Dev., 2005, 19, 1129-1155. 5. Kruger, G. M.; Mosher, J. T.; Bixby, S.; Joseph, N.; Iwashita, T.; Morrison, S. J. Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness. Neuron, 2002, 35, 657-669. 6. Stemple, L.; Anderson, D. J. Isolation of a Stem Cell for Neurons and Glia from the Mammalian Neural Crest. Cell, 1992, 71, 973-985. 7. Gauge, F. H. Mammalian Neural Stem Cells. Science, 2000, 287, 1433-1438.


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Peptide nanofiber substrates for long-term culturing of primary neurons

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