Peptide nanofiber substrates for long-term culturing of primary

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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

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Peptide Nanofiber Substrates for Long-Term Culturing of Primary Neurons Adam D. Martin,*,†,§ Sook Wern Chua,† Carol G. Au,† Holly Stefen,‡ Magdalena Przybyla,† Yijun Lin,† Josefine Bertz,† Pall Thordarson,§ Thomas Fath,‡,∥ Yazi D. Ke,† and Lars M. Ittner*,†,∥,⊥

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Dementia Research Unit, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia ‡ Neurodegeneration and Repair Unit, School of Medical Sciences and Neuronal Culture Core Facility, University of New South Wales, Sydney, NSW 2052, Australia § School of Chemistry, The Australian Centre for Nanomedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, University of New South Wales, Sydney, NSW, 2052, Australia ∥ Dementia Research Centre, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia ⊥ Neuroscience Research Australia, Sydney, NSW 2031, Australia S Supporting Information *

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 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 fibers which facilitate adhesion and development of primary neurons, their long-term survival (>40 days), synaptic maturation, and electrical activity. Furthermore, these substrates are permissive toward 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

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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 to provide a more realistic representation of the in vivo environment, as compared to immortalized neuronal cell lines such as HEK, PC12, and NT2 cells13−16 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 substrate is of paramount importance. Most commonly, primary neurons are seeded onto glass coverslips coated with a thin film of polylysine.19 While effective, variability between batches of polylysine owing to its © 2018 American Chemical Society

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 self-association, a number of other self-assembling peptides Received: May 8, 2018 Accepted: July 6, 2018 Published: July 6, 2018 25127

DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

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Figure 1. Lysine containing tetrapeptides (a) Fmoc-Phe-Phe-Lys-Lys and (b) Fmoc-Phe-Lys-Phe-Lys, (c and 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 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.

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.

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 Guler,33,34 among others. However, the majority of these examples employ neural stem cells (NSCs), which typically require expansion of cultures and chemical treatment to maintain stemness. NSCs are capable of proliferation which, while useful for cocultures 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, self-assembly into nanofibers is driven through hydrophobic interactions.35−37 The ease of synthesis 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 cells,42,43 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.

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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 selfassemble into nanofibers; however, the majority of these peptides requires 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 25128

DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

Research Article

ACS Applied Materials & Interfaces

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

k denotes the presence of a D-lysine) and Fmoc-Phe- D-LysPhe- D-Lys (Fmoc-FkFk), bearing two lysine groups (Figures 1a and b) were dissolved in water and spontaneously selfassemble into fibers with β-sheet secondary structures, according to circular dichroism, viscosity, and atomic force microscopy measurements (Figure S1 and 2, Supporting Information and Figures 1c and d). Fiber diameters for Fmoc-FFkk 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, Supporting

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. Here, D-lysines were selected to avoid matrix degradation at longer culture time periods through the action of proteolytic enzymes.51 Analogues bearing only one lysine residue were synthesized, but they were too insoluble to be investigated further. Peptides Fmoc-Phe-Phe-D-Lys-D-Lys (Fmoc-FFkk, where the lower-case 25129

DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

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ACS Applied Materials & Interfaces

Figure 3. (a) Primary neurons were cultured on PDL or peptide nanofibers and fixed at the times indicated above. Staining was performed with β3tubulin (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 PDL or peptide nanofibers and fixed at the indicated time points. Staining was performed with synaptophysin (green) marking the presynapse, PSD-95 (red) to stain the postsynapse, and the cell nucleus (blue). The density of synapses along dendrites was observed over days 10 to 30 (c) with evident marker colocalization. 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.

Information). Some lateral association of fibers is observed in Fmoc-FFkk; however, this may be due to drying effects.52 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, Supporting Information), which is then neutralized upon the addition of Neurobasal media when neurons are seeded atop the nanofibers. Similar charge and pH neutralization have been reported for other peptide nanofibers.53−57

Development and Maturation of Primary Neurons on Nanofiber Substrates. We used the tetrapeptides FmocFFkk 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 self-assembled 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 25130

DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

Research Article

ACS Applied Materials & Interfaces

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.

required here. PDL is generally prepared in borate buffer,1 which is incompatible with cell culture conditions. Thus, each coverslip must be washed multiple times, which is a timeconsuming and laborious process. Thus, the coating procedure for these tetrapeptide nanofiber substrates represents a considerable time savings. Primary murine neurons undergo well-defined developmental stages during their maturation in culture.58 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 Figure 1e. Cultures were fixed and stained every 24 h 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-Dlysine substrates (Figure S5, Supporting Information). 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. Because 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 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 toward 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 (Figures 2d and e and Figure S6, Supporting Information), 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. Because 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 three days;59 however, development of dendritic spines requires culturing times of at least two weeks.60 The formation of synapses in neurons cultured on coverslips coated with PDL or peptide nanofibers was visualized by immunostaining for presynaptic synaptophysin and postsynaptic PSD-95 at time points ranging from DIV10 to DIV30 (Figure 3b, Figures S7 and S8, Supporting Information). 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 FmocFFkk and Fmoc-FkFk substrates undergo normal maturation. 25131

DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134

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ACS Applied Materials & Interfaces Electrical Activity of Primary Neurons on Peptide Nanofiber Substrates. 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 to visualize the development of dendritic spines (Figure S9, Supporting Information). In all cases, the development of spines was evident at DIV14 and 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, Supporting Information) which have previously been reported for cultures using PDL coating.61 Notably, across primary neurons cultured on PDL or peptide nanofibers, similar transduction efficiencies were observed for AAVmediated 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-GCaMP6s-nls-dTomato) to visualize electrical activity. Upon binding of calcium, GCaMP undergoes a conformational change which results in fluorescence emission.62,63 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, Supporting Information), whereas primary neurons recorded at DIV15 fired in tandem (Figure 4a). Firing events for multiple neurons over time were quantified (Figure S12, Supporting Information 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 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 polylysine, 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, Supporting Information).

when dissolved in water. These nanofibers can be coated atop glass coverslips, which were 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 Banker53 before synapse generation occurs. This is followed by the appearance of dendritic spines and electrical activity of the cultured neurons, as confirmed through GCaMP 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.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

The following files are available free of charge: Supporting Information pdf file and supporting movie files showing . The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b07560. Further experimental details, including synthesis and characterization of peptide substrates, cultures and measurements with corresponding figures (PDF) Random firing of neurons cultured on Fmoc-FFkk nanofiber substrates at DIV8 (AVI) Random firing of neurons cultured on Fmoc-FkFk nanofiber substrates at DIV8 (AVI) Synchronous firing of neurons cultured on Fmoc-FFkk nanofiber subtrates at DIV15 (AVI) Synchronous firing of neurons cultured on Fmoc-FkFk nanofiber substrates at DIV15 (AVI)

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Adam D. Martin: 0000-0002-5445-0299 Pall Thordarson: 0000-0002-1200-8814 Lars M. Ittner: 0000-0001-6738-3825 Author Contributions

A.D.M., P.T., T.F., Y.D.K., and L.M.I. all equally contributed to the design of this work. A.D.M. synthesized and characterized the self-assembling peptides and performed immunostaining, viability measurements and electrical imaging. S.W.C., C.G.A., and H.S. prepared the primary neurons. Y.L. produced viral vectors containing GCaMP6s for use in this work. J.B. supplied AAV-GFP and performed these transfections. M.P. performed the quantification of neuronal firing at different time points. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 National Health and

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CONCLUSION In conclusion, we developed the two novel tetrapeptides Fmoc-FFkk and Fmoc-FkFk, which self-assemble into fibers 25132

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Medical Research Council (NHMRC) (1081916, 1132524) to L.I., (1083209) to T.F. and (1143848, 1143978) to Y.K., the Australian Research Council (ARC) (DP150104321) to Y.K., (DP180101473) to T.F., (CE140100036) to P.T. and (DP170100781, DP170100843) to L.I. A.D.M. is an ARCNHMRC Dementia Development Research Fellow (APP1106751), Y.K. is a NHMRC Career Development Fellow (1123564), and L.I. is a NHMRC Principal Research Fellow (1136241).

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ACS Applied Materials & Interfaces

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DOI: 10.1021/acsami.8b07560 ACS Appl. Mater. Interfaces 2018, 10, 25127−25134