Patterned poly(chlorotrifluoroethylene) guides primary nerve cell adhesion and neurite outgrowth

Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury. Recently it has been shown that in the developing CNS, a combination of cell-adhesive and cell-repulsive cues guide growing axons to their targets. We hypothesized that by mimicking these guidance signals, we could guide nerve cell adhesion and neurite outgrowth in vitro. Our objective was to direct primary nerve cell adhesion and neurite outgrowth on poly(chlorotrifluoroethylene) (PCTFE) surfaces by incorporating alternating patterns of cell-adhesive (peptide) and nonadhesive (polyethylene glycol; PEG) regions. PCTFE was surface-modified with lithium PEG-alkoxide, demonstrating the first report of metal-halogen exchange with an alkoxide and PCTFE. Titanium and then gold were sputtered onto PEG-modified films, using a shadow-masking technique that creates alternating patterns on the micrometer scale. PCTFE-Au regions then were modified with one of two cysteine-terminated laminin-derived peptides, C-GYIGSR or C-SIKVAV. Hippocampal neuron cell-surface interactions on homogeneously modified surfaces showed that neuron adhesion was decreased significantly on PEG-modified surfaces and was increased significantly on peptide-modified surfaces. Cell adhesion was greatest on CGYIGSR surfaces while neurite length was greatest on CSIKVAV surfaces and PLL/laminin positive controls, indicating the promise of peptides for enhanced cellular interactions. On patterned surfaces, hippocampal neurons adhered and extended neurites preferentially on peptide regions. By incorporating PEG and peptide molecules on the surface, we were able to simultaneously mimic cell-repulsive and cell-adhesive cues, respectively, and maintain the biopatterning of primary CNS neurons for over 1 week in culture.     

Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers

Embryonic hippocampal neurons cultured on surface modified fluoropolymers showed enhanced interaction and neurite extension. Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surfaces were aminated by reaction with a UV-activated mercury ammonia system yielding FEP-[N/O]. Laminin-derived cell-adhesive peptides (YIGSR and IKVAV) were coupled to FEP surface functional groups using tresyl chloride activation. Embryonic (E18) hippocampal neurons were cultured in serum-free medium for up to 1 week on FEP film surfaces that were modified with either one or both of GYIGSR and SIKVAV or GGGGGGYIGSR and compared to control surfaces of FEP-[N/O] and poly(L-lysine)/laminin-coated tissue culture polystyrene. Neuron-surface interactions were analyzed over time in terms of neurite outgrowth (number and length of neurites), cell adhesion and viability. Neurite outgrowth and adhesion were significantly better on peptide-modified surfaces than on either FEP or FEP-[N/O]. Cells on the mixed peptide (GYIGSR/SIKVAV) and the spacer group peptide (GGGGGGYIGSR) surfaces demonstrated similar behavior to those on the positive PLL/laminin control. The specificity of the cell-peptide interaction was demonstrated with a competitive assay where dissociated neurons were incubated in media containing peptides prior to plating. Cell adhesion and neurite outgrowth diminished on all surfaces when hippocampal neurons were pre-incubated with dissolved peptides prior to plating.     

B-50/GAP43 localization on membranes of putative transport vesicles in the cell body, neurites and growth cones of cultured hippocampal neurons.

We conducted an electron microscopic immunocytochemical study to locate B-50/GAP43 in various cellular elements of hippocampal neurons grown in dissociated cell culture. B-50 was detected with pre- and post-embedding immunoincubation, using affinity-purified B-50 antibodies and secondary antibodies coated on gold probes. For the first time ultrastructural evidence is presented for the location of B-50 on the membranes of electron-lucent transport vesicles with a diameter of 99.4 +/- 2.9 nm, present in the trans region of the Golgi apparatus, in neurites and in growth cones of cultured hippocampal neurons.     

Two-Dimensional Protein Micropatterning for Sensor Applications Through Chemical Selectivity Technique

Two-dimensional protein micropatterning with immobil-ization of IgG and poly (ethylene glycol) (PEG) on patterned Au and Si surfaces was performed through a new technique. The technique for micropatterning is based on a chemical selectivity method by creating chemical bonding between protein, self-assembled monolayers (SAMs) and substrates rather than physical means. The substrates used in this study are pre-fabricated with silicon wafer patterned with arrays of gold squares. The silicon regions of the substrate are modified with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. The gold regions on the substrate are first immobilized with bifunctional SAM layers that can covalently bound adhesion proteins for individual cell attachment against a PEG background. The surface coatings are characterized by contact angle measurement, ellips-ometry, and atomic force microscopy (AFM). The patterns of fluorescence-labeled proteins are examined using fluorescence microscopy. Our study demonstrated that the PEG modified silicon region showed an effective protein reduction while the gold regions were successfully covalently bonded with proteins. This technique also demonstrated a combined feature of ensuring the activity, selectivity, and stability of the immobilized proteins. A simple lift-off microfabrication process was introduced in this study to pattern metal on silicon substrates without using expensive metal etching.     

In vitro assessment of bioactive coatings for neural implant applications

Recent efforts in our laboratory have focused on developing methods for immobilizing bioactive peptides to low cell-adhesive dextran monolayer coatings and promoting biospecific cell adhesion for biomaterial implant applications. In the current study, this dextran-based bioactive coating technology was developed for silicon, polyimide, and gold, the base materials utilized to fabricate our prototype neural implants. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). We observed that surface-immobilized dextran supported very little cell adhesion in vitro (24-h incubation with serum-supplemented medium) on all base materials. Inactive nonadhesion-promoting Gly-Arg-Ala-Asp-Ser-Pro peptides immobilized on dextran-coated materials promoted adhesion and spreading at low levels not significantly different from dextran-coated substrates. Arg-Gly-Asp (RGD) peptide-grafted surfaces were observed to promote substantial fibroblast and glial cell adhesion with minimal PC12 (neuronal cell) adhesion. In contrast, dextran-coated materials with surface-grafted laminin-based, neurite-promoting Ile-Lys-Val-Ala-Val (IKVAV) peptide promoted substantial neuron cell adhesion and minimal fibroblast and glial cell adhesion. It was concluded that neuron-selective substrates are feasible using dextran-based surface chemistry strategies. The chemical surface modifications could be utilized to establish a stable neural tissue-implant interface for long-term performance of neural prosthetic devices.     

Corneal epithelial adhesion strength to tethered-protein/peptide modified hydrogel surfaces

In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP.     

Photolithographic patterning of polyethylene glycol hydrogels

A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor solution were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradiation time and precursor solution concentration on the levels of immobilized peptide were characterized, demonstrating that bound peptide concentration can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addition, 3D structures can be generated by incorporating PEG-diacrylate into the precursor solution. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnology.     

Preparation of uniaxial multichannel silk fibroin scaffolds for guiding primary neurons

Physical guidance cues have been exploited to stimulate neuron adhesion and neurite outgrowth. In the present study, three-dimensional (3-D) silk fibroin scaffolds with uniaxial multichannels (42-142 μm in diameter) were prepared by a directional temperature field freezing technique, followed by lyophilization. By varying the initial silk fibroin concentration, the chemical potential and quantity of free water around cylindrical ice crystals could be controlled to control the cross-section morphology of the scaffold channels. Aligned ridges also formed on the inner surface of the multichannels in parallel to the direction of the channels. In vitro, primary hippocampal neurons were seeded in these 3-D silk fibroin scaffolds with uniaxial multichannels of ～120 μm in diameter. The morphology of the neurons was multipolar and alignment along the scaffold channels was observed. Cell-cell networks and cell-matrix interactions established by newly formed axons were observed after 7 days in culture. These neurons expressed β-III-tubulin, nerve filament and microtubule-associated protein, while glial fibrillary acidic protein immunofluorescence was barely above background. The ridges on the inner surface of the channels played a critical role in the adhesion and extension of neurons by providing continuous contact guidance. These new 3-D silk scaffolds with uniaxial multichannels provided a favorable microenvironment for the development of hippocampal neurons by guiding axonal elongation and cell migration.     

Simple approach to micropattern cells on common culture substrates by tuning substrate wettability

The ability to spatially control cell adhesion and multicellular organization is critical to many biomedical and tissue-engineering applications. This work describes a straightforward method to micropattern cells onto glass, silicone rubber, and polystyrene using commercially available reagents. An elastomeric polydimethylsiloxane stamp is used to contact-transfer extracellular matrix protein onto a surface followed by blocking cell adhesion in the surrounding regions by the physisorption of Pluronic surfactants. Using self-assembled monolayers of alkanethiols on gold as model surfaces to control surface wettability, we found that protein printing was most effective at intermediate to highly wetting surfaces whereas Pluronic adsorption occurred at intermediate to low wetting surfaces. Within a regimen of intermediate wettability both techniques were applied in conjunction to restrict cell adhesion to specified patterns. Adjusting the wettability of common tissue culture substrates to the same intermediate range again allowed the micropatterning of cells, suggesting that this approach is likely to be generally applicable to many types of materials. This technique therefore may allow for wider adoption of cell patterning.     

Adhesion of alpha5beta1 receptors to biomimetic substrates constructed from peptide amphiphiles

Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III(9-10) sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air-water interface using Langmuir-Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated alpha5beta1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of alpha5beta1, to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to alpha5beta1 integrin was required for highest adhesion.     

Neurite outgrowth on well-characterized surfaces: preparation and characterization of chemically and spatially controlled fibronectin and RGD substrates with good bioactivity

Study of axonal growth and ligand-receptor interactions requires specificity and careful characterization of the biomaterial substrates to which the neurons bind. It would be impossible to predict the effects of important variables such as composition, surface density, spatial distribution, and conformation of the ligands on axonal growth of a neuron without highly specific surface characterization. Here, we compare two methods of surface modification (hereafter referred to as "Heterobifunctional Crosslinker" and "Pluronics" methods) used for immobilization of fibronectin (FN) and FN-derived, RGD-containing peptides to the substrates. We also characterized their performance in neurite outgrowth experiments. Various surface analytical techniques such as contact angle measurement, XPS, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used for the analysis of the substrates at each step of the two different chemistries involved. FN-patterned surfaces were created by micro-contact printing methods and confirmed by imaging TOF-SIMS, and AFM techniques. After immobilization of FN and/or FN-derived RGD-containing peptide, including the formation of micron-scale patterns of FN, the modified surfaces were plated with neurons from postnatal rat dorsal root ganglia (DRG) and incubated in serum-free medium. Both the peptide- and/or protein-modified substrates supported significantly greater neurite outgrowth than controls, and outgrowth on both substrate chemistries was inhibited by the addition of soluble RGD peptide. Patterned FN surfaces were successful in spatially controlling the neuron attachment and outgrowth.     

Behaviour of SH-SY5Y neuroblastoma cell line grown in different media and on different chemically modified substrates

Among the parameters that can be tested in experiments on neuronal cell culture the use of different culture media and substrates represents a powerful assay to influence cell adhesion and differentiation. In this work, plasma-enhanced-chemical vapour depositions (PE-CVD) from acrylic acid and allylamine vapours have been performed to deposit coatings bearing oxygen (O)- and nitrogen (N)-containing functional groups on polyethylenetherephtalate (PET) surface. Human neuroblastoma SH-SY5Y cells were grown on plasma modified substrates and in presence of media containing different amount of fetal calf serum (FCS) or in serum-free medium containing cAMP. Our results showed that N-containing substrates improved cell adhesion, while the neurites sprouting was influenced by cell culture media. Interestingly, the presence of carboxylic groups on the modified surface can influence the expression of a differentiation marker, neurofilament-200 (NF-H), in cells grown in serum-containing media.     

Guided cell patterning on gold-silicon dioxide substrates by surface molecular engineering

We report an effective approach to patterning cells on gold-silicon dioxide substrates with high precision, selectivity, stability, and reproducibility. This technique is based on photolithography and surface molecular engineering and requires no cell positioning or delivery devices, thus significantly reducing the potential damage to cells. The cell patterning was achieved by activating the gold regions of the substrate with functionalized thiols that covalently bind proteins onto the gold regions to guide subsequent cell adhesion while passivating the silicon dioxide background with polyethylene glycol to resist cell adhesion. Fourier transform infrared reflectance spectroscopy verified the successful immobilization of proteins on gold surfaces. Protein patterns were visualized by tagging proteins with Rhodamine fluorescent probes. Time-of-flight secondary ion mass spectrometry was used to characterize the chemistry of both the cell-adhesive and cell-resistant regions of surfaces after each key chemical reaction occurring during the molecular surface engineering. The ability of the engineered surfaces to guide cell adhesion was illustrated by differential interference contrast (DIC) reflectance microscopy. The cell patterning technique introduced in this study is compatible with micro- and photo-electronics, and may have many medical, environmental, and defense applications.     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

β淀粉样蛋白25～35急性给药和慢性孵育对神经元Ca2+非依赖性的K+电流作用的比较

目的 探讨β-淀粉样蛋白25～35（Aβ _25-35）急性给药和慢性孵育对神经元Ca ²⁺非依赖性的K+电流作用的区别。 方法 急性分离大鼠海马及培养皮层神经元; Aβ25-35急性给药(3min)或慢性孵育（24h）;利用全细胞膜片钳技术记录Ca2+非依赖性的K+电流以及Calcein-AM法检测神经元活力。 结果 Aβ _25-35急性给药使急性分离的海马神经元Ca ²⁺非依赖性的K+电流幅度明显降低(n=11),而慢性孵育则使培养的皮层神经元该电流幅度明显升高(n=11)。前者是Aβ _25-35通过对K+通道直接的效应发挥其抑制作用,而后者可能主要是通过Aβ _25-35上调通道蛋白,改变通道数量而发挥作用。 结论 不同的给药方式通过不同的机制对海马和皮层神经元的Ca ²⁺非依赖性的K+电流产生不同的作用。     

Surface patterning using plasma-deposited fluorocarbon thin films for single-cell positioning and neural circuit arrangement

Micropatterning glass substrates with a plasma-deposited fluoropolymer thin film was shown to be an efficient approach to manipulate cell positioning. The glass windows promoted cell adhesion, whereas the surrounding fluoropolymer displays a cell-repelling character. Herein, multiple micropatterned substrates were developed with pattern dimensions sufficient to host solely single-cells. These single-cell arrays would allow analysis of individual cell response to stimulation without interference from cell-cell interactions. Mouse myoblast C2C12 cells and cortical neurons from mice were examined, both for amenability to patterning, as well as success of cell adhesion and cell morphology. Both cell types were found to have optimal adherence and growth on the glass surface, while cell adhesion and function was inhibited on the fluoropolymer. The C2C12 cells conformed to the shape of the pattern, while maintaining a healthy structure. Moreover, the neuron cells followed the hexagonal grid patterns and formed circuits, wherein the complexity of the connections depended on incubation time.     

Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons

Self-assembled monolayers (SAMs) of omega-substituted alkanethiols on gold have been explored as well defined in vitro model surfaces for the investigation of neuronal growth and function. When used as cell culture substrates, surfaces with monolayers functionalized with terminal -COOH groups support neuron attachment and growth even without an intermediate protein layer. Addition of a poly-L-lysine layer (PLL) to the -COOH terminated monolayers significantly increases total neurite outgrowth. Mixed monolayers containing -COOH and -CH3 terminal groups in 1:10 and 1:100 ratios poorly support neuron adhesion and preclude neurite extension. A layer of PLL improves the ability of mixed monolayer surfaces to support neuronal growth in culture. The morphology of cultured neurons depends on the chemical composition of SAMs on the support surface. Using glass microelectrode intracellular recording, the properties of cell culture substrates modulate the dynamic properties of action potentials of cultured neurons. These findings provide insight into the cellular responses of excitable cells to the chemical details of a surface and, thus, may help direct the rational design of biologically active materials.     

Combined chemical and topographical guidance cues for directing cytoarchitectural polarization in primary neurons

Chemical and topographical cues can be used to guide dissociated neurons into user-defined network geometries on artificial substrates, yet control of neuron polarity (differentiation into axons and dendrites) remains an elusive goal. We developed a dual guidance cue strategy for directing morphological maturity in neurons in vitro using combined chemical and topographical guidance cues on glass substrates. The surface chemistry provides chemical attraction and repulsion for controlling neuron placement and outgrowth, while the topography provides additional surface area for neuron attachment. Poly-l-lysine (PLL) was adsorbed into etched trenches in glass substrates, and an acetone liftoff process was used to produce bifunctional surfaces with a hydrophobic hexamethyldisilazane (HMDS) background and trench patterns of PLL. We examined the cytoarchitectural polarization of dissociated hippocampal pyramidal neurons on guidance cues designed to promote rapid outgrowth of neurites onto continuous line features and delayed neurite outgrowth onto interrupted line features. An optimum distance of approximately 5 μm between the cell body attachment node and the first interrupted line guidance cue led to specific cytoarchitectural polarization of ≥60% of neurons by 3 days of culture in vitro.     

Micropatterned polymer substrates control alignment of proliferating Schwann cells to direct neuronal regeneration

Microcontact printed polymeric substrates were evaluated for their ability to control Schwann cell attachment and direct proliferation, as Schwann cell guidance is a crucial factor in directing peripheral nerve regeneration. Elastomeric stamps of poly(dimethylsiloxane) were "inked" with laminin, a permissive protein for Schwann cell adhesion, and stamped onto poly(methyl methacrylate) substrates to create patterns of lines and intervals varying from 10 to 50 microm wide. Schwann cells were seeded onto the substrates in serum-free media. After 4h, media was replaced with serum-containing growth media and changed daily thereafter. The addition of growth media to stimulate proliferation initially caused some loss in cell orientation relative to the laminin pattern, but when monolayer formation was complete, a high degree of cell orientation was observed. As both cell-cell contacts and surface coverage were maximized, the Schwann cells achieved an even higher order of orientation than observed during the early stages of proliferation. Significantly, smaller pattern widths increased the degree of orientation, regardless of interval width. Our results indicate that patterned polymeric substrates may enhance peripheral nerve regeneration by creating a highly ordered Schwann cell matrix for guidance of neurons.     

Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices

Current needs of central nervous system therapy urge for the identification of scaffolds supporting the generation and long-term maintenance of healthy and functional neuronal tissue. We compared for the first time the viability of hippocampal neurons and astrocytes grown on conventional 2-dimensional (2D) conditions with that of cells grown on an aragonite bioactive 3-dimensional (3D) scaffold prepared from coralline exoskeleton. Cultures in 3D showed significantly lower mortality rate and higher neurons/astrocytes ratio than 2D cultures. Moreover, whereas cell survival in 2D was arrested in the absence of the supporting substrates poly-D-lysine and laminin, these substrates had negligible effect on the 3D cultures. Furthermore, aragonite matrices supported cell survival and growth under conditions of calcium and nutrients deprivation, whereas in 2D such treatments led to death of all neurons and of almost all astrocytes. To show that the aragonite matrices are permissive for neural cells also in vivo, aragonite matrices having no substrate coating grafted into postnatal rat cortex were invaded by neurons growing on the surface and in multilayer structures resembling those seen in the 3D culture in vitro. Hence, culture of neurons and astrocytes on 3D aragonite coralline matrices is a novel mean for production of stable neuronal tissue, with significant implication to the field of neuronal tissue restoration.